Protein Engineering of Pyruvate Oxidase from Lactobacillus Plantarum for Application in Biosensors

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Doctoral Thesis

Protein engineering of pyruvate oxidase from Lactobacillus plantarum for application in biosensors

Author(s): Kramer, Matthias

Publication Date: 2008

Permanent Link: https://doi.org/10.3929/ethz-a-005708619

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ETH Library Doctoral Thesis ETH No. 17765

PROTEIN ENGINEERING OF PYRUVATE OXIDASE FROM LACTOBACILLUS PLANTARUM FOR APPLICATION IN BIOSENSORS

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of Doctor of Natural Sciences

presented by

MATTHIAS KRAMER

Eidg. dipl. Apotheker, ETH Zürich born 25.05.1975 citizen of Full-Reuenthal AG

deposited to examination

Prof. Dr. G. Folkers, examiner Prof. Dr. L. Scapozza, co-examiner Prof. Dr. U. Spichiger-Keller, co-examiner Prof. Dr. A. Schubiger, co-examiner

2008

Für meine Familie

“Nicht im Wissen liegt das Glück, sondern im Erwerben von Wissen.“

Edgar Allen Poe

Table of Contents

Table of Contents I

Abbreviations V

Summary IX

Zusammenfassung XI

1 Introduction 1

1.1 The Aim of the Work...... 3 1.1.1 Working Hypothesis ...... 3 1.2 Phosphate Analysis ...... 4 1.2.1 Chemical Properties and Deposits of Phosphate ...... 4 1.2.2 Phosphate Measurement in Water ...... 5 1.2.3 Phosphate in Clinical Diagnostics ...... 7 1.2.4 Phosphate Measurement in the Food and Pharmaceutical Industry ...... 9 1.2.5 Phosphate Measurement with Traditional Chemical Methods ...... 10 1.2.6 The Potential of Phosphate Biosensors...... 11 1.3 Pyruvate Analysis ...... 11 1.3.1 Pyruvate a Key Metabolite...... 11 1.3.2 Pyruvate Analysis in Clinical Diagnostics...... 12 1.3.3 Further Advantages of Pyruvate Sensors...... 13

2 Phosphate Biosensors 15

2.1 What are Biosensors ...... 15 2.1.1 Definition of Biosensors ...... 15 2.1.2 History of Biosensors...... 16 2.1.3 Phosphate and Pyruvate Analysis: Advantages of Sensors and Biosensors Compared to Conventional Analytical Techniques ...... 17 2.1.4 Disadvantages of Biosensors Compared to Conventional Analytical Tech- niques ...... 21 2.2 An Ideal Phosphate Sensor Setup...... 21 2.2.1 Recognition Elements for Phosphate Sensors...... 21 2.2.2 Recognition Elements for Pyruvate Sensors...... 34 2.2.3 Transducing Techniques in Biosensors ...... 35 2.2.4 Application of Pyruvate Oxidase from Lactobacillus plantarum in Biosensors...... 38

I 2.2.5 Electron Transfer in Bioelectronics ...... 42 2.2.6 Electron Transfer in Enzymatic, Amperometric Biosensors ...... 43 2.3 Setup of the Phosphate and Pyruvate Biosensor...... 45 2.3.1 Development of the Biosensor Device ...... 45 2.3.2 Composition of the Sensing Paste ...... 46 2.3.3 Stability of the Triple Mutant lpPOX in the Biosensor ...... 48 2.4 Conclding remarks ...... 49 2.5 Sensor Stability ...... 50 2.5.1 Definition of Sensor Stability ...... 50

3 Protein Stability 53

3.1 Enzyme Inactivating Processes...... 53 3.1.1 Common Protein Denaturation Processes...... 53 3.1.2 Common Extrinsic Factors that Destabilise Proteins...... 54 3.1.3 Common Intrinsic Factors that Destabilise Proteins...... 61 3.2 Protein Stabilisation Techniques...... 64 3.2.1 Extremozymes Show Different Stabilisation Concepts...... 67 3.2.2 Stability of Glucose Oxidase, an Enzyme Designed for Application in Biosen- sors? ...... 68 3.2.3 Stability of Structural Proteins...... 70 3.2.4 Conclusions taken from these Common Stabilising Techniques...... 71 3.3 Procedure in Biosensor Research in Cases of Stability Insufficiency...... 72 3.3.1 Stability Testing in the Sensor ...... 72 3.3.2 Screening for stable enzymes...... 73 3.3.3 Protein Stabilisation and Sensor Optimisation ...... 75 3.4 Structure and Stability of lpPOX...... 75 3.4.1 Structure of lpPOX ...... 75 3.4.2 Stability and currently known Stabilisation of lpPOX ...... 79 3.4.3 Protein Destabilising Extrinsic Factors in the Amperometric Biosensor .... 82 3.5 Most Promising Stabilising Methods for lpPOX in the Biosensor ...... 84 3.5.1 Extrinsic Protein Stabilisation Techniques for lpPOX ...... 84 3.5.2 Intrinsic Protein Stabilisation Techniques for lpPOX ...... 85 3.6 Conclusions and Hypothesis ...... 89

4 Expression and Purification of lpPOX 91

4.1 Introduction - The Challenge of Soluble Expression of lpPOX...... 91 4.1.1 Strategy for lpPOX Expression...... 91 4.1.2 Insoluble POX Precipitated in Inclusion Bodies ...... 92 4.1.3 In vitro Refolding of Unsoluble Protein ...... 93 4.1.4 Strategy of Fusion Proteins...... 93

II 4.1.5 Strategy of Controlled Induction Rate ...... 94 4.2 Materials and Methods...... 95 4.2.1 Materials ...... 95 4.2.2 Methods...... 98 4.3 Results...... 103 4.3.1 Expression Vector...... 103 4.3.2 Protein Expression ...... 104 4.3.3 Protein Purification ...... 106 4.4 Discussion ...... 110 4.4.1 Expression System...... 110 4.4.2 Equilibration with Cofactors and Purification ...... 111

5 Stabilisation of the Quaternary Structure and Cofactor Sites 113

5.1 Introduction...... 113 5.1.1 Stabilisation at the Tetrameric Interface...... 113 5.1.2 Effects of Cofactors on Protein Stability ...... 114 5.1.3 Selection of Promising Mutations Concerning Cofactor Binding ...... 115 5.2 Materials and Methods...... 116 5.2.1 Chemicals and Enzymes ...... 116 5.2.2 Methods...... 117 5.3 Results...... 119 5.3.1 Mutagenesis and Transformation...... 119 5.3.2 Expression...... 120 5.3.3 Purification...... 120 5.3.4 Trx Does not Influence the Activity of lpPOX...... 122 5.3.5 Specific Activity of Mutant Proteins ...... 122 5.3.6 Stability Testing Using Heat Inactivation...... 123 5.4 Discussion ...... 126 5.4.1 Quaternary Stabilisation...... 126 5.4.2 Cofactor Mutants ...... 127 5.4.3 Perspective on Further Cofactor Stabilisation ...... 128

6 Entropic Stabilisation 129

6.1 Introduction...... 129 6.1.1 Entropic Stabilisation by Proline ...... 129 6.1.2 Entropic stabilisation by Glycine Replacement...... 131 6.2 Methods...... 131 6.2.1 Selection of Stabilising Xaa-Pro Mutations...... 131 6.2.2 Selection of Stabilising Gly-Xaa Mutations ...... 133 6.2.3 Mutagenesis of Proposed Stabilising Mutants...... 133

III 6.2.4 Expression and Purification of the Mutants...... 135 6.2.5 Thermal Stability monitored by far-UV-CD...... 135 6.2.6 Kinetics of Protein Inactivation and Longterm Stability Experiments...... 136 6.2.7 Kinetics of Protein Inactivation ...... 137 6.3 Results...... 138 6.3.1 Mutagenesis and Transformation...... 138 6.3.2 Expression and Purification of the Mutants...... 138 6.3.3 Specific Activity of Mutant Proteins ...... 138 6.3.4 Stability Testing Using Heat Inactivation...... 140 6.3.5 Biophysical Characterisation of Selected Mutants ...... 141 6.3.6 Longterm Stability Data...... 144 6.4 Discussion ...... 146 6.4.1 Stability of Single Xaa->Pro Mutants...... 146 6.4.2 Discussion of CD Experiments...... 147 6.4.3 Stability of Double Mutant (L193P_S559P) ...... 147 6.4.4 Glycine to Alanine Mutations...... 148 6.4.5 Long-term Stability Experiments...... 149 6.4.6 Outlook ...... 149

7 Final Discussion and Outlook 151

7.1 Methods of Producing lpPOX...... 151 7.2 Stabilising Concepts...... 152 7.2.1 Quaternary structure Stabilisation...... 152 7.2.2 Stabilisation at the Cofactor Binding Site...... 152 7.2.3 Stabilisation by Backbone Rigidification ...... 153 7.3 Outlook ...... 153

Bibliography 157

Danksagung 175

Curriculum Vitae 177

IV Abbreviations

4-AAP 4-amino-antipyrine ∆G Gibbs free energy aa amino acid ALAT alanine aminotranferase (EC 2.6.1.2) anGOD Aspergillus niger glucose oxidase (EC 1.1.3.4) ADP adenosine diphosphate AMP adenosine monophosphate ATP adenosine triphosphate ASA solvent accessible surface area Å 1 Ångstrom = 0.1 nm bcGLUC Bacillus cereus oligo-1,6-glucosidase BL21-SITME. coli cellline for protein expression by the use of adjustable induction rate bp base pairs (unit for DNA lenght) BSA bovine serum albumin CCS Centre for Chemical Sensors, Technopark ETH, Zurich CD circular dichroism cDNA cyclic DNA CFSS continuous flow sensor system CE counter electrode CV cyclic voltammogram D denatured protein state DA-bipy diamino bipyridine DHS sodium-3,5-dichloro-2-hydroxy-benzolsulfonate DGM diffusion coefficient of ?-D-glucose in dialysis membrane [cm2 s-1] DGP diffusion coefficient of ?-D-glucose in the "paste" [cm2 s-1] DNA desoxyribonucleic acid DNAse DNA degrading enzyme DOPA L-3,4-dihydroxyphenylalanine dsDNA double strand DNA DSC differential scanning calorimetry DTT dithiothreitol E electromotive force of an electrochemical reaction Eh° standard electromotive force of an electrochemical reaction

V EC enzyme class EC European Comunity EDTA etylenediaminetetraacetic acid ELISA enzyme linked immuno sorbent assays Eq. equation ETH Swiss Federal Institute of Technology Zurich (Eidg. Techn. Hochschule) FAD flavinadenine dinucleotide FET field-effect transistor FPLC fast protein liquid chromatography GAPDH glyceraldehyde-3-phosphate-dehydrogenase GOD glucose oxidase H-bond hydrogen bond HRP horse-radish peroxidase (EC 1.11.1.7) IKC Institute for Clinical Chemistry at the University Hospital Zurich IPTG isopropyl-β-D-thiogalactopyranoside ISE ion selective electrode i.v. intra venous kcat catalytic constant (turnover number) kDa kilo Dalton Km Michaelis-Menten constant LB mediumLuria-Bertani medium LBON Luria-Bertani medium omited NaCl LDH lactate dehydrogenase (EC. 1.1.1.27) llDHOD Lactobacillus lactis dihydroorotate dehydrogenase (EC 1.3.99.11) lpPOX Lactobacillus plantarum pyruvate oxidase (EC 1.2.3.3) med medical application of proteins (also called „red“ biotechnology) mM millimole per litre MRD magnetic relaxation dispersion MWCO molecular weight cut off N native protein state NAD+ nicotineamide adenin dinucleotide (oxidiced form) NADH nicotineamide adenin dinucleotide (reduced form) NME new molecular entity NusA-tag NusA fusion protein tag OD600nm optical density at 600 nm wavelength

VI oiPOX pyruvate oxidase of Oceanobacillus iheyensis PCR polymerase chain reaction PDB protein data base PEG polyethylenglycol pET The pET system is a widely used systems for the cloning and in vivo expression of- recombinant proteins in E. coli. It is protected by U.S. Patent no. 4,952,496 3- Pi orthophosphate ion (PO4) pKa the negative logarithm of the ionization constant (K) of an acid pH negative logarhitmic concentration of H+ PMSF phenyl-methyl-sulfonyl fluoride (serin protease inhibitor) POX pyruvate oxidase (EC. 1.2.3.3) RB refractile or inclusion bodies RE reference electrode RNA ribonucleic acid RSCB Reserach Collaboration for Structural Bioinformatics SAEFL Swiss Agency for the Environment, Forests and Landscape (BUWAL) SDS sodium dodecylsulphate SPR surface plasmon responce SR Swiss law numbers (Systematische Sammlung des Bundesrechts) tech technical application of proteins (also called „white“ biotechnology) Td temperature of deactivation (temperature at which 50% of the protein is inactivated) ThDP thiamine diphosphate Tm melting temperature (temperature at which 50% of the protein lost his sec. structure) TrEMBL database of Translated nucleotide sequences of the Europ. Mol. Biology Laboratory Trx-tag Thioredoxin fusion protein tag TSR template suppression reagent U 1 unit is defined as the amount of enzyme, which converts 1 µmol substr/min (pH7, 25°C) vmax maximal enzyme reaction rate [U mg-1] VdW Van der Waals V141C valine to cysteine mutation at amino acid position no. 141 WE working electrode wt wild typ (in this thesis wt stands for a threefold mutated lpPOX (P178S, S188N,

VII A458V) provided by Roche Diagnostics) X irreversibly unfolded protein state

VIII Summary

he application of pyruvate oxidase (POX) to a biosensor allows the measurement of phosphate or T pyruvate in aqueous solution. These two substrates are very attractive analytes mainly in the environmental, medical and food industry. An advantage of using POX from Lactobacillum plantarum (lp- POX) in a biosensor is that the expensive and unstable cofactors flavinadenine dinucleotide (FAD), thiamine pyrophosphate (ThDP), and manganese-ion (Mg2+) need not be added to the sample solution because the cofactors are relatively tightly bound to the enzyme compared to other POX. The lpPOX was implemented in a previous project as an amperometric paste biosensor within a flow cell developed at the Centre for Chemical Sensors (CCS). Under continuous measurement conditions the lpPOX-biosensor showed a signal decrease of 85% after 30 h whereas a glucose-oxidase biosensor did not show any significant alteration of the signal after 10 days. The lifetime of the pyruvate oxydase sensor does not meet the requirement of less than 50% signal loss within two weeks corresponding to a lifetime of at least two weeks. In order to improve the lifetime, the enzyme structure has to be stabilised. The aim of this project was to develop and produce a stabilised lpPOX in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor would be less than 50%. A deep analysis of the structural details and the deactivation process led to the hypothesis that structural based rational protein design is a promising approach for enzyme stabilisation. Three different stabilisation strategies were tested. The first was focused on ameliorating the interactions between the protein monomers. The second was targeted to improve the interactions between cofactors and the protein and the third one was directed at backbone rigidification (entropic stabilisation). The starting template for further protein engineering was the threefold mutated lpPOX (P178S, S188N, A458V) with a higher tolerance to pH-values between 4.5 and 7.5 (Risse et al. 1992b). In order to produce the proposed mutant proteins in active, soluble form and in high yields, the expression and purification protocols were developed. The procedure developed allows for the production of His-tagged lpPOX in a fast, straightforward way. The problems realted to the production of low amounts, insoluble or inactive protein have been circumvented. Yield (up to 10mg per litre culture), purity (>95%) and activity (in the same range as the wt enzyme) of the protein comply with the requirements for an efficient protein production process. During development of the production procedure the implementation of a fine adjustable induction rate based on NaCl was the crucial step for expression of soluble and active protein in acceptable amounts. The new production procedure can be scaled up without too many modifications. Mutant L323Q designed to stabilise the tetramer interfaces led to a significant thermal stabilisation of ∆Td +1.4°C compared to the wt. This result supports the hypothesis of a possible stabilisation at the level of the quaternary structure by improving the interactions between the POX monomers. Two mutants (I221T and A373K), that theoretically should improve cofactor binding didn’t lead to measurable improvement of thermal stabilisation, despite the fact that one of these residues is found

IX at the same position in the POX of the extremophilic Oceanobacillus iheyensis. It turned out that a rational stabilisation strategy focused on cofactor binding sites was not successful with the current applicable modelling tools including orthologous alignement. Eight proline mutants Xaa->Pro and three Gly->Xaa mutants were proposed by Nagel (2004), nine of them were successfully expressed, purified and tested for activity and thermal stability measuring the temperature of deactivation (Td). L193P, V199P, S559P, A564P and G508A increase Td compared to the wt enzyme in phosphate buffer (0.2M pH=7) with ∆Td of +4.12°C, +2.66°C, +2.88°C, +1.22°C and +0.93°C respectively. The combination of two single mutations in the double mutant L193P_S559P did not show a cumulative thermal stabilisation with a ∆Td of -0.48°C. Although entropic favouring mutants lead to more thermostable enzymes in a predictive manner, the higher thermostability is generally not perfectly correlated to longer shelf life of the enzyme. Never- theless, S559P is the first mutant which fulfils the requirements defined in the research plan with a remaining activity of 60.6% after 14 days in phosphate buffer (0.2M, pH=6) at 25°C and without addition of cofactors. Thus, these results indicate that the aim of this project - to develop and produce a stabilised lpPOX in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor will be less than 50% - is partially met by the S559P lpPOX mutant. The goal was not only to further improve the stability of lpPOX but also to achieve that goal without loss of activity. This second objective is also fulfilled by the S559P mutant that has (2.24 U/mg) a similar activity to wt lpPOX (1.17 U/mg). The stability of the enzyme was improved and the enzyme activity was at least conserved. So one can conclude that protein engineering by rational protein design was a successful approach for stabilising lpPOX. However, so far, it´s not known whether these results can be translated into a more stable biosensor. The next step is to test the stability and activity of this first stabilised lpPOX enzyme (S559P lpPOX) in a biosensor measuring pyruvate or phosphate. In summary, an optimal enzyme mutant for further biosensor development has been found and a lot of consolidated findings about lpPOX stability and activity were made during this project.

X Zusammenfassung

ie Anwendung von Pyruvat Oxidase (POX) in einem Biosensor ermöglicht die Messung von DPhosphat oder Pyruvat in wässrigen Lösungen. Diese Substrate sind attraktive Analyten, in der Umwelttechnologie, in der Medizinischen Analytik und in der Lebensmittelindustrie. Der Vorteil der Anwendung von Lactobacillum plantarum Pyruvat Oxidase (lpPOX) im Biosensor ist, dass die teuren und instabilen Kofaktoren Flavinadenindinucleotid (FAD), Thiaminpyrophosphat (ThDP) und Magne- sium-ionen (Mg2+) nicht der Probelösung zugegeben werden müssen, weil die Kofaktoren im Vergleich zu anderen POX relativ stark ans Enzym gebunden sind. Die lpPOX wurde in einem vorhergehenden Projekt am Centre for Chemical Sensors (CCS) in einem amperometrischen Pasten-Biosensor mit Durchflusszelle eingesetzt. Der lpPOX-Biosensor zeigte allerdings unter kontinuierlichen Messbedin- gungen nach 30h einen Signalabfall von 85%, während ein Glucoseoxidase-Biosensor nach 10 Tagen keine signifikante Signalabnahme zeigte. Die Lebensdauer des Pyruvatoxidase-Sensors erfüllt somit die Anforderungen von weniger als 50% Signalverlust während zwei Wochen nicht, was einer Haltbarkeit von weniger als zwei Wochen entspricht. Das Enzym soll hinsichtlich einer längeren Haltbarkeit stabil- isiert werden. Das Ziel dieses Projektes war es eine stabilisierte lpPOX zu entwickeln und zu produz- ieren, wobei die Abnahme der POX-Aktivität, während zwei Wochen kontinuierlicher Messung im Biosensor, weniger als 50% beträgt. Eine vertiefte Analyse der strukturellen Eigenschaften und der Deaktivierungs-Kaskade führte zur Hypothese, dass strukturbasiertes rationelles Proteindesign der vielversprechendste Ansatz für eine En- zymstabilisierung ist. Drei verschiedene Stabilisierungsstrategien wurden getestet. Die erste war fokussiert auf eine Verbesserung der Interaktionen zwischen den Protein-Monomeren. Die zweite war auf eine Verbesserung der Interaktionen zwischen den Kofaktoren und dem Protein ausgerichtet. Die dritte war fokussiert auf eine Protein-Hauptketten-Rigidisierung (entropische Stabilisierung). Der Start- punkt für das weitere Protein-Engineering war die dreifache lpPOX Mutante (P178S, S188N, A458V) mit einer höheren Toleranz gegenüber pH-Werten von 4.5 bis 7.5 (Risse et al. 1992b). Es wurden Expressions- und Reinigungs-Protokolle entwickelt, damit die vorgeschlagenen Mu- tanten in aktiver Form, löslich und in hoher Ausbeute produziert werden konnten. Diese entwickelte Pro- zedur erlaubt eine rasche und unkomplizierte Produktion von lpPOX mit einem angehängten His-Tag. Die Probleme, verursacht durch die Produktion von kleinen Mengen unlöslichem oder inaktivem Pro- tein, konnten damit umgangen werden. Die Ausbeute (bis zu 10 mg pro Liter Medium), die Reinheit (>95%) und die Aktivität (im gleichen Bereich wie beim wt Enzym) des Proteins entsprechen den An- forderungen an einen effizienten Protein-Produktionsprozess. Bei der Entwicklung des Produktionsver- fahrens war die Einführung eines fein steuerbaren Induktionssystems, basierend auf NaCl, ausschlaggebend für die Expression von löslichem und aktivem Protein in akzeptablen Mengen. Das neue Produktionsverfahren kann einfach und ohne grosse Anpassungen auf grössere Ansätze übertragen werden.

XI Mutante L323Q, entworfen für eine Stabilisierung der Tetramer Kontaktfläche führte zu einer sig- nifikanten Temperatur-Stabilisierung von ∆Td +1.4°C verglichen mit dem wt. Dieses Resultat unter- stützt die Hypothese einer möglichen Stabilisierung auf der Stufe der quaternären Proteinstruktur durch Verbesserung der Wechselwirkungen zwischen den POX Monomeren. Zwei Mutanten (I221T und A373K), welche theoretisch die Kofaktor-Anbindung verbessern soll- ten, führten nicht zu messbaren Temperatur-Stabilisierungen, obwohl eine der vorgeschlagenen Seiten- ketten an derselben Position in der POX des extremophilen Oceanobacillus iheyensis vorkommt. Es stellte sich heraus, dass eine rationelle Stabilisierungs-Strategie bezüglich Bindungsstelle der Kofak- toren mit den heute zur Verfügung stehenden Protein-Modelling Werkzeugen inkl. Sequenzvergleich mit orthologen Proteinen nicht zum Erfolg führte. Acht Prolin Mutanten Xaa->Pro und drei Gly->Xaa Mutanten wurden von Nagel (2004) vorge- schlagen. Neun davon wurden erfolgreich exprimiert, gereinigt und unter Messung der Inaktivierungs-

Temperatur (Td) auf Aktivität und Temperatur-Stabilität getestet. L193P, V199P, S559P, A564P und

G508A erhöhten Td verglichen mit dem wt Enzym in Phosphatpuffer (0.2M pH=7) mit einer ∆Td von +4.12°C, +2.66°C, +2.88°C, +1.22°C und +0.93°C. Die Kombination von zwei Einfachmutationen in eine Doppelmutante L193P_S559P zeigte keine kummulative Temperatur-Stabilisierung (∆Td -0.48°C). Obwohl die Entropie-Mutanten zu voraussagbar thermostabileren Enzymen führten, ermöglichte die höhere Temperatur-Stabilität nicht zwangsläufig eine längere Haltbarkeit des Enzyms. Dennoch konnte mit S559P die erste Mutante gefunden werden, welche die Anforderungen im Forschungsplan mit einer Restaktivität von 60.6% nach 14 Tagen in Phosphatpuffer (0.2M, pH=6) bei 25°C ohne Kofaktoren er- füllt. Diese Resultate zeigen, dass das Projektziel (Entwicklung und Herstellung einer stabilisierten lp- POX, mit einer Aktivitäts-Abnahme von weniger als 50% in einem über zwei Wochen kontinuierlich messenden Biosensor) mit S559P mindestens teilweise erreicht werden konnte. Das Ziel war nicht nur die Stabilität der lpPOX weiter zu verbessern, sondern dies auch ohne Ver- lust an Aktivität zu erreichen. Dieses zweite Ziel wurde bei der Mutante S559P (2.24 U/mg) mit einer ähnlichen Aktivität wie beim lpPOX wt (1.17 U/mg) ebenfalls erreicht. Die Stabililtät des Enzymes wurde verbessert und die Enzymaktivität war zumindest im gleichen Bereich. Daraus lässt sich schlies- sen, dass Protein-Engineering durch rationales Design ein erfolgreicher Ansatz für die Stabilisierung der lpPOX darstellt. Indes ist noch nicht bewiesen, dass diese Resultate in einen stabileren Biosensor über- führt werden können. Der nächste Schritt ist die Prüfung der Stabilität und Aktivität des ersten stabilisierten lpPOX Enzyms (S559P lpPOX) in einem Biosensor unter Messung von Pyruvat oder Phosphat. Zusammengefasst wurde eine optimale Enzymmutante für die weitere Entwicklung des Biosen- sors gefunden. Während dieses Projektes konnten zahlreiche Erkenntnisse über die Stabilität und Aktiv- ität der lpPOX zusammengetragen werden.

XII

1 Introduction

n the area of analytical chemistry the demand for continuous control of process parameters I is increasing. The quality and the working load of industrial processes can be optimised by their on-line monitoring and direct feedback control by sensors. Optimisation allows for the reduction of costs and waste as well as reagent consumption. An exact monitoring of industrial processes is indeed relevant from both the economical and the ecological point of view. The development of biosensors involves a multidiciplinary approach which includes several disciplines (e.g. chemistry, engineering, physics, biochemistry) and research fields such as electronics, micro-fluidics, polymer chemistry, protein engineering (see figure 1-1). While working on a particular aspect e.g. the biochemical issue of enzyme stability in biosensors, questions related to the other disciplines, that may affect the strategical decisional pathway, have to be considered by the scientist during the whole project.

biochemistry recognising elements immobilisation transducing concepts techniques chemistry biosensor physics technology polymer chemistry electronics, optics...

micro micro-fluidics mechanics engineering

Figure 1-1: The multidisciplinarity of biosensor research. In the field of biosensor technology (grey zone) communication between the four disciplines biochemistry, physics, engineering and chemistry is very important. It is obvious that a successfully running biosensor can only be developed following this multidisciplinary approach.

Despite the advantages of sensor technology in comparison to standard analytical procedures as outlined in chapter two, there are still a few hurdles to overcome during the development of sensors. The major one is the stability of the biomolecules e.g. enzymes or antibodies introduced in the biosensor for specific recognition processes . This thesis explores the main issue of protein stabilisation with an example, pyruvate oxidase (POX, EC 1.2.3.3). The importance of protein engineering to increase protein stability is not only restricted to the field of analytics and diagnostics but extends to the fast growing field of biopharmaceuticals or technical production of enzymes e.g. for cleaning agents (see table 1-1 page 2). Within

1 1 Introduction the pharmaceutical industry, the market of therapeutic proteins and peptides has the greatest growth potential nowadays. Biopharmaceuticals provides opportunities to effectively treat up to now immedicable diseases and already represent about 25% of new molecular entities (NMEs). An increasing number of these therapeutic proteins are engineered. For example 29 of the 65 biopharmaceutical approvals (44%) in the period of 2000-2004 were modified in comparison to their natural template (Walsh 2005). The first chapter describes the background of phosphate and pyruvate measurements and the aim of the present work. An overview of state-of-the-art monitoring of the analytes and an abstract of biosensor theory in chapter two closes the introductionary explanations. In the second and main part of this work (chapter 3-6) the problematic of enzyme stability and different strategies to improve the latter as well as the experimental verification of the working hypothesis are given using pyruvate oxidase as a case study. Final discussion of the results and conclusions as well as an outlook complete the work in the seventh chapter.

signalling structural catalytic transport fields where protein proteins proteins proteins proteins engineering is applied (e.g. receptors, (e.g. gelatine) (e.g. enzymes) (e.g. albumin) antibodies) glue and adhesives tech. - - - artificial silk tech. - - - biodegradable polymers tech. / med. - - - food technology tech. tech. - - washing detergents - tech. - - chemical catalysis - tech. - - biosensors - tech. / med. tech. / med. - diagnostics (ELISA, SPR,...) - med. med. - vaccines - - med. med. biopharmaceuticals - - med. med.

Table 1-1: Application areas of protein engineering: tech. and med. refer to technical and medical application fields of the mentioned proteins. These two fields of biotechnology are also called “red“ (medical) and “white“ (technical) biotechnology.

2 1.1 The Aim of the Work

1.1 The Aim of the Work

The application of pyruvate oxidase (POX) to a biosensor allows the measurement of phosphate or pyruvate in aqueous solutions based on the following reaction eq. 1-1 catalysed by this enzyme.

pyruvate + orthophosphate + O2 + H2O acetylphosphate + CO2 + H2O2 (Eq. 1-1)

The two substrates pyruvate and orthophosphate are very attractive analytes mainly in the medical field, in food and environmental technology (see paragraph 1.2 on page 4 and paragraph 1.3 on page 11). In addition further enzymes, which release pyruvate, can be quantified using this sensor (see 1.3.2 on page 12). An advantage of using the pyruvate oxidase from Lactobacillus plantarum (lpPOX) in a biosensor is that no expensive and unstable cofactors have to be added to the sample solution, because cofactors flavinadenine dinucleotide (FAD), thiamine diphosphate (ThDP), and magnesium-ion (Mg2+) are relatively tightly bound to the enzyme compared to other POX. After a first stabilisation done by random mutagenesis and involving three point mutations, the structure of both, stabilised and wild-type POX from L. plantarum, were elucidated by Muller et al. (1994). First experiments with this enzyme in a biosensor at the Centre for Chemical Sensors (CCS), Technopark ETH Zurich, have shown that it is possible to measure pyruvate or phosphate with the stabilised pyruvate oxidase without external addition of cofactors. But the sensor could not be used for more than two days of continuous measurement due to the denaturation of the oxidase. The aim of this project was to develop and produce a stabilised lpPOX and to improve the biosensor in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor will be less than 50%. While pursuing the main aim, new technologies for stabilising enzymes and other proteins in general have been evaluated (see table 1-1 page 2).

1.1.1 Working Hypothesis

The random mutagenesis proved that it is possible to stabilise this enzyme. The working hypothesis is that the enzyme can be further stabilised in the most promising way by increasing the interactions between cofactors and protein at the active site of the enzyme. Stabilisation of the tertiary structure by new mutations, based on modelling studies, should result in new enzymes with higher stability (see chapter 3).

3 1 Introduction

There are mainly two strategies in enzyme engineering that can be pursued with techniques available today: - Random mutagenesis followed by a selection of stabilised mutants. - Structure based protein design combined with site directed mutagenesis. Both set-ups have been successfully applied to improve properties of enzymes or to answer questions concerning their functional principles and structures. When one considers that metric tons of engineered subtilisin with enhanced stability and activity under different conditions are produced industrially and added to washing agents, the principle proposed here of structure- guided protein engineering has already been demonstrated to be effective (Bryan 2000). The literature reporting on successful engineering of proteins with increased melting temperatures or other pH-ranges is tremendous and an overview is beyond the scope of this thesis. In chapter three a number of selected sources on this topic is reported and mentioned in the discussion. In this project, the enzyme has been stabilised by site-directed mutagenesis guided by theoretical calculations based on its known structures (structure-based protein design). Activity, stability as well as biophysical characteristics of several mutant enzymes has been determined. To study additivity effects of the combination of single mutants, double mutants of promising stabilising mutation sites were tested.

Before the whole protein engineering approach is discussed, the next paragraphs give a short overview of the analysis of phosphate and pyruvate and some background information about these analytes.

1.2 Phosphate Analysis

In this paragraph a number of fields where phosphate analysis plays a role and the conventional methods of phosphate measurement are outlined. At the end the potential of phosphate biosensors in the fields of phosphate analysis is discussed.

1.2.1 Chemical Properties and Deposits of Phos- phate

P4O6 and P4O10 represent the most stable oxidation state of phosphor, +3 and +5. In the ionic 3- form, it carries a -3 formal charge and is denoted PO4 . In a biochemical setting, a free phosphate ion in solution is called orthophosphate (Pi) to distinguish it from organic phosphates

4 1.2 Phosphate Analysis bound in the form of phosphor esters, phospolipides or phosphonates. For a whole overview of phosphorous species in water and the special role of orthophosphate see figure 1-2 on page 6. Due to its high reactivity, phosphor is never found as a free element in nature. It is commonly found in inorganic phosphate minerals and is an essential element for living organisms. In all living cells phosphate plays an important role in form of DNA, RNA and ATP molecules, as buffers or in the form of phospholipides - the main structural component of cellular membranes. In animals and humans calcium phosphate stiffens the bones. The most important commercial use of phosphorus and therefore sources of phosphate ions are fertilisers, whose global demand has led to growing increases in phosphate production in the second half of the 20th century. Phosphate refers to one of the three primary plant nutrients in addition to nitrogen and potassium. Also cleaning agents contain phosphates in the form of polyphosphates or phosphonates. In washing agents the phosphates act as water softeners to improve cleaning. Phosphorus is further widely used in explosives, firework, friction matches, pesticides, toothpaste and as stabilising agent in food technology.

1.2.2 Phosphate Measurement in Water

Phosphate is a limiting nutrient for plants in many environments. Introduction of non-naturally occurring levels of orthophosphate to stagnant waters causes an ecological imbalance, leading to algae booms. This excessive growth of algae leads to oxygen depletion and fish kills. Since the early 1970s phosphate pollution in water has led to an uncontrolled eutrophication of numerous lakes around the whole industrial world. In addition to the natural sources of inorganic phosphate (phosphate minerals and decomposition of organic material) there exist four anthropogenic sources; two of these are agricultural (animal urine and fertilizers), the other two are domestic (human urine and purifying agents). During the 1980s the main sources of phosphate pollution of lakes were leaching of fertilizers and liquid manure from farmland and phosphate-based purifying agents in the waste water.

5 1 Introduction

total phosphorus

inorganic phosphate organic phosphate

naturalanthropogenic natural anthropogenic

phosphatic minerals orthophosphate phosphor esters phosphonates (e.g. apatite: (out of vegetal (e.g. in purifying agents) (fertilisers, urine and animal sources) Ca5(PO4)3(F,Cl,OH)) and as decomposition- products of all other phospholipides phosphorous species) (out of vegetal degradation polyphosphates and animal sources) (purifying agents) slow degradation

Figure 1-2: Overview of different phosphorus species in water and the role of orthophosphate: Phosphatic minerals are not directly bioavailable and therefore do not predominantly cause eutrophication. Orthophosphate is the main cause for eutrophication of lakes. Orthophsophates derive from different sources like agricultural fertilisers, animal and human urine, or as end products of decomposition of all the abovementioned phosphorus species. The phosphate biosensor described in this thesis measures only orthophosphate ions; the most important analyte for monitoring open waters. If the total phosphate in water were to be measured, all other phosphorous species present would need to be decomposed. Polyphosphates are used in purifying agents, in food technology as stabilisers, emulgating agents or buffers, in fertilisers and in paper technology. They are degraded by microorganisms to orthophsophates. Organic phosphates from natural sources also need to be degraded to orthophosphate before they cause any eutrophication. Phosphonates are organic complexing agents and contain direct phosphorus carbon bonds. They degrade very slowly under the effect of light to orthophosphate.

The most prominent example of eutrophicated and so called „tilt lakes“ in Switzerland is the Baldegg lake. In 1975 a phosphate concentration of 500 mg/l was measured whereas concentrations of about 20-30 mg/l guarantee an ecologically healthy water system (SAEFL 2002a, SAEFL 2002b). The article written by Ambühl (1982) gives a broad overview of the problematic of eutrophication in Swiss lakes. The Baldegg, Sempach and Hallwil lakes still need venti- lation today in order to avoid fish kills, as a lot of phosphate is still liberated out of the lake sediment and the over-fertilised grounds in the catchment area. After detecting the cause of the nutrient pollution several measures were taken by the na- tional government. In 1980 the German government implemented the Phospathöchstmengen- Verordnung that restricted the phosphate content in washing agents. In Switzerland phosphates were prohibited in textile washing agents in 1986. Phosphates still plays a role in a few industrial products, for example in washing up liquids, but the amounts used today are far from those used in the eighties.

6 1.2 Phosphate Analysis

The phosphate burden of lakes is also reduced by precipitating phosphate during waste water treatment by addition of calcium hydroxide dissolved in water or iron(II)- or aluminium salts diluted in hydrochloric acid. Due to the high price of these chemical additives, the phosphate levels have to be controlled continuously in waste water treatment plants, so that the chemical additives are not under or overdosed and the phosphate elimination is effective. The phosphate limit after waste water treatment for sensitive waters is stipulated by the swiss law at a maximum of 0.8 mg total phosphorus (after disintegration) per litre water (SR 814.201, Gewässerschutzverordnung der Sch- weiz, appendix 2, cypher 3). Since the 1990s phosphate is also eliminated from waste water by biological procedures. Anaerobic and aerobic phosphate (in the form of polyphosphates) enrich- ing bacteria are applied to the sludge. In this case the phosphate concentrations in the exit water must also be continuously controlled. All regulations concerning the limitation of phosphates in chemical products, the phosphate separation in waste water treatment and restrictions in agriculture led to a decreased phosphate release to surface waters. Thus the eutrophicated lakes recover from year to year. An overview of phosphate concentrations in Swiss lakes is given in the SAEFL report (2002a). In drinking water, the swiss government limits the concentration of allowed phosphate to 1 mg/kg calculated as total phosphorus (SR817.021.23, Fremd und Inhaltsstoffverordnung, cypher 4, p.120). A phosphate sensor would not only be advantageous for drinking water control but also in the broad control of ground water and critical surface waters like lakes or rivers.

1.2.3 Phosphate in Clinical Diagnostics

A human consists of about 1% (m/m) phosphate, whereas the greatest amounts (85%) are deposited together with calcium in bones and teeth in the form of hydroxyapatite

Ca10(PO4)6(HO)2. About 14% are deposited in cells and only 1% is soluble in extra cellular liquids. In plasma, phosphate acts as buffer and exists in the two forms of hydrogen phosphate and dihydrogenphosphate (see eq. 1-2).

- + 2- H2PO4 H + HPO4 ; pKa = 7,21 (Eq. 1-2)

Besides these dissociated phosphate ions in plasma, organic bound phosphor acid also exists, on one side in the form of phospholipides and on the other side in the form of phosphor esters.

7 1 Introduction

At the Institute of Clinical Chemistry at the University Hospital Zurich and in many other hos- pitals, the phosphate concentration in plasma and in urine is measured using the molybdate reaction in a reducing system (IKC 1998) (see 1.2.5 on page 10). The reference intervals of phosphate in plasma are given in table 1-2. The values are elevated in the case of renal insufficiency, hypoparathyroidism, vitamin D intoxication, bone mes- tastases, myelomas and leucosis. Decreased phosphate concentrations are found in cases of hyperparathyroidism, tubular acidosis, malabsorption, alcoholism and i.v. glucose infusions. With regards to phosphate concentration in urine, the reference interval for adults is between 12.9-42 mmol/24h. Phosphate excretion is dependent on phosphate uptake via nutrition. There- fore, standardised nutrition is required for interpretation of renal phosphate elimination. Never- theless, tubular dysfunctions are difficult to diagnose because the ratio of phosphate elimination via stool is unknown in most cases (IKC 1998).

phosphate in phosphate in age of patients plasma plasma (mmol/l) (mg/dl) newborn (up to 4 weeks) 1.56-3.1 4.8-9.6 infants (up to 1 year) 1.56-2.54 4.8-7.9 children (older than 1 year) 1.09-2.0 3.4-6.2 adults 0.87-1.45 2.7-4.5

Table 1-2: Reference intervals of phosphate in plasma: source: Keller (1991) p218

Although enzymatic methods for phosphate measurements exist on the market, many labs still use the classical chemical methods due to lower costs in comparison to the new enzymatic tests. Other ions in the millimolar range are currently measured via indirect potentiometry using ion selective electrodes (ISEs). Examples are sodium, potassium, calcium or chloride ions. The advantage of the method using ISEs is the fact that the exact ion activity is measured. A cost effective phosphate sensor that can be calibrated and that measures over weeks can remove the time and reagent consuming method of phosphate measuring described above.

8 1.2 Phosphate Analysis

1.2.4 Phosphate Measurement in the Food and Pharmaceutical Industry

Phosphates exist as natural compounds in most food products but are also added to achieve a wide range of effects in the food processing industry. They are applied in different chain lengths, at different pH-values and with different counter ions. All these factors influence the chemical and physical characteristics of the phosphates and make them highly functional and indispensable aids in food technology. In the EC directive No 95/2/EC all phosphates allowed as food additives in the European Union are classified by E numbers and are listed in tables together with their maximum concentration levels. The Schweizerische Lebensmittelverordnung (SR 817.02) only enumerates the allowed phosphates in foodproducts without giving any guidelines on the maximum allowed levels. Up to 700 mg/l (expressed as P2O5) phosphoric acid is accepted in non-alcoholic flavoured drinks; this is an example taken from the abovementioned list. Phosphates are used as buffers, for binding polyvalent cations, or as polyanions that can increase solvation of some proteins. Phosphate salts of calcium, magnesium and iron are used as a mineral enrichment component within drinks e.g. in fruit juices. Polyphosphates (E 452) are used as preservatives against microbial contaminations and ammonium phosphatides (E 442) are used as antioxidants. Solid tricalciumphosphate, finally, is used as an anti-caking agent, improving the free flowing properties of hygroscopic powdered foods and acidic phosphates can release carbon dioxide together with sodium bicarbonate when heated during the baking process. In the food industry, phosphate is often determined in fruit juice or meat products. During the production of secondary metabolites in fermentation processes, such as strep- tomycin or tetracycline, the levels of nutrients like phosphate or glucose in the culture media has to be controlled. When the cells are hungry and can not grow for want of nutrients, they produce desired secondary metabolites such as the antibiotics mentioned previously. This phase of fermentation is called idiophase and has to be initiated after the logarithmic period of growth by a controlled reduction of nutrients. One of the most important nutrients that has to be reduced and therefore also has to be controlled in these fermentation processes is phosphate. The mentioned examples clearly show the need for phosphate sensors within the bioprocess technology.

9 1 Introduction

1.2.5 Phosphate Measurement with Traditional Chemical Methods

Three of the most prominent methods for phosphate measurement are given in this paragraph.

Gravimetric Determination of Phosphate

Phosphate ions form in the presence of molybdate ions in to hydrochloric acid molybdophos- phat ions, which can be precipitaded by addition of 8-hydroxychinolin (called HOx or oxin) to oxin-12-molybdo-1-phosphate (see eq. 1-3). Afterwards the precipitate is dried at 160°C and weighted. The dried precipitate conains only 1.37% phosphorus. Therefore, the method is suitable for the determination of micromolar amounts of phosphate. Unfortunately, the method is rather time and also reagent consuming.

3- + [P(Mo3O10)4] + 3 HOx + 3 H (H2Ox)3[P(Mo3O10)4](Eq.1-3)

Indirect Complexometric Titrations

The phosphate content of a sample can be determined by indirect titration of a defined excess of Mg2+, La3+ or Bi3+ ions. Thereby a defined amount of precipitating ions has to be added to the phosphate sample in the presence of ammonium ions. The phosphate precipitates in the form of insoluble salts and can be separated by filtration. In the flow through, the residual precipitating ions can be determined via complexometric titration with etylenediaminetetraacetic acid (EDTA) against eriochromschwarz. These method is disturbed by the presence of precipitating ions in the sample and is also rather time consuming.

The Photometric Molybdate Method

The molybdate method allows the measurement of phosphate photometrically and therefore is much faster than the first described method of indirect complexometric titration. The determination of phosphate is based on the principle that the orthophosphate ions form, together with molybdate(VI) ions, the heteropolyacid H3(P[Mo3O10]4). This heteropolyacid can be reduced to a blue phosphor-molybdate complex. In this reaction the reducing agent and the acidity of the solvent play an important role for the complex formation. At higher pH-values free molybdate ions may also give rise to a blue coloured phosphate free complex of molybdate that disturbs the measured absorption. In different protocols, stannous chloride (SnCl2) or ascorbic acid are used as reducing agents.

10 1.3 Pyruvate Analysis

Applying standard protocols with exact specified reaction times and temperatures, the phosphate concentrations can be calculated after the law of lambert beer (see eq. 1-4) using the absorption values.

A = ε ⋅⋅cd (Eq. 1-4)

A = absorption; ε = extinction coefficient (1 mol-1 cm-1); c = concentration (mol/l); d = path length (cm)

Another similar method is the photometric measurement of an orange coloured complex con- -5 sisting of ammonium molybdate, vanadate and phosphate ([PV2Mo10O40] ). The photometric methods are not as reagent-consuming as the gravimetric or complexometric titration. But they are also time-consuming because a complex sample preparation is required to avoid turbidity and interfering colouration, especially in clinical or food analytics.

1.2.6 The Potential of Phosphate Biosensors

In the fields of clinical analysis, in food and biotech plants and in waste water monitoring, exact and ongoing phosphate measurements would be very advantageous tools. Above all, in the waste water treatment and in fermentation processes, an on-line phosphate measurement would be a big advantage in comparison to the conventional, time consuming, discontinuous method of wet chemistry that has to be performed in batches. All advantages of biosensors per se are discussed in detail in chapter two.

1.3 Pyruvate Analysis

1.3.1 Pyruvate a Key Metabolite

Pyruvate is the anion of pyruvic acid with a pKa of 2.5. It is a key intermediate in the anaerobic and the aerobic metabolism of almost all living organsisms. Pyruvate is generated as an intermediate during glycolysis out of phosphenol pyruvate or via the catabolism of certain amino acids (e.g. serine, cysteine and alanine). In anaerobic glycolysis, pyruvate is further reduced to lactate by lactate dehydrogenase e.g. in strongly contracting muscles, in erythrocytes or in microorganisms. In anaerobic alcoholic fermentation of yeast, pyruvate is reduced to ethanol. In the aerobic metabolism, finally, pyruvate is oxidised to acetyl-coenzyme A and is introduced

11 1 Introduction

into the citric acid cycle where it is further oxidised to CO2 and H2O or consumed in fatty acid biosynthesis. In Lactobacillus casei, L. delbrückii, L.plantarum and E.coli, pyruvate can be oxidised via Pyruvate Oxidase to acetate. In gluconegenesis, pyruvate reacts first to phosphenol pyruvate via different bypass reactions. (see figure 1-3 on page 12)

glucose serine glycolysis cysteine gluco- neogenesis serine- desaminase cysteine- phosphenol pyruvate desulfhydrase alanine pyruvate- kinase alanine- bypass- OH O transaminase reactions O O fatty acid biosynthesis O pyruvate O pyruvate- pyruvate- pyruvate- dehydrogenase decarboxylase oxidase acteyl-coenzyme A lactate- dehydrogenase acetaldehyde acetylphosphate alcohol- acyl- lactate dehydrogenase phosphatase citric acid- ethanol acetate cycle

Figure 1-3: Pyruvate, a key intermediate in metabolism:

1.3.2 Pyruvate Analysis in Clinical Diagnostics

Pyruvate is measured in clinical laboratories via the enzymatic reaction of lactate dehydrogenase (LDH) see eq. 1-5. The reference range in whole blood is 41-67 µmol/l. The values are elevated in cases of terminal hepatopathia, heart insufficiency, intoxication with heavy metals, diabetic ketacidosis, rey syndrome and beriberi. A biosensor that can measure in complex ma- trices like whole blood would be a big advantage in clinical pyruvate analysis. In addition, enzymes which release pyruvate can be quantified using this sensor (e.g. alanine transaminase, pyruvate kinase, lactate dehydrogenase, glycerophosphate-kinase). In clinical routine analysis, the following enzymes play an important role: • Lactate dehydrogenase (LDH, EC 1.1.1.27) catalysed reaction eq. 1-5 can be measured by the initiated reverse reaction. Under addition of L-lactate to the LDH sample, pyruvate is produced and can be measured by the pyruvate sensor. LDH is currently meas-

12 1.3 Pyruvate Analysis

ured via a photometric enzyme kinetic test and plays a role in the diagnosis of the progress of megaloblastic anaemia.

LDH pyruvate + NADH L-lactate + NAD (Eq. 1-5)

• Alanine aminotransferase (ALAT, EC 2.6.1.2) catalyses the reaction eq. 1-6 and is measured photometrically via LDH that converts pyruvate under oxidation of NADH to NAD. ALAT is very important for the early diagnosis and prognosis of hepatopathia.

ALAT L-alanine + 2-oxoglutarate pyruvate + L-glutamate (Eq. 1-6)

The pyruvate biosensor may not be operating under ideal conditions for ALAT measurements due to the instability of this enzyme. In the conventional analysis of ALAT, pyridoxalphosphate is added to the sample avoiding protein inactivation. But phosphate that has to be added as a cosubstrate for the biosensor measurement inhibits the recombination of ALAT apoenzyme and pyridoxalphosphate and so inhibits ALAT activity (Keller 1991, p.318f).

1.3.3 Further Advantages of Pyruvate Sensors

Aside from the application of pyruvate sensors in clinical diagnoses, there are other fields where this device can be used. Due to the fact that pyruvate is a broadly used metabolite, many other enzymes that consume pyruvate can be incorporated together with POX in a multi enzymatic biosensor. An example of such a multi enzyme biosensors is described by Gajcovic et al.(1997) for a novel maleat sensor.

13 1 Introduction

14 2 Phosphate Biosensors

hich architecture of sensors guarantees a long life time and the best characteristics for Wan on-line phosphate measuring device? This chapter gives answers to these questions and a broad overview of the considerations for the development of the phosphate sensor.

2.1 What are Biosensors

2.1.1 Definition of Biosensors

A biosensor is a self-contained analytical device that incorporates a biological recognition element which is integrated or very close to a signal transducer, thereby facilitating direct or mediated signal transfer. Thus biosensors combine the selectivity of biological systems and the processing power of electronics or optics. A comprehensive definition of biosensors with exact differentiations to other analytical devices is given in a report from the International Union of Pure and Applied Chemistry (IUPAC, 1999).

sample solution biosensor

electrochemical - amperometrical -potentiometrical - coulometrical optical signal calorimetrical piezoelectrical …

mol ecul ar analyt transduction recognition

Figure 2-1: General schema of a biosensor: On the left side the sample solution with different chemical compounds (angular forms) including the analyte (black triangle) that is in close contact with the recognition elements (round forms in the middle). The biological recognition elements in the middle transduce the chemical information of the sample solution into technical readable quantity. The transducing element on the right side reads out the electrochemical information delivered by the biomolecule and transduces this information to a processing signal.

Biosensors may be classified according to the biological specificity-conferring mechanism -

15 2 Phosphate Biosensors namely catalytic or non-catalytic - or alternatively, the mode of physicochemical signal transduction. The catalytic group of biological recognition elements includes enzymes, whole microorganisms or tissue samples. Many enzymatic reactions are associated with the consumption or formation of electroactive species in concentrations proportional to the analyte. For this reason oxidoreductases, hydrolases, dehydrogenases and lyases are applied in sensing devices. In the group of non-catalytic biosensors, the recognition is based on affinity whereas antibodies, receptors or single strand nucleic acids act as recognition elements.

2.1.2 History of Biosensors

Prehistory

One can argue that the first precursors of modern biosensors were invented in the prehistoric era, when humans domesticated dogs for their purposes (hunting etc.). Measurements with olfactory meters showed that the olfactory sensitivity of dogs is up to one billion times better than that of humans. This characteristic determined their use as guard dogs or as beagles, for example, German shepherds have up to 220 billion olfactory cells in comparison to the 5 billion cells of humans. Dogs also have an additional olfactory organ, the so called Jacobsonsche organ, and their olfactory centre in the brain takes 10% of their brain mass compared to the 1% in humans. To this day humans make use of many animal senses for measuring chemicals in a quali- tative manner. Dogs are trained to find buried victims, illegal drugs or explosives, canaries in coal mines were used as indicators of carbon dioxide gas and trout control fresh water in Rome, for example. Of course these examples are not biosensors in our modern understanding, because they lack a transducing element generating an electronic signal. Nevertheless they show that the concept of using biological recognition systems for measuring chemical compounds is very old and established. In fact, the concept of combining biological recognition molecules with electronic systems exists has only existed for 40 years.

The Invention of Electrochemical Biosensors

The concept of an electrochemical, enzymatic biosensor was published the first time by L. C. Clark Jr. in 1962 at a New York Academy of Sciences symposium. In his address he described "how to make electrochemical sensors more intelligent" by adding "enzyme transducers as membrane enclosed sandwiches". An experiment in which glucose oxidase (GOD) was entrapped in a Clark oxygen electrode illustrated the concept. The measured oxygen decrease

16 2.1 What are Biosensors caused by the glucose oxidising GOD was proportional to the glucose concentration in the sample. Clark and Lyons coined the term “enzyme electrode“ in their paper (Clark and Lyons 1962) for the invented analytical device. This idea of biochemically modified sensors was the basis of numerous variations of biosensors with different sensing elements and transducers.

2.1.3 Phosphate and Pyruvate Analysis: Advantages of Sensors and Biosensors Compared to Con- ventional Analytical Techniques

Biosensors have major advantages over conventional analytical technologies because they are able to measure continuously, in real time and so allow on-line control of industrial processes. They are small, highly specific for selected analytes and do not consume reagents. These and further characteristics explain why a biosensor system for measuring phosphate and pyruvate can be superior to conventional analytical methods in many cases. Although biosensors are applied to many different fields of analytical chemistry, they have been found to be especially useful in process monitoring, biomedical and environmental analysis. As a summary of this paragraph, the general advantages of sensors and especially of biosensors are outlined in a schematic overview on figure 2-2 page 20.

On-line Measurement

In the fields of environmental and industrial process analysis, there is a great demand for continuous monitoring which only sensors can provide in such a direct and instantaneous way. Sen- sors are by definition devices that permit continuous measurement of a quantity of a physical parameter or an analyte. The miniaturisation of sensors together with their ability to measure continuously and their short response time allows the assembly of sensors directly into industrial systems. Sensors are thus a powerful tool for optimising industrial processes, despite more and more automated laboratory systems applying the so called sequential injection analysis (SIA) method (Ruzicka and Marshall, 1990) and competing with sensors for a large period of time. In SIA, a selection valve and a pump are used to draw up small volumes of sample and reagents. These volumes of samples are then admixed with corresponding reagents by pressing them through coils. At the end of the reaction, the resulting chemical species are pumped to a detector where they can be measured.

17 2 Phosphate Biosensors

Reagentless Chemistry

It is thought that worldwide more than 109 analyses are performed every day. This enormous number and the exponential increase of analyses threatens to create financial and ecological problems, since the most conventional chemical analyses produce chemical waste. With their reagentless recognition process, sensors produce great economical and ecological benefits. However, with sensors the specimen must also be conditioned, in many cases via the addition of buffers or salt solutions. These chemicals are in most cases low cost and nontoxic.

Fast Measurement

Due to the short response time of sensors and the whole simplification of the analytical process, it becomes possible to complete high throughput measurements. The short response time and the negligible sample preparation in combination with the defined reversibility of sensors allows on-line measurements as mentioned previously. The high throughput measurement and on- line process control saves human and technical resources and reduces costs. In principle, unstable analytes can be measured with higher precision using this faster analytical method. However, this does not play a crucial role in the case of phosphate or pyruvate detection.

Training Demand on Personnel and Accuracy

In clinical diagnoses, avoiding complicated sample preparation before measuring simplifies analyses with sensors in comparison to conventional methods. In addition, the handling of sensors is much easier than that of complicated ion chromatography apparatus for example. Thus personnel with less training can perform measurements with sensors. Secondly the accuracy of the results should improve because of the simplified of the analytical process.

High Selectivity and Design of Specificity in Biosensors

One of the most important advantages of biosensors compared to conventional analytical techniques and chemical sensors is their high selectivity (see paragraph 2.2.1 p. 21). Sensors with high selectivity for the analyte in the sample in most cases do not need a preceding sample sep- arating step. Thus, very complex samples including blood and other physiological fluids can easily be analysed. The specificity of biosensors can be influenced by the selection and engineering of appropriate recognition elements. The required high specificity can often be achieved by application

18 2.1 What are Biosensors of a selected recognition system. On the other hand, it is possible to develop screening biosensors with low specificity, where whole classes of chemicals such as organic phosphates can be measured as well . However, calibration is a constant problematic of such screening sensors.

Further advantages of Biosensors

Direct electronic processing is a further advantage of sensors and also of modern analytical methods in comparison to old techniques such as classical gravimetry, colorimetric titration and so on. Sensors measure the active molality of free analyte exactly and not the total amount of phosphate for example. This primarily plays a role in clinical analysis where blood samples are analysed and many analytes are at least partially bound to proteins. In most of the cases the activity of analytes is more relevant than the total amounts. Indeed, for every analytical problem the definition of an analyte has to be considered from case to case. In water analysis, for example, the concentration of orthophosphate and the total phosphorus are important parameters because after a period of time, all phosphates are degraded to orthophosphate and therefore become useable for plants (see figure 1-2 page 6). Also, when decomposition steps have to be performed first, a phosphate sensor can still perform the test due to the advantages outlined in comparison to conventional analytical techniques.

19 2 Phosphate Biosensors iue22 Characteristicsan of 2-2: Figure in in grey. Griffiths from Hall (1993). has beenadapted and This overview characteristics leadtothemany advantages can measure multipleanalytes small sample volume sample small in a small sample a small in high sample througput sample high fast measurement field useare possible compact designsfor directin complex measurements preparationprior to analysis ideal electrochemical biosensor. samples (blood, food, (blood, samples no need for sample no needfor waste water etc.) of biosensors incomparison toconventi on-line measurements response time small size short industrial processes An idealelectrochemical biosensor can beoptimised alyte withhighspecificityorawhole designed to detect either a single an- a single either designed todetect a biosensor can be group of analytes onal analytical techniques listedontheout high selectivity regenerative biosensor ideal continuous measurements processing and control shows different keychar direct electronic of calibration reagentless test can be performed by op- by beperformed can test erators with less training ecological benefit er circle. The most important points arehighlighted points important Themost er circle. risk ofprocessing mistakesand false islower calculations ed per data point generated point ed perdata cost effectiveness calculat- acteristics listed in the inner circle. These circle. theinner in acteristics listed devices allows actions tobe tak- actions devices allows en automatically in response to inresponse en automatically biosensor measurements ported and displayedin integration into other into integration processed, stored, ex- different ways data canbe

20 2.2 An Ideal Phosphate Sensor Setup

2.1.4 Disadvantages of Biosensors Compared to Conventional Analytical Techniques

The main disadvantage of biosensors is their relatively unstable biological recognition component; biosensors often show a limitation in the accuracy of measurements due to electrode response drift. Because of this, many biosensors are designed as single use test strips e.g. test kits for gravidity, hand-held blood glucose meters etc. Another concept addressing this difficulty is the stabilisation of the biological component. This represents the main part of the work performed during this thesis, as well as the physico-chemical characterisation of the molecules. The stability and activity of biological macromolecules depend on many different parameters such as the aqueous environment, ionic strength, pH-value, temperature, light and redox potential in the environment. All these parameters have to be considered during biosensor development.

2.2 An Ideal Phosphate Sensor Setup

2.2.1 Recognition Elements for Phosphate Sensors

2.2.1.1 Recognition Elements in Nature

Complex recognition processes in nature in most cases are effected by three dimensional molecules like proteins or RNA molecules. The only exception to this rule are DNA strands that recognise their counterpart on the basis of one dimensional sequences. Only proteins or ribozymes are considered for the recognition of phosphate molecules because the required specificity can only be guaranteed by a well defined three dimensional recognition processes.

2.2.1.2 Ribozymes are inappropriate for Phosphate Recognition

RNA molecules with catalytic activity, so called ribozymes, use mainly the same strategies for catalysis as enzymes, lowering the energy barrier between the transition state and the ground state of the reactants. During catalysis in ribozymes, nucleobases and metal ions play a role in general acid-base and electrophilic catalysis, substrate orientation and proximity are also important factors for rate enhancement of chemical reactions (Lilley 2003, Fedor and Williamson

21 2 Phosphate Biosensors

2005). The currently identified ribozymes predominantly have nuclease or nucleotidyl trans- ferase activity with some minimal esterase activity. In the majority of cases the substrates of ribozymes are RNA molecules. With their limited repertoire of functional groups, ribozymes are less versatile catalysts than proteins which are based on 20 different amino acids. Therefore proteins are the obvious choice for the analytical problem of phosphate and pyruvate detection.

2.2.1.3 Different Types of Proteins as Recognition Elements

The recognition process induced by the active site of enzymes, antibodies or receptors is extremely selective, permitting differentiation even between chiral isoforms of molecules. Recog- nition in these proteins demands a perfect three dimensional geometrical and physicochemical fit to permit binding of the analyte to the sensing molecule. Biomolecules like enzymes, antibodies or receptors fulfil these requirements of recognising elements in an unique way. Biolog- ical evolution designed optimal binding partners or catalytically active partners to many molecules in innumerable small adaptation steps. When considering proteins as recognition elements for a sensor, one has to distinguish between the main types of recognising proteins; enzymes, antibodies, ion channels, receptors and transport proteins. Mammalian blood transport proteins, for instance, were withdrawn from the selection for phosphate recognising proteins because of their unspecific binding of different chemical species. Due to the difficulties in handling transmembran proteins, Pi-carriers (PIC) originating from renal tissue or from mitochondria (Takeda et al. 2004, Schroers et al. 1997) are not the first choice as phosphate recognising elements. Furthermore, phosphate carrier molecules work only as co-transporters depending on a gradient of other ions (sodium-, hydrogen ions, or protons). An other species of phosphate recognising proteins is the class of bacterial periplasmatic binding proteins (bPBP). They are an interesting alternative to enzymatic biosensors, although the required coupling of recognition and detection makes the system of bPBP more complex. One of the following paragraphs outlines a short-list of different applications of bPBP in biosensors in detail. Designing a phosphate binding antibody is assumed difficult and inexpedient because of several reasons. In every cell which produce antibodies, Pi exists as a physiological buffering substance. Therefore, no natural anti-Pi-antibodies exists. An evolutionary protein engineering approach for the design of such an antibody would be very difficult, because a phosphate free natural expression system for the following selection would not be operable. The construction

22 2.2 An Ideal Phosphate Sensor Setup of a phosphate biosensor using antibodies is further unsuitable because these affinity sensors detect concentrations in the nano- and subnaomloar range, which is not the requested range of Pi concentrations, found in samples at milimolar concentrations. Detection of antigen binding by antibodies requires optical, high cost transduction systems and rinsing after each measurement, this complicates on-line measuring. While antibodies or ionophores need to be rinsed after a sensing cycle, the catalytic function of enzymes is not “consumed“. This represents a strong argument in favour of developing a Pi-sensor based on enzymes. Secondly, the performed chemical reaction can be easily transduced into an electric signal detectable by an electrical device. This is much more difficult in cases of antibody binding. Under these circumstances oxidoreductases are the most suited enzymes, generating chemical redox species which can be easily transduced into electronic devices. Thirdly, the turnover number of these enzymes situated between 10-2 and 103 [sec-1] and

Km values in the millimolar range are optimal for the detection range required within Pi-sensors. Thus enzymes are the best applicable proteinogenic recognition elements for phosphate biosensors.

Enzymes as Recognition Elements for Phosphate Sensors

Enzymatic Pi-biosensors are reviewed by Fernandez et al. (1998), Engblom (1998) and Karube and Nomura (2000). Most Pi-biosensors have been developed based on enzymatic cascades where a first enzyme uses Pi as cosubstrate, delivering a product, which is the substrate for an oxidase in a second reaction. The Pi concentrations are measured electronically (see paragraph 2.2.3 p. 35) on the basis of O2 consumption, the generation of H2O2 or other electroactive species. Optical Pi-biosensors have also been developed on the basis of chemilu- minesecence (see no. 5b on table 2-1). The most prominent enzyme cascades which have been investigated during the last 30 years of phosphate sensor development are listed in table 2-1 on page 25.

The first approach carried out in enzymatic Pi measurement was the enzymatic cascade described by Guilbault and Nanjo (1975) (No. 1 in table 2-1). The active principle of this first

Pi-biosensor was the inhibitory effect of phosphate on alkaline phosphatase activity followed by a second reaction catalysed by glucose oxidase. The weak point of this biosensor was its low selectivity.

Another mentionable approach are Pi amplifying sensors which lower the detection limit (Wollenberger and Scheller (1993) table 2-1, no. 2a; Wollenberger et al. (1992)). Thus the de-

23 2 Phosphate Biosensors tection limit of the phosphate electrode could be shifted to 25 nM. Even though numerous different enzymatic cascades for phosphate biosensors have been investigated, none of them accomplished the breaktrough to the sensor market. The difficulty of such complicated enzymatic cascades are the need for expensive cosubstrates or the low operational stability of various enzymes. Establishing the optimal reaction conditions for four or more different enzymes working in the same biosensor is indeed a challenge.

24 2.2 An Ideal Phosphate Sensor Setup Wollenberger and Scheller and Wollenberger (1993) Parellada et al. (1998) Male and Luong (1991) D‘Urso & Coulet (1990), (1993) Müller (2000) Guilbault and Nanjo (1975) Kutuzov and Andreeva (2001) + ion. Less glucose formation in ion. Less glucose -glucose-1phosphate -glucose-1-phosphate 2 α 2 2 α O i O O 2 i 2 2 lower oxygen consumption in reaction II) and this D-glucono-1,5-lactone-6-phosphateH NADPH + + Reactions References -glucose + P -glucose + -glucose + P β -glucosyl)n-1 + β -glucosyl)n-1 + α α G6PDH -glucose-6-phosphate

and thus lowers glucose format lowers glucose and thus + (1,4- α ALP (1,4-

ALP XOD uricacid + 2H O 2 O 2 2 phate biosensors: phate biosensors: D-glucono-1,5-lactoneH + 5.4.2.2 2.4.1.1 2.4.1.1

-glucose i i β O + 2O O + 2 GOD GOD inhibits reaction I) D-glucono-1,5-lactone + H + D-glucono-1,5-lactone 2 i MR 2 2.4.2.1 ribose-1-phosphate + hypoxanthine + ribose-1-phosphate i ades used in phos ades used analysis possible. analysis -glycosyl)n + P -glycosyl)n + P i α α glucose -glycose-6-phosphate + NADP -glucose + O -glycose-1-phosphate -glucose + O -glucose-6-phosphate + H -glucose-6-phosphateH + β α− Ι) β I) (1,4- + P I) inosine I) (1,4- II) hypoxanthine + 2H reaction I) can be measured amperometricly due to amperometricly due measured reaction I) can be P makes ΙΙ) β P The presence of III) ΙΙ) β ΙΙΙ) α ΙΙ) α IV) Enzymes and EC. numbers EC. and

(ALP) 3.1.3.1 glucose oxidase (GOD) 1.1.3.4 phosphoglucomutase 5.4.2.2 glucose-6-phosphate dehydrogenase (G6PDH) 1.1.1.49 2.4.2.1 xanthine oxidase (XOD) 1.17.3.2 alkaline phosphatase (ALP) 3.1.3.1 5.1.3.3mutorotase (MR) glucose oxidase (GOD) 1.1.3.4 No 1 alkaline phosphatase 2b phosphorylase2.4.1.1 A 3 nucleoside phosphorylase 2a phosphorylase2.4.1.1 A Table 2-1:casc enzymatic Most prominent

25 2 Phosphate Biosensors Table 2-1: Most prominent Most enzymatic casc 2-1: Table 4a maltose phosphorylase phosphorylase maltose 4a 5b pyruvate oxidase (POX) oxidase pyruvate 5b (POX) oxidase pyruvate 5a phosphorylase maltose 4c phosphorylase maltose 4b No 1.1.3.4 (GOD) glucose oxidase (MP) 2.4.1.8 (HRP) 1.11.1.7 (HRP) peroxidase horseradish 1.2.3.3 1.2.3.3 1.1.3.4 (GOD) glucose oxidase 3.1.3.2 (aP) acid phosphatase (MR) mutorotase 5.1.3.3 (MP) 2.4.1.8 1.1.3.4 (GOD) glucose oxidase (MR) mutorotase 5.1.3.3 (MP) 2.4.1.8 and EC. numbers Enzymes III) III) III) II) luminol +H luminol II) IV) ΙΙ) ΙΙ) II) II) II) I) maltose I) maltose + P I) pyruvate+P P + pyruvate I) maltose + P I) maltose + P α− glucose + glucose O α− β β β -glucose-1-phosphate -glucose +O -glucose -glucose + O -glucose + glucose glucose glucose glucose ades used in phos i + O i i i

i 2 2 + O + D-glucono-1,5-lactone D-glucono-1,5-lactone + H O MR MR MP 2 MP MP 2 2 2 GOD + H + + 2OH 2 + H GOD GOD β β 2 glucose + α α acetyl-phosphate + CO O -glucose -glucose 2 -glucose + -glucose +

O acetyl-phosphate + acetyl-phosphate CO O - N phate biosensors: POX

D-glucono-1,5-lactone + H + D-glucono-1,5-lactone D-glucono-1,5-lactone + H D-glucono-1,5-lactone HRP aP POX β -glucose-1-phosphate

β β β -glucose + P -glucose + -glucose-1-phosphate -glucose-1-phosphate 2 +4H 2 ecin References Reactions O +h i 2 ν O 2 2 2 O O 2 2 2 2 +H + H + 2 2 O O 2 2 (2005b); etal.(2005) Rahman (2005b); (2005a) al. et Kwan (2003); al. et Mak (2001); Chaniotakis and Gavalas (2000); tani et al. Gajovic etal. (1999); Mizu- Mascini andMazzei(1997); etal. (1991); Kubo Zapata andBurstein(1987); Conrath et al.(1995) et (2001) al. Mousty (1997) etal. Hüwel Nakamura (2001) Nakamura Nakamura et al. (1997) (1999 (1997) al. et Nakamura (1996b) etal.(1996a) Ikebukuro )

26 2.2 An Ideal Phosphate Sensor Setup

All examples listed in table 2-1 are well known enzymatic cascades for phosphate measurement. But do other enzymes exist in nature, that use phosphate as substrate? A search through the brenda enzyme database yielded the following list of phosphate dependent oxidases (see table 2-2). As described below in paragraph 2.2.3 p. 35 oxidases are the most appropriate class of enzymes for application in biosensors. Nine oxidases were found in that database search. Two of them (no 8 and 9) need thioredoxin as a coenzyme and are therefore not suitable for a simple sensor architecture. Six out of nine (no 1-6) need nicotinamide-adenin-dinucleotid-phosphate (NADP+) or nicotinamide-adenin-dinucleotid (NAD+) as a cosubstrate for their catalytic reaction. In a potential biosensor using one of these six oxidases, the cosubstrate has to be immobilised in the sensor or added to the sample solution. Aspects in the disfavour of cosubstrate immobilisation is their short shelf life due to high redox activity. This requirement complicates the construction of sensors with enzymes no 1-6 or needs large amounts of expensive cosubstrates. The most simple reaction mechanism with no need of NADP+ or NAD+ as cosubstrate or thioredoxin as coenzyme can be realised by applying pyruvate oxidase (no 7 in table 2-2). The cosubstrate pyruvate is not too expensive and can easily be added to the sample solution.

No Enzyme and EC. number Reaction + 1 aspartate-semialdehyde dehydrogenase L-aspartate 4-semialdehyde + Pi + NADP 1.2.1.11 L-4-aspartyl phosphate + NADPH + 2 glyceraldehyde-3-phosphate dehydrogenase D-glyceraldehyde 3-phosphate + Pi + NAD (phosphorylating) 1.2.1.12 3-phospho-D-glyceroyl phosphate + NADH + 3 glyceraldehyde-3-phosphate dehydrogenase D-glyceraldehyde 3-phosphate + Pi + NAD (NADP+) (phosphorylating) 1.2.1.13 3-phospho-D-glyceroyl phosphate + NADH + 4 N-acetyl-gamma-glutamyl-P reductase N-acetyl-L-glutamate 5-semialdehyde + NADP + Pi 1.2.1.38 N-acetyl-5-glutamyl phosphate + NADPH + 5 glutamate-5-semialdehyde dehydrogenase L-glutamate 5-semialdehyde + Pi + NADP 1.2.1.41 L-5-glutamyl phosphate + NADPH + 6 glyceraldehyde-3-phosphate dehydrogenase D-glyceraldehyde-3-phosphate + Pi + NAD (NAD(P)+) (phosphorylating) 1.2.1.59 1,3-diphosphoglycerate + NADPH

7 pyruvate oxidase pyruvate + Pi + O2 + H2O 1.2.3.3 acetyl-P + CO2 + H2O2 8 sarcosine reductase N-methylglycine + Pi + thioredoxin 1.21.4.3 acetyl-P + methylamine + thioredoxin disulfide

9 betaine reductase N,N,N-trimethylglycine + Pi + thioredoxin 1.21.4.4 acetyl-P + trimethylamine + thioredoxin disulfide

Table 2-2: Phosphate dependent oxidases: The table enumerates all phosphate dependent oxidases that are found in the brenda database.

27 2 Phosphate Biosensors

Conclusions Concerning Pi-Recognising Enzymes Summarising the large list of published phosphate biosensors and the list of phosphate dependent oxidases, the most straightforward and most investigated enzymatic phosphate recognising cascade is the one applying pyruvate oxidase (no. 5a in table 2-1 and no 7 in table 2-2). In this approach no other enzymes than POX and pyruvate as a cosubstrate are needed for phosphate measurements.

Other Proteinogenic Phosphate Recognising Elements

Campanella et al. (1992) and other authors developed phosphate sensors based on plant tissue intending to produce a stable and cheap biosensor. Enzymes in their natural surroundings are generally more stable than enzymes that are immobilised alone in a biosensor. Furthermore, enzyme purification can be a time consuming and expensive step during the production of biosensors. In this way, acid phosphatase in potato tissue used as a phosphate recognising element and GOD as the second oxidising enzyme are combined in the sensor, analogous to the enzymatic cascade described in table 2-1, no 1. With a storage stability of about two weeks and an easy production method, the tissue sensor is an interesting alternative setup to pure enzymatic biosensors. Nevertheless, due to unknown operational stability, interferences caused by glucose and the difficult standardisation of potato tissue, this sensor setup was not considered further during this project. Demuth et al. (2001) designed an oligopeptide to mimic the phosphate binding site of pu- rine nucleosid phosphorylase (PNP, EC. 1.2.4.2, see table 2-1 no 3). CD measurements in methanol showed significant spectral changes when sulphate ions were added, but only a small effect to the addition of phosphate ions. NMR experiments further indicated, that the peptide-phosphate complex is (helix)-structured in comparison to the peptide alone. One can conclude that the recognition of phosphate and simultaneous discrimination of other oxianions seems to be difficult and requires a large network of chelating functional groups similar to these on enzyme’s active sites. It is essential that the functional groups in active sites are exactly oriented. The connection of only a few functional groups of the active sites does not fulfil the requirements of specific phosphate recognition.

Bacterial Periplasmatic Binding Proteins

Bacterial periplasmatic binding proteins (bPBP)s consist of two domains connected by a hinge region, with a ligand-binding site located at the interface between the two domains. The two do-

28 2.2 An Ideal Phosphate Sensor Setup mains clamp together like a Venus flytrap when specific ligand molecules are engaged (see figure 2-3). This conformational change during phosphate binding allows the construction of optical and electrochemical biosensors based on bPBPs. Optical biosensors operate by coupling ligand binding to changes in fluorescence intensity by positioning single, environmentally sensitive fluorophores in locations that undergo conformational changes (Brune et al. 1994, Salins et al. 2004). Electrochemical biosensors act by modulating electronic coupling between a covalently conjugated redox-active reporter group and the surface of an electrode (Benson et al. 2001). bPBPs originally mediate chemotaxis and solute uptake and are specific for a wide variety of small molecules, including carbohydrates, amino acids, inorganic ions, dipeptides and ol- igopeptides. Over 100 bPBP structures have been determined so far and biosensors for maltose, glucose, ribose, arabinose, glutamine, glutamate, histidine, Fe3+, Ni2+, phosphate, sulphate and dipeptides are described in literature but are not yet commercially available. (see reviews of De Lorimir et al. 2002, Dwyer and Hellinga 2004)

The phosphate binding protein (PBP), which is a member of bPBPs, binds Pi in the peri- plasm and transfers it to a membrane protein which transports it into the cytoplasm. (Arsenate is transported by the same system, but binds to PBP five orders of magnitude less tightly than phosphate.) PBP consist of a single polypeptide chain (Mr: 34.400 Da) with one phosphate binding site. The phosphate, bound in the hinge region, is totally dehydrated, completely sequestered below the protein surface and is held in place by 12 hydrogen bonds. Asp56 plays an important role in phosphate binding and in discriminating sulphate or similar tetrahedral fully ionised divalent oxianions (Luecke and Quiocho 1990), (Wang et al. 1997). An A197C mutant of E.coli PBP, labelled with a coumarin fluorophore [N-[2-(1-maleim- idyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) at Cys197, was developed as a fluorescent probe (MDCC-PBP) (Brune et al. 1994). Pi binding to MDCC-PBP causes cleft clo- sure with a simultaneous increase in the hydrophobicity around the coumarin and hence the changes in fluorescence intensity at 465 nm. MDCC-PBP sensitivity for Pi is in the sub micromolar range (Hirschberg et al. 1998, Brune et al. 1998, Salins et al. 2004). The stability of the system was unchanged after four months storage at 4°C in the dark.

29 2 Phosphate Biosensors

a) b)

Figure 2-3: Phosphate binding in PBP; Proteinstructures of periplasmatic PBP mutants T141D from E. coli., where structures without (a) 1OIB) and with (b) 1IXG) complexed phosphate ions are available on the PDB database. The mutation T141D in the binding site introduces an additional hydrogen bond with the phosphate O2. The two domains clamp together like a Venus flytrap, when they engage phosphate ions (marked by the red arrows). References: a) 1OIB: Yao et al. 1996, b) 1IXG: Wang et al. 1997.

The required coupling of recognition and detection makes the system of phosphate binding proteins more complex than an electrochemical enzymatic biosensor. Furthermore, PBP-biosensors have to be rinsed like other bioaffinity sensors after measurement and this protein also has a fi- nite lifetime. These facts show that PBPs are not the first choice for phosphate recognising elements in biosensor development.

2.2.1.4 Non-Proteinogenic Phosphate Recognising Elements

Engblom (1998) gives a broad overview of the field of phosphate sensors. Aside from enzymatic biosensors, ion selective electrodes (ISE), amperometric plant-tissue electrodes and other devices in the form of integrated probes used for determining Pi concentrations are discussed. In the ISE field, solid state electrodes and polymer membrane electrodes are differenti- ated:

30 2.2 An Ideal Phosphate Sensor Setup a) Solid State Electrodes

Xiao et al. (1995), Chen et al. (1997) and Engblom (1998) developed phosphate sensors based on metallic cobalt oxide (CoO). The CoO layer at the electrode surface responds to dihydrogen phosphate ions in a kind of host-guest chemistry, due to Co2+ imperfections. The sensors showed reliable selectivities in comparison to other ions. But the cobalt electrode was also sensitive to oxygen, and could be used as O2-sensors too. A decreasing sensitivity in electrode re- - 2- sponse at pH values higher than 5 was observed due to conversion of H2PO4 to HPO4 . Furthermore, Engblom reported signal dependency on other oxidising substances like Fe3+.

This dependency on O2 and on other oxidising agents in sample solutions and the suboptimal pH-ranges of the cobalt sensors exclude this recognising element from application in this project. Coated wire electrodes containing cobalt phtalocyanine reported by Liu et al. (1990) exhibit variable results, but suffer from limited selectivity and therefore are inapplicable as phosphate recognising elements. A phosphate sensor containing hydroxyapatite described by Petrucelli et al. (1996) showed a lifetime of at least four months with daily use, but the electrode was only usable in the pH-range of 3-6. Further investigations are needed to exclude interactions of real samples with hydroxy apatite before this sensor can be further applied. b) Polymer Membrane Electrodes

Polymer membrane ISEs work on the basis of measuring the electric potential generated across a polymeric membrane by “selected“ ions. Organic ion exchangers or chelating ion carrier agents, also called ionophores, in the ISE membrane selectively bind certain small ions from the sample solution and exchange them across the membrane creating a potential. Whereas a broad + + + range of cation-selective electrodes are commercially available, such as for K , Na , NH4 , Ca2+, Mg2+, Pb2+, Cu2+ etc., the design of sensory molecules for anion-selective electrodes is still a challenging subject. Ion selective electrodes for lipophilic anions are usually based on ion exchangers and in this case their selectivity pattern follows the Hofmeister series, which originates from the rank- ing of various ions toward their ability to precipitate proteins (Kunz et al., 2004);

2- 2------Hofmeister series for anions: SO4 > HPO4 > acetate > Cl > Br > I > NO3 > ClO4 > SCN 3+ 2+ + + + + + - Hofmeister series for cations: Al > Mg > Li > Na = K > NH4 > H(CH3)4 > SCN

31 2 Phosphate Biosensors

The hydration of ions decreases from the left to the right in the Hofmeister series, therefore the solubility of ions in hydrophobic membranes increases from the left to the right. Due to the high hydration energy of hydrophilic anions such as phosphate and sulphate on the left side of the Hofmeister series, it is more difficult to force these strongly hydrated anions to be dissolved in the lipophilic membranes of ISEs. The high hydration of phosphate is one of the major problems for the development of chemical phosphate sensors. Highly selective and rather strong binding phosphate chelating agents are therefore required to catch the few phosphate ions dissolved in the membrane itself. Metal-ion containing ionophores as anion carriers in ISEs may result in anion selectivity patterns significantly different from the Hofmeister selectivity series. A few problems relating to phosphate selective potentiometric sensors that have been reported in literature are outlined in the next paragraphs to give a small insight into the story of

Pi-selective ionophores; Organic tin(IV) compounds have traditionally been used as phosphate selective ionophores; they are the most promising ionophores. A study by Chaniotakis et al. (1993) led to the development of a phosphate ISE based on a multidentate-tin(IV) carrier incorporated into a liquid polymeric membrane. Liu et al. (1997) used bis(tribenzyl)tin oxide, which exhibited a selective response to hydrogen phosphate at a neutral pH. The weak point of all these tin-based phosphate ionophores are their short lifetimes, due to ligand exchange with anions in solution. A zwitterionic bis(guanidinium) ionophore described by Fibbioli et al. (2000) has been used in potentiometric membrane electrodes. Detection limits in ambient water were in the micromolar range. In unbuffered solutions the detection limit was even in the range of 10-8 (mol/ l) due to the lack of OH- interactions. But the bis(guanidinium) ISEs displayed poor selectivity ------2- for phosphate against most common anions such as Cl , Br , I , NO3 , ClO4 , acetate , CO3 and 2- SO4 .

Phosphate-selective electrodes containing N3-cyclic polyamins described by Carey and Riggan (1994) and further developed by Goff et al. (2004) achieved lifetimes of 40 days due to covalent immobilisation of the ionophore in polymer membranes. But during immobilisation the good selectivity coefficients for chloride, sulphate and nitrate dropped to rather low values. Molecularly imprinted polymers (MIPs), fully synthetic binding molecules, can in principle be generated for any analyte molecule. MIPs are intrinsically stable, robust, cheap to produce and have been developed for small organic molecules, like pesticides, amino acids, steroids, sugars, but also for proteins and cells (Haupt and Mosbach 2000). The majority of experimental results in real samples, however, still exhibit drawbacks with regard to affinity,

32 2.2 An Ideal Phosphate Sensor Setup cross-reactivity and unspecific binding. The main problems in most ISEs for phosphate are the insufficient selectivity compared to other anions and/or the short lifetime of the electrodes of only a few days. A few remarkable exceptions circumventing the difficulties mentioned above are listed in the next paragraphs: Firstly, Antonisse et al. (1998) used chemically modified field effect transistors (CHEM- FETs) with ion-selective membranes containing uranyl salophene derivatives (figure 2-4a). The CHEMFET-sensors described by Worbelewski et al. (2000, 2001) also based on uranyl salophene molecules, showed high selectivity and lifetimes of up to 1 month. But these electrodes - are only selective for H2PO4 -ions and therefore are not suitable for orthophosphate measuring beyond the pH range of 3-6. Ganjali et al. (2003) reported on a phosphate sensing electrode based on vanadyl-salen (figure 2-4b). This electrode was highly selective to phosphate and showed good slope stability over 8 weeks at a pH of 8.2. But the results were not reproducible (oral comunication with Prof. U. Spichiger at CCS) Secondly, Gupta et al. (2005) recently showed a new and promising species of ionophores with their concept of calixarenes that can be used in ISEs for Pi measurements (figure 2-4c). The carbamoyl methoxy groups combine hydrogen-bond functions, electrostatic and dipole–dipole interactions, thus imitating natural receptors for nucleosides where phosphate is also complexed by similar interactions. The selectivity coefficient values indicate good selectivity against phosphate over a large number of anions. But a storage lifetime of about one month has been identified and therefore operational stability can not be estimated.

33 2 Phosphate Biosensors

a) R3 R3

c) H H N N UO2 R2 O O R2

R1 R1 O b) H H H N O N N 2 V 6 O O O

Figure 2-4: Formula of phosphate ionophores in ISEs: a) Template of an uranyl salophen complex used by Antonisse et al. (1998) and Worbelewski et al. (2000, 2001) b) Vanadyl salen complex described by Ganjali et al. (2003) c) Calixarenes developed by Gupta et al. (2005). The carbamoyl methoxy groups combines hydrogen-bond functions, electrostatic and dipole–dipole interactions, thus imitating natural receptors for nucleosides where the phosphate is also complexed by similar interactions.

2.2.1.5 Conclusions regarding Pi-Recognising Elements For years, great efforts have been made to design artificial recognition molecules for several analytes that circumvent the disadvantages of biomolecules. The results with uranyl salophenes, vanadyl-salen and calixarenes recently published by Antonisse et al. (1998), Worbelewski et al. (2000, 2001), Ganjali et al. (2003) and Gupta et al. (2005) showed interesting alternative phosphate recognising molecules for Pi-sensors. Because these artificial phosphate recognising molecules were not published at the beginning of the phosphate sensor project in 1998 and due to the lack of capacities for new synthesis of such compounds, POX was chosen as the recognising element for phosphate sensors in this project. Furthermore, redox-enzymes are still the most appropriate recognition elements because they combine high chemical specificity and easy signal transduction.

2.2.2 Recognition Elements for Pyruvate Sensors

Different enzymes could be applied as recognition elements for pyruvate sensors (see pyruvate metabolism on figure 1-3 page 12). But again, the approach based on oxidoreductases like LDH

34 2.2 An Ideal Phosphate Sensor Setup or POX is the most promising one. Pyruvate sensors using LDH as recognising elements depend on NAD+ addition during measurements (see reaction 1-5 p. 13). The pyruvate sensors described in literature are not tested with regards to long operational lifetimes. The results from storage stability studies in optical and amperometric detecting biosensors based on LDH do not promise good operational stabilities (Warriner et al. 1997; Zhang et al. 1997; Gerard et al. 1999). Pyruvate sensors based on POX not originating from Lactobacillus plantarum need flavinadenine dinucleotide (FAD), thiamine diphosphate (ThDP), and manganese-ions (Mn2+) or magnesium-ions (Mg2+) as cofactors and are therefore not suitable for on-line measurements. Aside from this, the pyruvate sensors described by Arai et al. (1999), Gajovic et al. (2000), Revzin et al. (2002) and Situmorang et al. (2002) are not tested for operational stability. The storage stability varies between less than 50 and about 95% residual activity after 7 days. Examples from literature where lpPOX was applied, did not need any addition of cofactors during measurements but showed weak operational stabilities (Kulys et al. 1992; Bergmann et al. 1999). The sensor described by Bergmann et al. was interesting, as the lpPOX was coupled to a second enzyme, horseradish peroxidase (HRP; EC 1.11.1.7; see eq. 2-1) and the influence of additives like raffinose, lactitol and poly-L-arginine on operational stability was analysed. The additives led to higher selectivity and stability of the sensor but they were rapidly eluted out of the sensing carbon paste.

HRP donor + H2O2 oxidised donor + H2O(Eq.2-1)

A pyruvate sensor based on corn tissue coupled to an optical CO2-sensor is also described in literature (He and Rechnitz (1995)). But due to its long response and recovery time of up to 30 minutes, short lifetime of 7 days and complex architecture, this sensor is not appropriate for medical pyruvate measurements. POX, selected as the most promising recognising element for a phosphate sensor, also seems to be the most promising candidate for pyruvate measurement.

2.2.3 Transducing Techniques in Biosensors

A list of the most applied transducing techniques in biosensors is given in figure 2-1 page 15. In this paragraph the specific advantages and disadvantages of amperometric transducing techniques are discussed in detail. Other widly used techniques such as potentiometry and optics are

35 2 Phosphate Biosensors briefly explained. In conclusion the rationale for chosing the amperometric transducing technique for the sensor setup in this project is presented.

Amperometry

Amperometrical sensors monitor currents generated when electrons are exchanged between the active site and the electrode itself. An external potential which does not correspond to the potential of the thermodynamic equilibrium is applied and the activation energies of oxidation and reduction become unequal. As a result of this disequilibrium, the product is converted. After the enzymatic turn over of the substrate, electrons are transmitted to the electrode and can be measured as current. The activity of the corresponding enzyme determines the turn over rate of substrate and also the generated current. In order to control the applied potential between the counter electorde (CE) and the working electrode (WE), a third electrode called reference electrode (RE) has to be introduced to close the circuit in the sensor. Small variations of the potential between working and counter electrode do not alter the rate of the redox reaction. Therefore, the reference electrode need not be perfectly drift-free to achieve stable measurements as with potentiometric sensors. Theoretically, the current obtained in a quiescent solution with a conventional set of electrodes decays to zero according to the spread of the diffusion layer into the sample solution. This behaviour is described in the Cottrell equation (Rieger 1994). Two mechanisms are used in amperometry to control this unstable diffusion profile, firstly, the movement of the solution relative to the electrode and secondly, the imposition of a diffusion barrier in the form of a membrane. Nagel describes the exact electrochemical behaviour of the flow through sensor developed for this project in his thesis in chapter two (Nagel 2004). Another reason why many enzymatic biosensors are based on amperometric transduction technique is the fact that electron transfer plays a central role in the energy conversion of oxidoreductases, which transfers electrons during their catalytic activity. Examples where electrons are transferred in natural systems are photosynthesis, the respiratory chain in mitochondria or metabolic pathways such as glycolysis or the citric acid cycle. One can find a broad variety of enzymes able to exchange electrons here, and on the other hand, amperometric transducing elements are easy to handle and are a well established transducing technique, which can be combined in an excellent way with oxidoreductases. A disadvantage of amperometrical biosensors is the fact that the signal is dependent on the mass transfer of analyte to the sensor surface. Because of this, amperometric sensors are not

36 2.2 An Ideal Phosphate Sensor Setup appropriate within bioreactors or other environments where fouling on the sensor often occurs. Adsorption to the sensor surface causes a loss in sensitivity. Nagel (2004 p.65ff) shows in experiments with glucose oxidase (GOD), that the problem of fouling seems to be negligible in the flow through sensor device developed at the CCS (Centre for Chemical Sensors; Techn- opark; ETH Zürich). A dialysis membrane was introduced in front of the enzyme containing layer to prevent enzyme leaching. The small leaching of mediator during prolonged measurements and therefore the lower transfer of electrons to the electrode, leads to a signal decrease of the GOD-sensor during the first ten hours of measurement. (Nagel 2004, p.66) Nevertheless, Griffits and Hall (1993) state amperometrical biosensors are to be the technique with the highest profile. In fact, amperometric sensors with their simple and therefore low cost technique, high sensitivity, good selectivity and broad dynamic ranges remain the most commonly used sensor technique, even though optic sensors use has increased in both research and industry in recent years.

Potentiometry

Potentiometric sensors measure the difference in potential between the working electrode and a reference electrode under conditions of zero current flow. Different types of potentiometric sensors are; 1) ion-selective electrodes (ISEs), 2) field-effect transistors (FETs) and 3) gas-sensing electrodes. All these potentiometric sensors show a logarithmic relationship between the potential generated and the activity of analytes and thus lead to sensors with a wide dynamic response range up to eight orders of magnitude of analyte activity (Spichiger 1998, p.199). In amperometrical and optical sensors the dynamic range is generally lower than this. A further advantage of this technique is that during potentiometric measurements there is no consumption of analyte, as occurs in the catalytic technique of amperometric sensors.

Optical Transduction

Rich and Myszka (2005) give a broad survey of commercial optical biosensors found in the literature of 2003. One of the advantages of optical sensors are their very low detecting limits and their board versatility. In research optical methods such as surface plasmon resonance (SPR) in BIAcoreTM analysers (Pharmacia Biosensor AB, Sweden), they play an important role in, for example the study of ligand-macromolecule interactions. The challenge in the development of

37 2 Phosphate Biosensors optical sensors in most cases is combining the recognising process to an optical detectable process either by combination of these two principles in one molecule or by co-immobilisation of two molecules with the required properties. For a phosphate or pyruvate sensor, the application of optical transducing techniques is not necessary and more costly in comparison to electrochemical techniques (see also Griffiths and Hall (1993)).

Comparison of Transducing Techniques with Regard to Phos- phate Sensors

Amperometry is a straightforward, cost effective and well established technique with no need for expensive equipment like light sources and optical detecting devices. The detection limits and dynamic response ranges requested for phosphate sensors do not justify the use of optical or potentiometrical techniques which are more complex and more expensive. Thus, amperometry is the most promising transducing technology for phosphate and pyruvate sensors. This is reinforced by the presence of oxidoreductases that use these analytes as natural substrates and due to their reaction mechanisms, can be used in amperometric sensors.

2.2.4 Application of Pyruvate Oxidase from Lactoba- cillus plantarum in Biosensors

Selection of Pyruvate Oxidase from Lactobacillus plantarum

Different pyruvate oxidases, found in various organisms, have been described in literature and tested for their applicability in biosensors. The most prominent POXs derive from Pediococcus sp. (Mascini and Mazzei 1997; Zapata and Burstein 1987; Mizutani et al. 2000; Mak et al. 2003; Kubo et al. 1991; Ikebukuro et al. 1996a 1996b; Nakamura et al. 1997), from Aerococcus sp. (Kwan et al. 2005a 2005b; Rahman et al. 2005; Nakamura et al. 1999; Nakamura 2001) and from Lactobacillus sp. (Gajovic et al. 1999; Gavalas and Chaniotakis 2001). A broad list of other pyruvate oxidases from other organisms is given in chapter three in the context of the analysis of structural details of different POXs relevant for stability. The main reason for the selection of Lactobacillus plantarum POX for use in the on-line biosensor is their reported independence of cofactors (Sedewitz et al. 1983, 1984, 1986, Elstner 1987). The cofactors FAD, ThDP and divalent metal ions are relatively tightly bound to the lp- POX apoprotein. Furthermore, stabilised mutants of lpPOX showed significantly higher inactivation temperatures and tolerated higher urea concentrations and pH values respectively

38 2.2 An Ideal Phosphate Sensor Setup

(Schumacher et al. 1991, 1992; Risse et al. 1992a, 1992b). Nagel compared commercially available pyruvate oxidases from Aerococcus viridans, Lactobacillus sp. 722T4 and Lactobacillus plantarum in order to check their suitability for biosensor application. He concluded that lpPOX had the best properties for application in a cofactor free biosensor (Nagel 2004). At the beginning of the project lpPOX was the only pyruvate oxidase with a known three dimensional structure (Muller et al. 1994). Recently the structure of Aerococcus viridans POX was also elucidated and deposited in the PDB database (Hossain et al. 2002).

Properties of Lactobacillus plantarum Pyruvate Oxidase

Pyruvate oxidase from Lactobacillus plantarum (lpPOX, EC. 1.2.3.3) is a homotetrameric fla- voenzyme with a subunit molecular weight of 65.6 kDa. One monomer is composed of 603 amino acids and consists of three domains. In the active conformation there is one molecule of each cofactor (FAD, ThDP, and Mg2+) attached to one monomer. After a first stabilisation done by random mutagenesis and involving three point mutations, the structure of both stabilised and wild-type POX from L. plantarum was elucidated by Muller et al. (1994). A deepened discussion of stability and relevant structural details of lpPOX compared with pyruvate oxidases from other species is outlined in chapter three.

Reaction Mechanism of lpPOX

The reaction mechanism of lpPOX is explained for a better understanding of the reactions in the biosensor containing pyruvate oxidase. LpPOX catalyses the oxidative decarboxylation of pyruvate with simultaneous phosphorylation resulting in the energy rich acetylphosphate eq. 1-1. Lactobacillus plantarum further utilises this acetylphosphate for ATP production catalysed by acetate kinase (EC. 2.7.2.1) (see eq. 2-2 or figure 1-3 page 12)

2.7.2.1 ADP + acetylphosphate ATP + acetate (Eq. 2-2)

The catalytic mechanism of lpPOX was elucidated by Tittman et al. (1998) and is displayed in figure 2-5. (1) The 4‘imino-function of the enzyme bound ThDP deprotonates the C2-ThDP forming an ylide species. This is the initial step shared by all ThDP dependent enzymes. (2) The carbonyl-group of the substrate pyruvate undergoes nucleophilic attack by this ylide. (3) The lactyl-ThDP decarboxylates to hydroxyethyl-ThDP containing an α-carbanion. Tittmann et al.

39 2 Phosphate Biosensors

(2000) suggested that the acceleration of decarboxylation by six orders of magnitude compared to free ThDP, might result from a nonpolar environment within the active site caused by Phe479 and Val394. (4) The HEThDP is subsequently oxidised by FAD via a two step single electron transfer mechanism. It is still unclear by which pathway the electrons are transferred between HETdDP and FAD in the enzyme active site, but it was suggested that this transfer may be re- layed by the benzene ring of Phe479 or Phe121 or both (Muller and Schulz (1993)). (5) The reduced flavin transfers the two electrons to the final electron-acceptor O2 in cells releasing H2O2 or in the biosensor to the mediator with a lower redox potential than O2, releasing the reduced mediator respectively. (6) Finally, the acetyl-ThDP is cleaved phosphorolytically to acetylphos- - 2- phate and ThDP. In this way, Glu483 can abstract one proton from H2PO4 resulting in HPO4

40 2.2 An Ideal Phosphate Sensor Setup

, which is a better nucleophile for phosphorolysis (Tittmann et al. (2000)).

- O HO O E59 R FAD P - + O O COO N lpPOX FAD acetyl-P E59 R + S R COO- N lpPOX O acetyl-ThDP 6 H S R H O - 2 2 H2PO4 5 1 O2 R H E59 FAD R + N N O COOH N lpPOX - N S R E59 R + N H - H+ COO N O lpPOX H O - O FADH2 S R 2 O O pyruvate acetyl-ThDP 4 FAD E59 R + COOH N lpPOX R 3 S R N N O HO O- N O E59 R + N H lactyl-ThDP CO2 COOH N lpPOX O - FAD S R OH HEThDP (α-carbanion) Figure 2-5: Catalytic cycle of lpPOX elucidated by Tittman et al. (1998): The Cofactors ThDP and FAD are bound non covalently to lpPOX represented by dotted lines. In the active tetrameric form of lpPOX Glutamate 59 (E59) from the opposite monomer is important for the activation (see also figure 2-6).

Kern et al. (1997) and Tittmann et al. (1998) showed that the H-exchange of enzyme bound C2- ThDP is strongly affected by the presence of FAD and the substrate phosphate. Only the holoenzyme deprotonation of C2-ThDP is fast enough to account for catalysis (314 s-1 at 4°C compared to 0.001 s-1 of free ThDP in solution). One reason for this accelerated deprotonation rate

41 2 Phosphate Biosensors of C2-ThDP is the V-conformation of ThDP in the lpPOX tetramer. Fast deprotonation of C2- ThDP requires the interaction of glutamate 59 of the adjacent subunit with N1‘in the pyrimidine ring, leading to an increased basicity of the 4‘ amino group (Kern et al. 1997) (see figure 2-6). Theoretical studies of Friedmann and Neef (1998) emphasise this manner of ThDP activation in enzymes.

O ThDP a) E O - + O O OH Glu59 HN1‘ N P P 2 - - 4‘ S O O O O N NH H

E OH + O O OH Glu59 N 1‘ N P P 4‘ 2 - - - S O O O O N NH2 ThDP

b) 2.66 Å Glu59 3.14 Å Mg2+ ThDP

Figure 2-6: Activation of ThDP in lpPOX (Kern et al. 1997): The figures show ThDP molecules from chain A and Glu59 from chain C of the tetrameric form of lpPOX a) Two tautomeric forms of the ThDP-Glu59 system in the V-conformation. b) The same ThDP molecule and the Glu59 in a three dimensional sight extracted from the elucidated structure of lpPOX is shown for a better understanding of the V-conformation. The V-conformation of ThDP in the lpPOX tetramer accelerates the deprotonation rate of C2-ThDP due to the near basic imino group. But fast deprotonation of C2-ThDP requires the interaction of glutamate 59 of the adjacent subunit with N1‘ in the pyrimidine ring, leading to an increased basicity of the 4‘ amino group.

The next paragraphs describe how the electrons gained are transported through the sensor paste to the electrode surface after the reaction of pyruvate and phosphate catalysed by POX.

2.2.5 Electron Transfer in Bioelectronics

Electronic coupling and communication between a biomolecule and an electronic material is a fundamental requirement of biosensors and other bioelectronic systems. For instance, coupling of neurons to electronic devices could lead to the development of neuronal networks that can be

42 2.2 An Ideal Phosphate Sensor Setup used as bio-computers. Perhaps one day enzymes will be used in biofuel cells or actin-myosin complexes in biomotors. Understanding charge transport through biological materials has been enhanced during the last twenty years by intensive experimental work and theoretical discussions in the fast growing discipline of bioelectronics. In natural systems such as cells, in most cases no free electron transfer is intended. In fact many natural processes like signal distribution in nerves, energy production via glycolysis or beta-oxidation operate with ion or proton gradients and not via direct electron transfer. Numerous redox enzymes exchange electrons with other biological molecules such as other redox proteins, cofactors or molecular substrates. But unfortunately most proteins lack pathways that can transport electrons from their embedded redox sites to electrodes. The barri- ers for charge transport out of and between redox proteins are explained by the Marcus theory (Marcus 1985) and are essential in nature for controlled redox reactions in cells or compart- ments. This lack of communication between biomolecules and electronic materials presents one of the fundamental difficulties in developing electronic biosensors. Many methods of electrical contact of biomolecules with electronic units were developed in recent years (Willner and Katz 2000).

2.2.6 Electron Transfer in Enzymatic, Amperometric Biosensors

The first amperometric biosensor invented by Clark was based on a oxygen electrode. The reaction of glucose and oxygen catalysed by immobilised GOD (see eq. 2-3) leads to a reduction of oxygen diffusing to the oxygen electrode which is measured in a system consisting of a Pt- electrode and a Ag/AgCl reference electrode.

GOD β-D-glucose + O2 D-glucono-1,5-lactone + H2O2 (Eq. 2-3)

The rate of this reduction, therefore, depends on the rate of diffusion of new oxygen from the bulk solution. Thus, enzymatic sensors based on the oxygen electrode measure a process far from equilibrium and are therefore sensitive to temperature fluctuations and to varying oxygen concentrations in the sample solution. The direct measurement of hydrogen peroxide inciden- tally applying another potential that converts the oxygen electrode to an hydrogen peroxide electrode can not abolish dependence on the dissolved oxygen concentration in the sample solution. Sensors where the electronic coupling between enzyme and electrode has been based on

43 2 Phosphate Biosensors the electroactivity of the cosubstrate or product are called first generation biosensors. The problem of dependency on oxygen concentration in the sample can be overcome by the use of mediators in so called second generation biosensors. Mediators are small redoxactive molecules that react with the active site of the enzyme, diffuse to the electrode and react with the electrode surface, thus shuttling the electrons between the enzyme and the electrode. The first applied mediators were ferrocenes. Today, different compounds are used as mediators: fer- ricyanide, organic salts like tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ), phe- nothiazine and phenoxazine or quinone derivatives. Mediators have to react with the reduced form of the enzyme at a smaller redox potential than oxygen or hydrogen peroxide to be used in biosensors. The redox mechanisms of lpPOX are discussed here as an example: POX catalyses the reaction eq. 1-1. This reaction can be divided into two discrete redox reactions eq. 2-4 and eq. 2-5 (The whole reaction mechanism of lpPOX is described in figure 2-5):

After binding of pyruvate and decarboxylation of CO2 (see steps 1-3 in figure 2-5) the hydroxyethyl-ThDP is oxidised by FAD to acetyl-ThDP (see eq. 2-4 or step 4 in figure 2-5):

- + hydroxyethyl-ThDP + FAD + H FADH2 + acetyl-ThDP (Eq. 2-4)

oxidation: hydroxyethyl-ThDP acetyl-ThDP+ +H++ 2e- + - reduction: FADox + 2H + 2e FADH2 red

The reduced FADH2 transfers two electrons to the final electron-acceptor O2 which is reduced to H2O2 (see eq. 2-5 or step 5 in figure 2-5):

FADH2 + O2 FAD + H2O2 (Eq. 2-5)

+ - oxidation: FADH2 red FAD +2H + 2e + - reduction: O2 + 2H + 2e H2O2 stand. red. pot.: +680 mV Finally, acetyl-ThDP is cleaved phosphorolytically to acetyl phosphate and ThDP (6). In biosensors that use mediators, the final electron acceptor is not oxygen but a mediator molecule. This molecule must have a lower standard reduction potential than O2, so that the electrons are no longer transferred from FADH2 to oxygen but to the mediator. All the processes in the sensor paste can then be summarised in eq. 2-6 similar to reaction eq. 1-1:

- 2- + POX pyruvate + orthophosphate + 2 mediatorred - acetyl phosphate + CO2 + 2 mediatorox (Eq. 2-6)

44 2.3 Setup of the Phosphate and Pyruvate Bio-

The use of mediators not only solved the problem of oxygen dependency but allowed a decrease in the applied potential, thus decreasing bias signals in real samples caused by easily oxidisable interfering compounds such as ascorbic acid. In third generation biosensors, the electrons are transferred directly from the enzyme active site to the electrode. The redox enzymes are therefore directly fixed on the electrode and no mediators are used. These sensors should have superior selectivity as they can operate in a potential window closer to the enzyme´s natural potential. As described above, lower potentials in the sensor leads to less interfering reactions with other redox compounds. Moreover the whole system is simplified with the absence of mediators. Unfortunately only a restricted number of enzymes show the phenomenon of direct electron transfer (DET). An overview of the small number of redox enzymes, for which DET has been shown, is given in Scheller et al. (2005) p.106f. It is not surprising that most of these enzymes have their redox centre near the surface of the protein and therefore are able to commu- nicate with other enzymes or with the electrode. Most of them originate from cascades where electrons have to be transferred to other molecules and almost all contain heme in their redox centres. Apart from the rare DET in enzymes, direct electron transfer is generally slower than mediated transfer. Prof. Lo Gorton from Lund University (SWE) also doubted that lpPOX could show DET, due to their natural function (personal communication, 2004). LpPOX was not selected during evolution in nature to transfer electrons. It is more a catalytic enzyme than an electron carrier. In addition, it contains no heme group and the active sites are buried in the interface between enzyme monomers. The most promising biosensor setup for lpPOX is thus a mediated one.

2.3 Setup of the Phosphate and Pyruvate Biosensor

2.3.1 Development of the Biosensor Device

In a foregoing project J. Müller (2000) developed a continuous flow system sensor (CFSS) based on an amperometric, enzymatic biosensor. In primary experiments, glucose oxidase (GOD) (EC 1.1.3.4) from Aspergillus niger was applied as a model enzyme in that biosensor due to its high stability. The second step in that project was the implementation of a phosphate sensing bienzymatic cascade in the CFSS with the intention of developing a phosphate sensor.

45 2 Phosphate Biosensors

Experiments with the bienzymatic biosensor based on nucleosid phosphorylase (EC 2.4.2.1) and xanthin oxidase (EC 1.17.3.2) showed the possibility of measuring phosphate by an enzymatic biosensor. One weak point of this sensor was its dependency on inosin as cosubstrate, but its main problem was its poor operational stability (Müller 2000). Replacing the complex bienzymatic cascade, S. Nagel introduced the enzyme pyruvate oxidase (EC 1.2.3.3) into the CFSS. Pyruvate oxidase catalyses reaction eq. 1-1 or eq. 2-6 respectively in biosensors and therefore can be used to monitor phosphate or pyruvate in the sample solution with the addition of the corresponding co-substrate. But due to the low stability of the enzyme the whole biosensor showed poor operational stability (Nagel 2004) like the bienzymatic biosensor used by Müller (2000). The exact sensor setup is described in detail in Nagel´s Ph.D. thesis (2004). To aid understanding, the sensor components are briefly summarised in the next paragraph.

2.3.2 Composition of the Sensing Paste

The most important factor of the sensing element of the biosensor is the phosphate and pyruvate converting enzyme pyruvate oxidase from Lactobacillus plantarum. This enzyme, catalysing reaction eq. 1-1, was embedded in a silicon oil matrix consisting of polyphenyl-methyl siloxane (formula see figure 2-7 a). An organic salt composed of tetrathiafulvalene (TTF) and tetracyanoqinodimethane (TCNQ), which change their charge upon electron transfer, was selected as a mediator. The mediator is responsible for the re-oxidation of the active centre of lpPOX. A hypothesis about the mechanism of electron transport in the paste via the mediator molecules TTF/TCNQ is discussed by Müller 2000 p.63ff. The paste consists of mass proportions of 20% enzyme, 50% silicone oil and 30% mediator.

CH3 CH3 CH3 S S NC CN

H3C Si O Si O n Si CH3 S S R R R NC CN

R = CH3 or C6H5 a) polyphenylmethyl siloxane b) TTF c) TCNQ

Figure 2-7: Formula of biosensor paste components; a) polyphenylmethyl siloxane also called silicon oil. b) tetrathiafulvalene (TTF). c) tetracyanoqinodimethane (TCNQ)

46 2.3 Setup of the Phosphate and Pyruvate Bio-

The paste was filled into the cavity of the working electrode. Enzyme leaching was prevented by covering the paste with a dialysis membrane. A CelluSeptTM T1 membrane (Orange Scien- tific, Belgium) with a 3.5 kDa cutoff was used. The reaction catalysed in the biosensor (eq. 2-6) is similar to the one catalysed in nature (eq. 1-1).

a) b)

2e-

2mediatorred 2mediatorox 2e-

lpPOXox lpPOXred 2e-

pyruvate- orthophosphate2- CO2 acetylphosphate-

Figure 2-8: Schema of the phosphate and pyruvate biosensor a) LpPOX embedded in a silicon oil matrix together with mediator molecules and prevented by a dialysis membrane from wash out to the sample solution converts pyruvate and Pi from the sample solution to CO2 and acetylphosphate. The sensing paste is in direct contact with the platinum electrode. During the reaction of lpPOX in the sensing paste, electrons are transferred from the substrates via FAD in the active site of lpPOX and via mediator molecules to the platinum wire. b) Shows the flow of electrons in the biosensor during measurements. In this process, the FAD in the enzyme is first reduced. In a second step, the reduced FAD in the enzyme is regenerated via reduction of mediator molecules as electron accep- tors. The two electrons that are transferred from the analyte to the mediator molecules are then transferred to the electrode surface, forced by the external applied potential between the working electrode and the counter electrode (not shown on the picture). The generated current is recorded by a potentiostat.

47 2 Phosphate Biosensors

2.3.3 Stability of the Triple Mutant lpPOX in the Bio- sensor

These experiments were done by Nagel (2004) and are presented here for a better understanding of the lpPOX stabilisation project; The triple mutant pyruvate oxidase (P178S; S188D; A425V) from Lactobacillus plantarum was tested using the biosensor described above (paragraph 2.3.1 p. 45). During these experiments, phosphate but no cofactors (ThDP, FAD, Mg2+), were added to the sample buffer. The sample buffer contained 51.4 mmol/L imidazole, 51.4 mmol/L phosphate and 21 mmol/L sodium chloride. It was adjusted to a pH-value of 7. Measurements were done at 25°C and a potential of +300 mV was applied against the reference electrode. The on-line measurement was simulated by continuously varying pyruvate concentrations between 0 and 1 mmol/L in the sample solution. As a control experiment, the same biosensor was run with GOD from Aspergil- lus niger. The conditions were exactly the same but the continuous measurement was simulated by varying glucose concentrations between 0 and 30 mmol/L. In figure 2-9, the resulting sensing curves show significantly different operational stabilities for the two enzymatic setups. Whereas the GOD sensor response did not change significantly during the 48h of measurement, the POX sensor showed a remarkable decrease of response over that time. The operational stability of the POX biosensor is obviously much lower than that of the GOD sensor. These experiments using the triple mutant lpPOX in a biosensor showed a signal decrease of 85% after 30 h. A minimal lifetime of two weeks is a prerequisite for commercial use of a sensor. In view of an improved operational lifetime, the enzyme structure has to be stabilised in order to fulfil the requirements for commercial applications. Electrode instabilities caused by mediator leaching to the sample solution are negligible with the stable glucose sensor in mind (see figure 2-9b).

48 2.4 Conclding remarks

a) b)

Figure 2-9: Stability Comparison of GOD (a) and lpPOX (b). The data were measured in the paste biosensor developed at the Centre for Chemical Sensors, ETH Zürich under identical conditions. The signals due to changing concentrations from 0.0 to 30 mM glucose (a) and from 0.0 to 1.0 mM pyruvate (b) were recorded. A different loss of signal over two days is clearly visible (Nagel 2004, reprint with permission of the author?).

2.4 Conclding remarks

Biosensors have major advantages over conventional analytical technologies as they are small, able to measure continuously, in real time, are highly specific and work without reagents. They have been found to be especially useful in process monitoring, biomedical and environmental analysis. Their main disadvantage is their relatively unstable biological recognition components. In the phosphate sensor research field, great efforts have been made to design artificial, stable recognition molecules. But redox-enzymes remain the most appropriate recognition elements for orthophosphate combining high specificity, easy signal transduction and a catalytic mode of operation, which allows for real time on-line measurements. Amperometry is the most promising transducing technology for phosphate sensors with regard to detection limits, dynamic response ranges, costs and technical requirements. The most straightforward and most explored enzymatic phosphate recognising cascade is the one applying pyruvate oxidase where only pyruvate is needed as a cosubstrate. The application of POX to a biosensor allows the measurement of orthophosphate or pyruvate, both attractive analytes in the medical field, in food and environmental technology. An advantage of using the POX from Lactobacillus plantarum is that no expensive and unstable cofactors have to be added to the sample solution, this is because cofactors are relatively tightly bound to the enzyme compared to other POXs. Primary experiments with this enzyme in a biosensor at the Centre for Chemical Sensors at ETH Zurich have shown that the sensor could not be used for more than two days of contin-

49 2 Phosphate Biosensors uous measurement due to the instability of the oxidase (see figure 2-9). The aim of this project was to develop and produce a stabilised lpPOX and to improve the biosensor in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor would be less than 50%.

2.5 Sensor Stability

The short shelf life of biosensors, as mentioned in the previous paragraphs, is a well known problem. Possible solutions for the enzyme instability problem are given in the main part of this thesis. The following short definition of sensor stability concludes the introduction to biosensors.

2.5.1 Definition of Sensor Stability

Due to the lack of a general definition for "sensor stability", direct comparisons of sensor stability data obtained from different research centres are difficult. To resolve this uncertainty, a few sensor stability definitions are outlined in this paragraph. Stability describes the property of a sensor to generate accurate results over a certain time, not the vulnerability of the analytical process to interferences (i.e. operator, contaminants in the sample, temperature etc.). The durability of an analytical method against interferences is described by parameters of reliability, recovery rate, reproducibility and specificity. Secondly, one should distinguish between operational (use) and storage stability (shelf life). For the evaluation and the assessment of experimental data, however, additional parameters (as listed below) should be mentioned when talking about stability. Stability is well defined within the IUPAC definitions for electrochemical biosensors (IUPAC, 1999);

"The operational stability of a biosensor response may vary considerably depending on the sensor geometry, method of preparation, as well as on the applied receptor and transducer. Furthermore, it is strongly dependent upon the response rate- limiting factor, i.e. a substrate external or inner diffusion or biological recognition reaction. Finally, it may vary considerably depending on the operational conditions. For operational stability determination, we recommend consideration of the analyte concentration, the continuous or sequential contact of the biosensor with the analyte solution, temperature, pH, buffer composition, presence of organic solvents, and sample matrix composition. (…)

50 2.5 Sensor Stability

For storage stability assessment, significant parameters are the state of storage, i.e. dry or wet, the atmosphere composition, i.e. air or nitrogen, pH, buffer composition and presence of additives. (…) Biosensor stability may also be quantified as the drift, when the sensitivity evolution is monitored during either storage or operational conditions. The drift determination is especially useful for biosensors whose evolution is either very slow or studied during a rather short period of time." (IUPAC 1999, pp. 2345-6)

In the same paper (IUPAC 1999) the term "lifetime" is used in the same sense as stability and is defined as follows;

"We recommend the definition of lifetime, denoted tL, as the storage or operational time necessary for the sensitivity, within the linear concentration range, to decrease

by a factor of 10% (tL10) or 50% (tL50). For the determination of the storage lifetime, we suggest comparison of sensitivities of different biosensors, derived from the same production batch, after different storage times under identical conditions." (IUPAC 1999, p. 2346)

A point which must be considered is the influence of the environment in which stability values are measured. Whether they are evaluated under laboratory bench conditions or real industrial processes, where the sensors sometimes have to work under rough conditions, leads to different data. Of course a biosensor containing a biological recognition element can only work properly under conditions where these molecules do not degrade. Because an enzyme has different reaction velocities at different temperatures, the working temperature of a biosensor has to be controlled by a thermostat. If a biosensor is to be used without a thermostat (i.e. in field measurements), the sensor has to be calibrated at different temperatures and the output signal has to be calculated knowing the actual temperature. This thesis is focussed on the improvement of the low operational stability of the recognition enzyme in the biosensor itself and not on the implementation of the sensor to an industrial process. The sensor hardware system has been well established (Müller 2000, Nagel 2004) under laboratory conditions. Investigations of long-term stability in real samples cannot be done until the instability of lpPOX in a biosensor has been resolved. For a comparison of different sensors (aside from stability and lifetime) other parameters have to be assessed. Calibration characteristics (sensitivity, linear concentration range, limit of detection (LOD) and limit of quantification (LOQ)), selectivity, specificity, response time, sam-

51 2 Phosphate Biosensors ple throughput, reproducibility, reliability, are the most important key figures of a sensor. Knowledge of the biosensor rate-limiting step or factor is an important factor needed to understand stability properties. Finally, the lifetime assessment procedure should be specified, i.e. by reference to initial sensitivity, upper limit of the linear concentration range for the calibration curve, accuracy or reproducibility.

52 3 Protein Stability

his chapter deals with the main challenge in biosensor research - operational protein sta- Tbility. Different stabilisation techniques are explained and outlined based on prominent examples of stable enzymes like glucose oxidase or extremozymes. The attempts made so far to stabilise POX are summarised briefly in paragraphs 3.4.2 and 3.5. New concepts for further stabilisation of this interesting protein are given in the working hypothesis at the end of the chapter.

3.1 Enzyme Inactivating Processes

The crucial factor in successful enzyme stabilisation is knowledge of the actual enzyme inactivating process under conditions of use. Of course, some common deactivation processes are play a role in most cases of protein instability, but each protein has its own individual sensitivities that have to be considered for a satisfactory stabilisation (see paragraph 3.4).

3.1.1 Common Protein Denaturation Processes

Proteins normally exists in their native, active state (N), which is in equilibrium with the partially or completely unfolded and inactive state (U). Starting from the unfolded state but also partially from the native state, proteins can undergo covalent or non covalent irreversible denaturation processes (see figure 3-1) which result in aggregated, precipitated or chemically inactivated proteins. A simple model showing unfolding of a monomeric protein is given in figure 3-1. aggregation t scrambled len structures (incorrectly folded) ku non cova ki chemically altered cova structures kn - oxidation l ent - deamidation - Maillard reaction - proteolysis - racemisation c ov - hydrolysis ale nt - ... NUX

D Figure 3-1: Model of Protein Unfolding: N: folded, native protein; U: unfolded protein; X: irreversibly unfolded state; D: denatured protein (this fraction consists of the unfolded (U) and of the irreversibly unfolded protein fraction (X))

53 3 Protein Stability

The difference in Gibbs free enthalpy ∆G between the two states N and U presents a measure of the conformational and thermodynamical stability of a protein respectively. It normally amounts to about -20 to -80 kJ/mol. A comparison with association energy values of -10 kJ/mol between two single benzene rings or of -22 kJ/mol between two water molecules (Böhm et al. 1996) shows how unstable the equilibrium between the two states N and U is. The equilibrium between the native and unfolded protein states may be shifted to the unfolded, inactive state by elevated temperatures, extreme pH-conditions, organic solvents, chaotropic salts, hydrophobic surfaces, air-liquid interfaces, interactions with metal ions or shear forces due to agitation. The non-covalent irreversible inactivation of unfolded proteins is mainly characterised by a change of physical conformations caused by factors such as elevated temperatures, extremes of ionic strength, hydrophobic surfaces or high pressure. Whereas, the covalent inactivating processes involves chemical protein modifications when sensitive amino acids are exposed (at the protein surface in state N or exposed via unfolding) and react with oxidants, reducing agents, acids, bases or other modifying reagents (see figure 3-2).

3.1.2 Common Extrinsic Factors that Destabilise Proteins

The following extrinsic factors (see table 3-1) cause the protein inactivation processe by pro- moting either the unfolding, aggregation, precipitation or the covalent modification of proteins:

Table 3-1: Common extrinsic factors that destabilise proteins: The list is not exclusive but gives the most important factors that destabilise proteins. (1) extremes of temperature (9) oxidising and reducing agents (2) extremes of pH ranges (10) detergents in high concentrations (3) extremes of ionic strength (11) proteases (4) chaotropic agents (12) hydrophobic surfaces (5) interactions with metal ions (13) air-liquid interfaces (6) electromagnetic waves (14) shear forces due to agitation (7) high water activity (15) extremes of pressure (8) organic solvents

Strategies to prevent these factors are discussed in paragraph 3.2. How these factors force protein deactivation is showed in figure 3-2 and listed in detail in the following paragraphs.

54 3.1 Enzyme Inactivating Processes

(1, 3, 12, 13, 14, 15) aggregation, scrambled ent l structures ova c (incorrectly folded) (1, 2, 4, 8, 10) non ((4), 10) chemically altered structures (possibly 15) cov - oxidation a le - deamidation nt - Maillard reaction (1, 2, 5, 6, 7, 9, 11) - proteolysis co - racemisation va len - hydrolysis t - ... NU X

D Figure 3-2: Model of Protein Unfolding: N: folded, native protein; U: unfolded protein; X: irreversibly unfolded state; D: denatured protein; The numbers near the individual processes indicate which factors mentioned in the list above influence the protein denaturation process: (1) extremes of temperature; (2) extremes of pH ranges; (3) extremes of ionic strength; (4) chaotropic agents; (5) interactions with metal ions; (6) electromagnetic waves; (7) high water activity; (8) organic solvents; (9) oxidising and reducing agents; (10) detergents in high concentrations; (11) proteases; (12) hydrophobic surfaces; (13) air-liquid interfaces; (14) shear forces due to agitation; (15) extremes of pressure;

Extremes of Temperature

High temperatures influence the balance between folded and unfolded protein fractions, favouring the unfolded state, because the increased thermic energy within the protein allows it to overcome the stabilising interactions energy present in the protein. Furthermore, higher temperatures also increase the reaction rate of irreversible inactivation processes. Interestingly, proteins are also denatured partially at low temperatures in the presence of water or during freezing (Graziano et al. 1997) because they are exposed for a limited but sufficient time period to extremes of salt concentration and pH ranges. The reason for this is that first only water freezes, increasing the salt and buffer concentrations in the residual liquid fraction. Thus, rapid freezing and thawing is suggested for protein storage because it limits the time that proteins are exposed to these extreme conditions.

Extremes of pH Ranges

Extremes of pH ranges can change protein charges by either protonation or deprotonation of amino acid side chains and therefore influence the equilibrium between N and U or catalytic activity when amino acids in the active site are incorrectly charged in respect to their catalytic function. Especially high pH ranges can also enhance covalent inactivation reactions like deam-

55 3 Protein Stability idation, Maillard reactions, proteolysis, racemisation and hydrolysis.

Extremes of Ionic Strength

High ionic strength leads to salting out as ions near the protein surface bind water in their solvation shells and therefore remove water molecules from the protein solvation shell. The dehydrated proteins tend to aggregate and precipitate, but, as outlined in chapter 2.2.1.4.b the hydration of ions varies depending on the ion itself. The hydration of ions decreases from the left to the right in the Hofmeister series as mentioned in chapter 2.2.1.4.b. The Hofmeister series ranks various ions using their ability to precipitate hen egg proteins and appears as follows;

2- 2------Hofmeister series for anions: SO4 > HPO4 > acetate > Cl > Br > I > NO3 > ClO4 > SCN 3+ 2+ + + + + + - Hofmeister series for cations: Al > Mg > Li > Na = K > NH4 > H(CH3)4 > SCN

Using this list of ions it is clear why NaCl, with its moderately solvated ions, is much better suited as a stabilising salt than (NH4)2SO4, which is used as a protein precipitating agent due to high solvated sulphate ions. A very low ionic strength or the lack of ions in a protein solution can also lead to protein precipitation because proteins lack counter ion binding and therefore have a lower net charge and solvation. The increase of protein solubility upon the addition of low level of salt is called “salting in“.

Chaotropic Agents

High concentrations of chaotropic agents such as urea or guanidinium chloride (GdmCl) cause denaturation of proteins. There is controvery surrounding urea over whether it acts directly by binding to the protein or indirectly by perturbing solvent mediated hydrophobic interactions. In their study, Halle et al. (2005) showed by magnetic relaxation dispersion (MRD) that proteins denatured by urea participate in clusters and not in total random coils, and that these clusters are penetrated by large numbers of water and urea molecules. An explanation of the denaturating effect of urea is that urea, with its 2.5-fold larger volume than water, reduces the entropic pen- alty for confining a certain volume of solvent to such a cluster. Thereby the hydrogen bonding capacity per unit volume is similar for both water and urea. Guanidinium chloride has a high hydrogen-bonding potential and - as a salt - has a weaker affinity than urea to the less polarisable protein core. However, the underlying mechanisms of

56 3.1 Enzyme Inactivating Processes protein denaturation by GdmCl are the same as for urea; a disruption of bonds which hold the protein in its unique native structure.

Interactions with Metal Ions

Metal ions e.g. Cu2+ or Fe2+ can activate molecular oxygen - and as a result - accelerate the oxidation rate of thiol groups in proteins. Therefore, they cause stability problems due to this covalent, irreversible protein denaturating process. Applying heavy metal free water or the addition of EDTA or a reducing agent such as β-mercaptoethanol or dithiotreitol (DTT) to the protein solution can help in preventing such protein degradation. It should be noted that “metal“ ions such as Ca2+ can also stabilise certain proteins (Maurer and Hohenester 1997, Akhtar et al. 2002; Durrschmidt et al. 2005; Vanbelle et al. 2005).

Electromagnetic Waves

Non-ionising or ionising radiation can enforce the chemical protein degradation processes. Thereby tryptophane can be decomposed to kynurenin and tyrosine can be degraded to L-3,4- dihydroxyphenylalanine (DOPA) respectively. Cystine is also sensitive to photo degradation. For example it is common knowledge that insulin preparations are sensitive to visible light or UV-radiation. Diabetics have to protect their insulin preparations not only form high temperatures, freezing and strong agitation but also from sunlight and UV-radiation.

High Water Activity

High water activity in the protein environment enhances the irreversible, chemical protein degradation. Pure hydrolysis reactions on proteins are only possible in water. Nevertheless, analogue protein degrading reactions take place in other polar and protic solvents such as methanol or ethanol. Removal of all water out from a protein formulation e.g. during freeze drying, prevents the chemical protein degradation processes and also the destruction of proteins by proteases or microorganisms. On the other hand, water also stabilises folded globular proteins via the well known hydrophobic effect. A certain amount of water surrounding the enzyme seems to be essential for enzyme catalysis (see paragraph about organic solvents below).

Organic Solvents

Proteins dispersed in pure organic solvents without traces of water tend to unfold mainly because of the absence of a hydrophobic effect, one of the most important stabilising forces of

57 3 Protein Stability globular proteins. Thereby, it should be noted that the hydrophobic effect is not an intrinsic mechanism of protein stabilisation based on protein properties, but that water as a solvent with its unique characteristics like polarity, high dielectricity constant and high rate of hydrogen exchange generates this hydrophobic effect. The correct interpretation of the phase separation of aliphatic compounds (paraffins) and water has to be credited to Hartley (1926) who postulated:

"Separation is based not upon aliphatic-aliphatic attraction, nor on water-aliphatic repulsion, rather it is based on water-water attraction"

"The antipathy of the paraffin chain for water is, however, frequently misunder- stood. There is no question of actual repulsion between individual water molecules and paraffin chains, nor is there any very strong attraction of paraffin chain for one another. There is, however, a strong attraction of water molecules for one another in comparison with which the paraffin-paraffin or paraffin-water attractions are slight."

This is the theoretical explanation of why proteins in hydrophilic, nonpolar organic solvents should denature or be inactivated. But surprisingly, enzymes behave in a more versatile way than the theory suggests. In their review, Tuena de Gómez-Puyou and Gómez-Puyou (1998) describe how enzymes can work in the unnatural conditions of organic solvents. It seems indispensable for enzymatic functionality to have water in minimal quantities in the enzyme surrounding matrix. They have also shown that enzymes are quiet versatile and some of them, surprisingly, work in organic solvents even at high temperatures, or they express superactivity or catalyse reactions that had not been seen in water. Therefore, enzyme catalysis in low water containing organic media has found an increasing number of applications during the recent years in the field of biosensors. Water is an essential element for catalysis and at optimal water contents, enzymes in organic solvents achieve activities ranging from 20 to 40% of those in aqueous solutions. How- ever, under optimised conditions some enzymes in organic solvents can reach activities that are remarkably comparable to those in water (Zaks and Klibanov 1988). Many causes of lower enzymatic activity in organic solvents compared with that in water and ways to overcome these insufficiencies are enumerated in Kilbanov´s review (1997). A question that arises is whether or not enzymes are surrounded by a shell of water in organic solvents. If this is the case, enzymes would not work in organic solvents but in water cages. Zaks and Klibanov (1988) showed that in hydrophobic solvents less water was required

58 3.1 Enzyme Inactivating Processes to reach maximal enzyme activity than in their hydrophilic counterparts. They concluded that the enzymatic activity depends on the amount of water bound to the protein, and not on the content of water in the whole system. Furthermore, they showed that enzymes in nonaqueous solvents need water shells to achieve a sufficient conformational flexibility required for catalysis. Hence they concluded that enzymes work better in hydrophobic (rather than hydrophilic) organic solvents because of the lower capacity of hydrophobic solvents to compete for the water around the enzymes. Finally, one can conclude that organic solvents can have destabilising as well as stabilising effects on proteins, depending on the enzyme itself and on the water content of the organic solvent.

Oxidising and Reducing Agents

Oxidation first of all affects the sulphur containing amino acid residues cysteine, cystine and methionine. The thiol group of free L-cysteine is unstable in air and is therefore rapidly oxidised to cystine in neutral or alkaline aqueous solutions. Methionine contains a sulphur atom bound via a thioether group and is therefore less susceptible to oxidation than cysteine. Nevertheless, this amino acid can also be oxidised to the corresponding sulphoxide or sulphone. The aromatic amino acids tryptophane, tyrosine, histidine and phenylalanine are less sensitive to oxidation. All the other proteinogenic amino acids are more or less insusceptible to redox reactions. All these amino acid residue oxidising reactions proceed via electrophilic and radical addition respectively and are, therefore, pH-independent. It is evident that an oxidation of the above mentioned amino acids can cause enzyme inactivity where the active site is concerned or can lead to irreversible degraded proteins when structurally important properties of the whole protein are affected. A reducing environment can also damage proteins for example, when structurally important disulphide bridges in small proteins are reduced to cysteines. An other reaction that is based on reduction is the so called Maillard reaction. Reducing sugars such as glucose can cause gly- cation of lysines. This protein degrading mechanism was first described by Maillard (1912) and leads, by means of the reaction of an aldehyde and a free primary amino group via a rather an unstable Schiff base, to complex carbohydrate-protein constructs. The Maillard reactions are favoured in higher temperatures or higher pH-values because the latter enlarge the collective of unprotonated amino groups of lysine residues (pKa = 10.5) that are involved in the initial reaction steps.

59 3 Protein Stability

Detergents in High Concentrations

Although detergents are applied in protein solubilisation or crystalisation to prevent protein aggregation, they cause unfolding of proteins at high concentrations. Anionic and cationic detergents typically modify protein structures to a greater extent than non-ionic or zwitterionic and are therefore not suitable for protein solubilisation. The most prominent example is sodium dodecyl sulphate (SDS) which is applied in denaturating gel electrophoresis of proteins in order to complete protein denaturation.

Proteases

Proteases inactivate and degrade proteins by cleaving them into small fragments. Flexible parts of proteins located at the surface are predestinated for proteolytic cleavage. The rate of proteolysis also depends on the specificity and the activity of the protease, the temperature, pH-value and also on the presence of metal ions depending on the type of protease. The folding state of a protein is important for the proteolysis rate, because proteases can only attack amide bonds that are exposed to solvent. Buried bonds in the protein core are protected from proteolysis by proteases. Apart from proteolysis, non-enzymatic hydrolyses of the polypeptide chain can also occur at extremes of pH and elevated temperatures. Asn-Gly or Asp-Pro bonds especially seem to be sensitive to these reactions.

Hydrophobic Surfaces, Air-Liquid Interfaces and Shear Forces due to Agitation

Agitation or shaking are the most common physical stress factors causing protein aggregation (Henson et al. 1970; Maa and Hsu 1997). Protein aggregation induced by agitation is the con- sequence of protein interactions at interfaces and concomitant affecting shear forces, for example at the air-water or at the solution-vial surface. Enzymes are denatured at these phase boundaries caused by the lopsided lapse of the important water shell. Thereby, the stabilising hydrophobic effect induced by water molecules decreases (see paragraph on organic solvents above).

Extremes of Pressure

In many cases high hydrostatic pressure denatures proteins, whereas oligomeric proteins can dissociate reversibly even at pressures below 30 MPa. At very high pressures, over 50 MPa, pro-

60 3.1 Enzyme Inactivating Processes tein aggregation and loss of secondary structure mainly occurs because of increased protein ex- posure to solvent. A broad overview of the major effects of pressure on molecular interactions causing dissociation of oligomeric proteins and subunit denaturation is explained in the review written by Boonyaratanakornkit et al. (2001). Pressure, however, has more than simply inactivating or denaturating influences on proteins. Many authors (Sun et al. (2001), Athes et al. (1999), Mozhaev et al. (1996) and Hei and Clark (1994)) showed that pressure in a moderate range of about 0.2 up to 100 MPa can also stabilise enzymes against temperature inactivation mainly from thermophilic organisms. The activity of some enzymes can also be accelerated by pressure Mozhaev et al. (1996).

3.1.3 Common Intrinsic Factors that Destabilise Pro- teins

Assembling enzymes out of amino acids also introduces some intrinsic weak points, where a chemical alteration favoured by external factors (see 3.1.2) can be initiated. Firstly, amide bond between amino acids is susceptible to hydrolysis. Secondly, all functional side chains are also vulnerable to different chemical reactions. The aliphatic amino acids like Ala, Val, Ile, Leu, Pro and Gly are not reactive under natural conditions. The following amino acids are especially reactive and therefore also responsible for irreversible protein denaturating effects (see figure 3-1 page 53);

Table 3-2: Especially reactive amino acids responsible for irreversible protein denaturating effects: amino acid reactivity sulphur containing Cys, cystine, Met oxidation aromatic Trp, Tyr, Phe, His oxidation polar Asn, Gln deamidation basic Lys Maillard reaction covalently conected side chains Pro, cystine Isomerisation

The exact mechanisms of side chain oxidation and of the Maillard reaction are already described in the paragraph 3.1.2.

Deamidation

Deamidation reactions convert the uncharged amino acids Asn and Gln via a hydrolytic mechanism to the negatively charged Asp and Glu. The effect of introducing a negative charge to protein molecules can be instability, inactivity or both. The reaction does not require special

61 3 Protein Stability chemicals but is increased with increasing temperature and pH-values (Ahern and Klibanov 1985).

Racemisation

Racemisation can convert natural L-amino acids to the D-form and vice versa. It can theoretically occur with each amino acid in proteins except glycines. The rate determining step in racemisation of amino acids is the production of a tertiary carbanion intermediate. In proteins, the protonation of the α-carboxyl and amino groups is prevented by peptide bonds. Therefore, racemisation rates are dependent mainly upon the electron-withdrawing abilities of the residue groups. Because of this, Asn and Asp are the most susceptible amino acids to racemisation with their β-carbonyl and β-carboxyl groups respectively (Zhao et al. 1989). The racemisation process takes place spontaneously at extremes of pH-values and at high temperatures. It can also be caused by different isomerases. Racemisation leads to protein unfolding and enzyme inactivity due to the alteration in the configuration of the protein backbone, which induces conformational disorders in the folded protein.

Isomerisation

The peptide bond in the protein backbone shows a partial double bond character. Therefore, the rotation around the torsion angle ω is constricted and allows only two conformations. Relating to the arrangement of the two vicinal Cα-atoms, one distinguishes between the cis conformation (ω=0°) and the trans conformation (ω=180°) (see figure 3-3 a). Due to the sterical hindrance of the neighbouring Cα-atoms and the corresponding amino acid side chains in the cis conformation in peptides and proteins generally, only the trans conformation is found (>99.95% in the

62 3.1 Enzyme Inactivating Processes trans conformation). O a) R O R H R Cα N α NH N C ω CCαα H N Cα ω O R H O trans cis b)

R O Cα R N N Cα ω Cα N H N Cα ω O H O O

Figure 3-3: Cis-trans Isomers of Amid Bonds in Proteins a) Cis-trans isomers of an amide bond in a peptide or a protein. The repulsion of the two side chains in the cis conformation is marked by two circles. b) Cis-trans isomers of a peptidyl-prolyl amid bond. The energy difference between trans and cis for peptidyl-prolyl is much smaller compared to other peptide bonds.

Proline with its rigid pyrolidine ring takes a special position in the list of proteinogenic amino acids. As a result of its unique three dimensional configuration, peptidyl-prolyl (-Xaa-Pro-) amide bonds in proteins exist up to a rate of 6% in the energetically unfavourable cis conformation (Lehninger et al. 1993a, p187). It has been shown that the participation of the cis/trans isomerisation can act as the rate limiting step during protein folding (Mayr et al. 1993). Correct isomerisation of peptidyl prolyl bonds has a great influence on the protein folding (thermodynamical and kinetic) and can therefore affect protein activity and stability. Isomerisation of peptidyl-prolyl amide bonds can occur spontaneously or be catalysed by the so called peptidyl prolyl cis-trans isomerases (PPIasen) first isolated by Fischer et al. (1984). The isomerisation of disulphide bridges in proteins with more than two cysteine residues depends strongly on the oxidative environment of the protein or on the presence of specific isomerases. Spontaneous isomerisation can virtually be excluded. Therefore this effect, which can also destabilise proteins, has to be classified as an extrinsic effect rather than an intrinsic one (see paragraph on oxidising agents above). After reading this paragraph one could tend to conclude that it is impossible to develop stable biomacromolecules. But surprisingly, nature has developed myriads of stable proteins over time, each adapted in a unique way to the corresponding environment. Recently, chemical and biotechnological techniques have been applied for protein stabilisation. Most of them are based on natural stabilising concepts.

63 3 Protein Stability

3.2 Protein Stabilisation Techniques

Enzymes are widely used in a large variety of industrial applications and in diagnostic instru- ments as reviewed in chapter two. The most challenging aspect of the application of enzymes in biosensors is their insufficient operational stability. In principle, this problem can be solved in two totally different ways, in a random approach using evolutionary techniques or in a knowledge based rational way. Both approaches have their advantages and disadvantages and before deciding to apply one technique or both, one has to consider different factors. The approach of random stabilisation has proven to be successful for the stabilisation of proteins in a more or less physiological environment. One advantage of this approach is, that no information about the structure of the enzyme has to be available. By application of several rounds of small random changes and following selection of the most stable protein, in many cases this iterative approximation led to a good result faster than the rational process of enzyme stabilisation. Two interesting reviews dealing with these techniques are representatively mentioned here, as an introduction to this new and potent methodology (Rubingh 1997, Eijsink et al. 2005). The problematic applying this method is the choice of a reliable selection mechanism that is sufficiently discriminative. Especially in this project, where the conditions in the biosensor determine the lifetime of the enzyme, a simulation of the selecting conditions is impossible. The knowledge based way of enzyme stabilisation as explained below is more suitable in the case where the enzyme application environment can not be simulated in an easy way. It is further more suitable when the rough conditions of enzyme application do not allow selection with living organisms, such as cells or with phage display, for instance, at low pH-values, in organic solvents or at very high temperatures. Additionally the rational based approach is more promising in cases where a lot of information about the protein in question is already available, for example, the protein structure or the degradation process. Broad discussions and reviews about rational driven protein design are given in the following articles; Van den Burg and Eij- sink 2002, Gaseidnes et al. 2003 and Eijsink et al. 2004. A few examples taken from the exten- sive variety of experiments where proteins are stabilised by rational design, are given in the reports of Godette et al. 1993, Mansfeld et al. 1997 and Bryan 2000. Several strategies are available to improve the stability of enzymes using the knowledge based approach (figure 3-4). On one hand, methods of extrinsic stabilisation such as the immobilisation of enzymes or the use of stabilising additives exist, which are based on optimisation of the protein environment. On the other hand, intrinsic stabilisation techniques like rational

64 3.2 Protein Stabilisation Techniques protein engineering or specific modifications of enzymes have also proven to be successful in developing more stable enzymes (see figure 3-4). Because lpPOX has to withstand unphysiological conditions in the biosensor (low salt concentration in the sample solution, voltage, silicon oil and mediators in the proximity), the approach using rational protein stabilisation was chosen to improve insufficient operational stability. Furthermore, good structural data and theories about the degradation process already exist with regard to lpPOX and this knowledge can be applied in the rational design of more stable enzymes. (Risse et al. 1992) Before presenting the exact concept for lpPOX stabilisation, a few considerations relating to general protein stabilisation techniques are discussed based on selected examples.

65 3 Protein Stability iue34 Protein Stabilisatio 3-4: Figure physical not the operational stability of enzymes inbiosensors) ofenzymes stability theoperational not but thestoragestability improves *(lyophilisation lyophilisation electromagn. waves immobilisation pressure optimal from protection optimal temperature non covalent covalent extrinsic * n Techniques in Biosensors chemical additives protection from from oxidation protection concentration salt optimal bufferoptimal pH-value optimal optimal solvent proteins (e.g. BSA) proteins polyoles /aminoacids protein stabilisationtechniques The figure shows the most important protein stabilisation techniques currently applied in in biosensor technology. applied currently techniques stabilisation protein important the most The shows figure modifications chemical covalent linking covalent acylation crosslinking PEGylation of cofactors of proteins of the protein modification modifications biotechnological stabilisation tags stabilisation ends flexible addition of of truncation glycosylation intrinsic amino acid sequence acid amino change inthe helix dipole stabilisation dipole helix monomers between interactions backbone of the entropy lower pairs ion H-bonds interactions hydrophobic disulphide bridges

66 3.2 Protein Stabilisation Techniques

3.2.1 Extremozymes Show Different Stabilisation Concepts

Extremophiles are microorganisms that are found in environments of extreme temperature (-2 to 15°C, 60-110°C), ionic strength (2-5M NaCl) or pH ranges (<4, >9) (Hough and Danson, 1999). The molecular mechanisms allowing organisms to live at these extreme conditions are diverse, including the structure of macromolecules, metabolism and regulation of gene expression. The aspect of macromolecule stability is of special interest in this context. Indeed, these remarkable organisms can be sources of enzymes - also called extremozymes - with enormous stability and sometimes novel activities or they can show new concepts of protein stabilisation. The structural basis of enzyme stability has been identified in several studies by comparison of homologous enzymes from both mesophiles and extremophiles. However, it seems that an uni- versal rule for the structural basis of stability does not exist. Rather, many different mechanisms are responsible for the outstanding stability of these extremozymes. However, extremozymes are an interesting source of stabilising concepts that might be implemented into the mesophilic enzymes of choice to adapt them to rough technical environments. The concepts of protein stability discovered in extremozymes are well outlined in the review article written by Danson and Hough (1998) and discussed briefly in the following paragraphs. Hyperthermophilic microorganisms developed both intrinsic and extrinsic concepts of protein stabilisation. An intrinsic stabilisation concept is the increased number of ion-pair networks at the surface and at the intermolecular interface, first described by Perutz and Raidt (1975). But the association between a tight ion-pair network and thermostability is not univer- sal. There are extremozymes with fewer ion pairs than their mesophilic counterparts but with an increased number of hydrophobic interactions. Thermozymes also showed increased compactness based on shorter surface loops, reduction of cavities in the core, increased internal packing and higher complementarity at the interfaces. But these properties can, on the other hand, decrease the catalytic activity of thermozymes. Further, there are thermozymes which have a reduced number of thermolabile amino acids such as Asn and Gln that are endangered by deamidation, or Cys and Met that are sensitive to oxidation. Another structural property of thermophilic enzymes are solvent filled hydrophilic cavities in the protein core, which led, together with tight ion-pair networks, to elastic-string proteins which can further withstand high temperatures. The accumulation of organic solvents such as bisphosphoglycerate or trehalose that stabilise proteins in general are extrinsic factors in thermophilic microorganisms.

67 3 Protein Stability

Enzymes taken from psychrophiles are cold-active, show more flexibility, and have active sites that are more accessible to substrates than those of mesophiles. Phsychrozymes with more intramolecular ion-pairs than hyperthermozymes exist, which these prevent cold denaturation caused by a diminished hydrophobic interaction at low temperatures. Russel et al. (1998) postulated a strategy on how cold stabilisation could work, based on structural comparison. Extremozymes from halophilic organisms are highly acid and therefore bind hydrated ions at their negatively charged surface, this reduces the surface hydrophobicity thus reducing the tendency to aggregate at high salt concentrations. No structural data is currently available for extremozymes at high pH values and so their basis of stability can not be elucidated. One can conclude that extremozymes show a broad range of stabilisation concepts against rough physical or chemical conditions. But these proteins are not predominantly stabilised with regard to long-term operational stability. Obviously, an application of the concepts of extremozymes for mesophiles should lead to better operational stability, but this must be demonstrated seperately for each protein.

3.2.2 Stability of Glucose Oxidase, an Enzyme De- signed for Application in Biosensors?

Glucose oxidase (GOD, EC 1.1.3.4) shows outstanding long-term stability in comparison to most of the other enzymes applied in biosensors. The success of biosensors on the market is mostly based on the incomparable stability of GOD. Many biosensors that are developed in the lab for the detection of a variety of analytes can never be introduced to the market due to the weak long-term stability of the corresponding biological recognition molecules. GOD is a FAD-dependent enzyme catalysing the oxidation of β-D-glucose by molecular oxygen. GOD from Aspergillus niger is a homodimer with a molecular weight of 150 to 180 kDa containing one tightly bound FAD molecule per monomeric subunit (Pazur and Kleppe 1964). The enzyme is highly glycosylated whereas the carbohydrate content accounts for about 9 to 12% of its molecular weight (Hayashi and Nakamura 1981). Its deglycosylated crystal- structure was refined at 2.30 Å resolution (Hecht et al. 1993, PDB-ID: 1GAL).

Glycosylation

It is believed that the carbohydrate moiety of many glycoproteins plays a role in the folding mechanism and therefore also in the stability of the enzymes. But a comparative study by Nak- mura and Hayashi (1974) of native and periodate-oxidised and therefore partially deglyco-

68 3.2 Protein Stabilisation Techniques sylated GOD did not show any significant alteration in catalytic properties, conformation or stability against heating. The only detectable change was a reduction of stability when heated in the presence of SDS. Therefore one can conclude that the carbohydrate moiety protects the enzyme from the influence of detergents. Kalisz et al. (1991) also showed, that the three dimensional structure of GOD was not affected by deglycosylation. Only a small change of secondary structure and an increase in the compactness of tertiary structure could be observed in their experiments. An influence of the carbohydrate content on the reaction-kinetics and the stability at low pH-values cannot be ruled out. However, the thermal stability, pH and temperature optima as well as activity were not affected by deglycosylation. It should be noted that these experiments only compare the short-term stability of GOD with or without glycans under rough conditions in so called stress tests. The more interesting long-term stability of different GOD forms has not been tested so far. The simulation or the en- couragement of a water shell around the enzyme by the hydrophilic glycans could be the key factor in the important intrinsic long-term stability of GOD.

The Cofactor FAD in Glucose Oxidase

The non covalently bound cofactor FAD plays a critical role in structure and activity in GOD. Dissociation of FAD from the holoenzyme is one of the first steps during the thermal inactivation of GOD. Gouda et al. (2003) showed in their experiments, that the midpoint for thermal inactivation and the dissociation of FAD was 3°C lower than the corresponding midpoint for loss of secondary and tertiary structure. This loss of structure after the dissociation of FAD led to enzyme unfolding and aggregation. Binding of FAD to the apoenzyme of GOD can change the conformation of the protein and it has been put forward that FAD stabilises the tertiary structure of the enzyme. The main interactions of FAD to GOD are 23 potential hydrogen bonds, mostly involving the ribose and pyrophosphate groups of FAD (Swoboda 1969).

The Dimer Interface

Hecht et al. (1993) described the dimer interface in their article on the structure of GOD as follows; contact between the two monomers is confined to a long but narrow stretch. A lot of contact points are centred around 11 residues, which either form hydrogen bonds or salt bridges. A lid formed by residues 75 to 98 (marked in blue in figure 3-5) in the dimer interface couples the

69 3 Protein Stability dimer formation with the FAD binding. This segment covers the FAD binding channel in the monomer and has to be bent up before FAD can be released or bound. FAD binding returns the lid to the closed conformation. The formation of the dimer buries part of this segment and thereby prevents the opening of this lid and therefore also a loss of FAD. On the other hand the open lid in the monomeric apoenzyme prevents dimer formation by elimination of some major contact points and allows only two monomers with bound FAD to build an active dimer.

Gouda et al. (2003) further showed that K2SO4 enhances thermal stability by making the GOD holoenzyme a more compact dimeric structure. Dissociation of the two subunits is possible at a pH-value of 5 or below and is accompanied by the loss of FAD (Jones et al. 1982).

a) b)

FAD disulphide bond

Figure 3-5: View of the Dimeric Interface of Glucose Oxidase a) Shows a GOD monomer with its structural characteristics. Helices are marked in purple, β-sheets are marked in yellow, the only disulphide bridge in the monomer is marked by a red arrow and the cofactor FAD is marked by a red circle. b) Shows the same view of a GOD monomer but in the surface view. In this picture one can see how deep the cofactor FAD is buried in he protein. In both figures the lid formed by residues 75 to 98 is coloured in blue. This segment covers the FAD binding channel in the monomer and has to be bent up before FAD can be released or bound (Hecht et al. 1993). The data-set for the figures is based on the PDB entry 1GAL (Hecht et al. 1993).

It can therefore be concluded that both factors together, the FAD binding and the stabilisation of the dimeric state of GOD, stabilise this protein. The influence of glycosylation and the disulphide bridge are probably also responsible for stabilisation, but these factors were not especially investigated for their influence on long term stability.

3.2.3 Stability of Structural Proteins

An extraordinary case of a long term stable protein was the discovery of a single silk thread from a spider's web that has been preserved in amber dating from the Early Cretaceous period about 130 million years ago (Zschokke, 2003). Of course, this protein was conserved mostly due to

70 3.2 Protein Stabilisation Techniques the inert surrounding of amber and not only due to its intrinsic stability. This example led me to the idea, of shortly overview protein stability in nature per se. My intention was to get an overview of principles that evolved in nature to stabilise proteins, that are not only conserved by optimal buffers and additives but withstand rough conditions, dissolved or in solid state in analogy to conditions prevailing in biosensors. Structural proteins are the most stable class of proteins in terms of long-term stability, for instance in feathers, claws, horns, hoofs, sinews, silk and so on. Three important mechanisms give them their outstanding stability; firstly, the principle of hydrophobicity on the protein surface, which prevents structural proteins from swelling in contact with aqueous solutions. α-Ker- atin for example is composed of hydrophobic amino acids such as Phe, Ile, Val, Met and Ala. Secondly, simple helical structures such as α-helices in α-keratin, the triple helix in collagen (with the sequence Gly-X-Pro or Gly-X-Hyp) or the molecular nano springs in spider silk (with the sequence GPGGX; Becker et al. 2003) that led to high tear strength or elasticity. And thirdly, covalent crosslinking between single protein strands generates strong materials such as α-keratin with its rate of up to 18% Cys that forms disulphide bridges or 2D elastic materials such as elastin (Lehninger et al. 1993b). Of course the approach with hydrophobic, insoluble structures cannot be applied to engineer stable enzymes because these molecules have to be soluble to easily contact their analyte. However, the other two principles should be considered during enzyme stabilisation. It may be possible to introduce more helical structures into the enzymes of interest and perhaps a better cross-linking between different domains or subunits will lead to more robust proteins.

3.2.4 Conclusions taken from these Common Stabil- ising Techniques

Although the concepts of extremozymes against denaturation are of an overall interest and show a big impact in the technical application of enzymes, their effect on the better operational stability of enzymes has to be demonstrated separately for each species. The stabilisation concepts of GOD and of structural proteins in general present other approaches for long term protein sta- bilisataion specifically. However, not all stabilisation concepts can be implemented in every case. The following paragraphs describe in detail which stabilisation techniques are applicable in the case of lpPOX in the biosensor.

71 3 Protein Stability

3.3 Procedure in Biosensor Research in Cases of Stability Insufficiency

The standard procedure in biosensor research in cases of stability insufficiency must involve the following points: 1.) Ascertaining the main factors causing instability (see figure 3-2 page 55) 2.) Screening for stable enzymes from different organisms, primarly from extremophiles 3.) Protein stabilisation (see figure 3-4 page 66) and sensor optimisation

3.3.1 Stability Testing in the Sensor

It is not possible to directly determine an exact degradation rate of the enzyme immobilised in the sensor. On one hand, the amounts of enzyme or paste in the sensor itself are rather small (less than one milligramme). On the other hand, the techniques used for the analysis of enzymatic integrity and functionality are not applicable in the sensor itself because of the imperme- ability of the sensor matrix to electromagnetic waves. Fluorescence spectrometric measurements which confirm correct binding of the cofactors and also the tertiary protein structure or also circular dichroism readings for the determination of secondary and tertiary structures are impossible in the opaque, black enzyme paste in the sensor. An extraction of the enzyme out from the sensor matrix would not be reliable because the equilibrium between the folded, native state (N) of the enzyme and the unfolded state (U) is highly dependent on the surrounding environment. Investigations on the basis of enzyme extracted from the sensor would never reflect the real situation in the sensor itself. Therefore, only measurements made with the working sensor are reliable to predict sensor stability data. A simple and fast simulation in a model system is not significant, because a lot of specific factors could not be included and simulated in a model system; the sensor geometry, the enzyme environment dependent on the interaction of the sample solution and sensor matrix, potential leaching of cofactors, presence of mediator molecules, the electric field and so on. Hence, the optimisation of a stable pyruvate oxidase formulation has to be done outside the sensor, based on well-known mechanisms that destabilise lpPOX. The main factors causing instability of lpPOX are discussed in detail in paragraph 3.4.

72 3.3 Procedure in Biosensor Research in Cases

3.3.2 Screening for stable enzymes

In each biosensor project, the selection of the most promising enzyme or enzyme cascade is a key step and has to be done with greatest care. Generally, enzymes from extremophile microorganisms - the so called extremozymes - are a good starting set for the development of stable enzyme formulations, preselected by evolution. A search on the SwissProt database (http:// www.expasy.org) outputs two gene sequences coding for pyruvate oxidase (> 210´000 entries) and 41 out of the TrEMBL database (>2´600´000 entries). These gene sequences originate form twelve different species (Azoarcus, Bacillus, Bradyrhizobium, Escherichia, Lactobacillus, Lac- tococcus, Listeria, Oceanobacillus, Pseudomonas, Shigella, Staphylococcus and Streptococ- cus). Further, the search revealed seven putative pyruvate oxidase genes, which were not further investigated. However, the pyruvate oxidase from Oceanobacillus iheyensis is currently the only one with known sequence originating from an extremophile organism so far. Oceanobacillus iheyensis HTE831 was isolated from deep-sea sediment at a depth of 1050m below sea level. It shows extremely halotolerant and facultative alkaliphilic properties. This species employs a large number of osmo- and alkaliprotectant transporter systems in its metabolism in order to survive in hypersaline environments that surpass 3 M NaCl (Lu et al. 2001; Takami et al. 2002). Comparing the pyruvate oxidase amino acid composition of Oceanobacillus iheyensis (oiPOX) with Lactobacillus plantarum, a few noticeable differences are seen (see figure 3-6 and table 3-3); oiPOX contains 3.2% more Glu, 3.4% more Lys but 1.3% less His than lpPOX. These charged amino acids content differences in 5.2% more charged amino acid side chains. This is one well known strategy used by halotolerant organisms to survive in high osmotic media (see paragraph 3.2.1). OiPOX also contains 3.9% less Ala, 1.2% more Met and 1.4% more Ser than the mesophilic lpPOX. Finally, the alkaliphilic enzyme also contains 1.7% less Gln and 0.8% less Asn, which are susceptible to deamidation at high pH-ranges (see paragraph 3.1.3). Due to the fact that Oceanobacillus iheyensis enzyme is neither expressed nor characterised in detail, lpPOX seems to be a much more reliable starting point and source for further biosensor research. How and why the pyruvate oxidase from Lactobacillus plantarum was selected for this project is described in detail in paragraph 2.2.4 p. 38. The main reason for the selection of lpPOX for use in the on-line biosensor was the reported independence on cofactors and the good knowledge of its structural properties.

73 3 Protein Stability

>CORE domain sec st S S1HHHHHHHH HH1 SSS 2 HH2HHH HHHHH3 ββ SSS3 β HH HHHHHHHHHH HH4 SSSS cofact ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 60 70 80 LACPL MVMKQTKQTN ILAGAAVIKV LEAWGVDHLY GIPGGSINSI MDALSAERDR IHYIQVRHEE VGAMAAAADA KLTGKIGVCF OCEIH ----MFEDR- --AGKVLVDL LSEWGVDHIY GMPGDSINQL MEELRKEKDK MKFIQVRHEE AGALAAASYA KLTGKLGVCL

sec st 4 HHH5 HHHHHHHH6 SSSSSS5 ββ β β HHH7 ββ βSSS6 ββ HHHHH HHHHHHHHH8 cofact L ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 90 100 110 120 130 140 150 160 LACPL GSAGPGGTHL MNGLYDARED HVPVLALIGQ FGTTGMNMDT FQEMNENPIY ADVADYNVTA VNAATLPHVI DEAIRRAYAH OCEIH SIAGPGAIHL LNGLYDAKKD SAPVLVLAGQ VPTDQIGTDA FQEINMQRMF EDVAVFNEQV VSEEQLPALV NQAVRTAYAE

>FAD binding domain sec st SSSSSS7β HH9 SS8ββ H10 HH HHHHHHHHH1 1 β SSSS9 HHHH12HHHH HHHHH13___ cofact L LL ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 170 180 190 200 210 220 230 240 LACPL QGVAVVQIPV DLPWQQIPAE DWYASANSYQ TPLLPEPDVQ AVTRLTQTLL AAERPLIYYG IGARKAGKEL EQLSKTLKIP OCEIH KGPAVLTIPD DIPAAKIKKH VQKNAAIYEE PEQV--PSST EMEKALELIG NAKKPIILAG TGTKGAKQEL EQFSDKIAAP

sec st S10 H14β β β β ββγ HHH HHHH15 βSS S12 β β ββ β β β SSSS13 H1 6β β βS cofact LLL LLLL LLLLLL LLL L ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 250 260 270 280 290 300 310 320 LACPL LMSTYPAKGI VADRYPAYLG SANRVAQKPA NEALAQADVV LFVGNNYPFA EVSKAFKNTR YFLQIDIDPA KLGKRHKTDI OCEIH VIVSLPAKGV IDDDHPNCLG NLGMIGTKPA YEAMEDSDLV ILIGTSFPYV EFL--PEG-V SYIQIDNDPL KIGKRYPVNV

>ThDP domain sec st S14 HHHHH HHHH17 HHHHHHH HHHHHHH18H HH18 HHHHHHHH H19 β SS 15 HHHHH cofct LLL L L L ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 330 340 350 360 370 380 390 400 LACPL AVLADAQKTL AAILAQVSER ESTPWWQANL ANVKNWRAYL ASLEDKQEGPL QAYQVLRAV NKIAEPDAIY SIDVGDINLN OCEIH GIAGDSQKSL TRLNERITRT ENRDFLENSQ EKMAKWWKEM EEDEQQTSTPI KPQQIIAEV QKIAEDDAVL SVDVGNVTVW

sec st HH20 β S16 γββ β HHHHHH HHH21 β SSSS17HHHH H22H23HHHH 24 γ SSS S18 HHHH cofact LL L L LMLL M M M LLLLL ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 410 420 430 440 450 460 470 480 LACPL ANRHLKLTPS NRHITSNLFA TMGVGIPGAI AAKLNYPERQ VFNLAGDGGA SMTMQDLATQ VQYHLPVINV VFTNCQYGFI OCEIH AARHFRMKTT QKFLTSSWLA TMGCGLPGAI ASKMAYSDRQ AIALCGDGGF SMNMQDFLTA VKYNLPMVVV VFNNERIGMI

sec st HHHHH25 β γHHHHHH26 SSS19 H2 7HHHHHHHHH 28 SSS S20 ββ ββ cofact ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 490 500 510 520 530 540 550 560 LACPL KDEQEDTNQN DFIGVEFNDI DFSKIADGVH MQAFRVNKIE QLPDVFEQAK AIAQHEPVLI DAVITGDRPL PAEKLRLDSA OCEIH KYEQEAKGNL DYK-TNLESF NFAQFAENCG GKGYRVENFE DLGPSIRAAA SF--NKPVIV DVCIEDEPPI PGKLSYEQVT

sec st HHHHHHH HH29 β HHHHH30 . cofact ....|....| ....|....| ....|....| ....|....| ....|....| 570 580 590 600 610 LACPL TSSAADIEAF KQRYEAQDLQ --PLSTYLKQ FGLDDLQHQI GQGGF OCEIH SYSKQTIKKL FEKGK---LE VPPIRKGLKR M

Figure 3-6: Sequence Alignment of lpPOX and oiPOX The Lactobacillus plantarum POX sequence (Swiss- Prot entry P37063) is on the first line and the POX of Oceanobacillus iheyensis (TrEMBL entry Q8EL23) is shown on the second line. Identical residues are coloured black, conserved residues are grey. The secondary structure elements of lpPOX are shown above the residue numbers. Helices are abbreviated by „H“, β-sheets by „S“, β- and γ-turns by „β“ and „γ“. The interactions between lpPOX and the cofactors is shown above the numbering line; „L“ indicates interactions between the protein and the FAD and ThDP cofactors, „M“ represents interactions between the protein and the metal ion. The secondary structure elements are based on the PDB entry 1POX.

74 3.4 Structure and Stability of lpPOX

lpPOX oiPOX diff. lpPOX oiPOX diff. amino acid amino acid numb. (%) numb. (%) (%) numb. (%) numb. (%) (%) Gly 40 6.6 42 7.3 +0.7 Ser 22 3.6 29 5.1 +1.4 Ala 78 12.9 52 9.1 -3.9 Thr 29 4.8 24 4.2 -0.6 Val 43 7.1 42 7.3 +0.2 Asn 31 5.1 25 4.4 -0.8

Leu 52 8.6 45 7.9 -0.8polar Gln 40 6.6 28 4.9 -1.7 Ile 40 6.6 39 6.8 +0.2 Tyr 23 3.8 16 2.8 -1.0

non polar Met 13 2.2 19 3.3 +1.2 Cys 2 0.3 6 1.0 +0.7 Pro 30 5.0 30 5.2 +0.2 Lys 52 8.6 45 7.9 -0.8 Phe 19 3.2 20 3.5 +0.3 Arg 23 3.8 18 3.1 -0.7 Trp61.050.9-0.1 His142.361.0-1.3

charged Asp 39 6.5 33 5.8 -0.7 Glu 30 5.0 47 8.2 +3.2

Table 3-3: Comparison of amino acid contents of lpPOX and oiPOX: The analysis is based on the sequences published in the SwissProt database (entry P37063; lpPOX) and on the sequence based in the TrEMBL database (entry Q8EL23; oiPOX) see figure 3-6.

3.3.3 Protein Stabilisation and Sensor Optimisation

Different protein stabilisation techniques for pyruvate oxidase summarised in figure 3-4 are discussed in detail in the following paragraphs. A possible sensor optimisation and the improvement of the enzyme matrix in the biosensor should be investigated in a parallel project and is not topic of this thesis. An exact analysis of the weak points of lpPOX should first be the basis for further rational protein stabilisation approaches.

3.4 Structure and Stability of lpPOX

In the next paragraphs, currently known facts relating to the structure and stability of lpPOX as well as the conditions that influence protein stability in the biosensor are discussed. New concepts for further stabilisation of this enzyme are given in paragraph 3.5.

3.4.1 Structure of lpPOX

The crystal structure of lpPOX stabilised by the three point mutations (P178S; S188N; A458V)

75 3 Protein Stability and the wild-type enzyme have been refined by Muller et al. (1994) at 2.1 and 2.5 Å resolution respectively. The coordinates and structural factors from residues 9 to 593 are deposited in the Protein Data Bank (PDB) provided by the Research Collaboratory for Structural Bioinformatics (RSCB) under PDB-ID 1POX and 1POW respectively (www.pdb.org). As mentioned previously and described in literature, lpPOX is a homotetrameric enzyme (Götz and Sedewitz 1991, Risse et al. 1992a), each monomeric subunit consists of 603 amino acids, one FAD, one ThDP and one divalent metal ion (Mn2+ or Mg2+). The interfaces between the four subunits differ in such a way that the whole tetramer is more easily described as a dimer of dimers (αI and αII; αIII and αIV; see figure 3-7), i.e. a loose association of two tightly bound subunit dimers Muller et al. (1994). The catalytic centres of each monomer are at the subunit interface within the tight dimers (see figure 3-7). . ThDP

αI αII

FAD

αIII αIV

Figure 3-7: Tetrameric structure of lpPOX (Muller et al. 1994) The four monomeric subunits are displayed in different colours. The corresponding FAD and ThDP cofactors are superimposed in the same colour and the secondary structural elements are shown as cartoons with α-helices represented as cylinders and β-sheets as arrows. The gap between the two dimers is visible in the middle of the figure. The quarternary structure of lpPOX has been described as loose dimer of tightly bound dimers (αI-αII and αIII-αIV). The four active sites are located at the interfaces between the tightly bound monomers.

The contact surface area between the tightly bound monomers is about 3800 Å2 and more than

76 3.4 Structure and Stability of lpPOX

2 two times as great as the surface area of about 1500 Å buried between the subunits αI - αIII or

αII - αIV respectively. The interaction cross-over (from αI - αIV and from αII - αIII) is marginal and focused on a surface area of about 900 Å2. A similar distribution results from the comparison of all polar interactions between the subunits; 66 polar interactions are possible between the tightly bound monomers, 30 between the subunits αI - αIII or αII - αIV and 12 cross over between αI - αIV and αII - αIII respectively. Each monomer can be divided into three domains; The CORE domain (residues 9 to 191), the FAD domain (residues 192 to 342) and the ThDP domain (residues 343 to 595). The CORE domain at the centre of the homotetramer is the most rigid, as expected from the domain arrangement and shown by comparison of B-factors for each domain separately. Each domain is formed by a central six-stranded parallel β-sheet surrounded by α-helices. The β-sheet topology of the FAD domain is that of a double Rossmann fold, in which only two of the five connections between β-strands contain α-helices. A Rossmann fold is a βαβαβ-motif in which the strands form a parallel β-sheet with (+1x, +1x) connectivity, and the helices lay anti parallel to the strands on one side of the sheet. The βαβ-units are right handed as in most of the Rossmann fold proteins. Astonishingly the CORE and the ThDP domains show similar β-sheet topologies. In a comparison, Muller et al. (1994) discovered that the structure of these two domains is congruent over a wide range, whereas sequence homology is only 13% in these intervals. Muller further showed, that ThDP binding domains corresponds to folds of two other ThDP proteins whose structure is known. However, the fold of the FAD domain is different from those of other known flavoproteins.

77 3 Protein Stability

a) b)

Figure 3-8: Monomer of lpPOX: On both pictures a) and b) one monomeric subunit of lpPOX with its corresponding cofactors is shown. Colour code: CORE domain: purple (residues 1 to 191), FAD domain: pink (residues 192 to 342), ThDP domain: red (residues 343 to 595), cofactors FAD and ThDP: usual chemical colour code, CORE domain of chain C: green silhouette. a) In all three domains the central six-stranded parallel β-sheet (arrows) surrounded by α-helices (cylinders) can be seen. b) The surface view of the three domains shows how the cofactors are embedded into the corresponding domains. In front of that active site the CORE domain of the opposite monomeric subunit covers the cofactors (indicated by the green line). The direct contact of the CORE domain of chain C forces the ThDP to the V-conformation (see figure 2-6 page 42) and activates ThDP via interaction of Glu59. (The data-set for the figures is based on the PDB entry 1POX.)

The two cofactors FAD and ThDP are embedded into the corresponding lpPOX domains, whereas the divalent metal ion compensates the charges of the two phosphate groups of ThDP and acts as a mediator of salt bridges from the protein to the cofactor. FAD is well embedded in the FAD binding site of the corresponding domain. However, it is not as buried and therefore not as fixed as the FAD molecule in GOD (compare to figure 3-5 page 70). The ThDP molecule is anchored in the corresponding domain with the phosphate end via the divalent metal ion. A major part of this cofactor molecule protrudes out of the monomeric protein. Only the CORE domain of the second monomer forces the ThDP to the V-conformation (see figure 2-6 page 42) and activates ThDP via the interaction of Glu59 (Tittmann 2000) through direct contact. Close contact of the CORE domain with the neighbouring subunit is therefore essential for an active pyruvate oxidase and should prevent the loss of both FAD and ThDP cofactors from the first monomeric subunit.

78 3.4 Structure and Stability of lpPOX

3.4.2 Stability and currently known Stabilisation of lpPOX

3.4.2.1 Stability of lpPOX wild type (wt)

The enzyme first isolated and characterised by Sedewitz et al. (1984) showed rather tightly bound cofactors in comparison to other pyruvate oxidases. Therefore, an addition of expensive cofactors in the sample buffer can be avoided in commercial phosphate biosensors applying lp- POX. Risse et al. (1992a) elucidated the stabilising effects of cofactor binding and subunit interactions in the wt enzyme. A kinetic analysis revealed a sequential mechanism of cofactor binding and tetramer formation. The release of cofactors destabilises the native quaternary structure, whereas the secondary and tertiary structures are not influenced by the presence or absence of cofactors. Thermal deactivation experiments also confirmed, that the transition midpoints depended on the concentration of enzyme and of FAD, whereas ThDP had no influence. Enzyme anf FAD concentrations have been shown to determine the state of association of the enzyme. The apparent binding constants of both FAD and ThDP cofactors increased in the presence of the other cofactor. One can conclude that insufficient cofactor binding, especially FAD but also ThDP, affects lpPOX stability via shifting the equilibrium from the tetrameric to di- and monomeric states. The fact that high protein concentrations, which shift the equilibrium to the tetrameric side, stabilise lpPOX, suggests that another kind of tetramer-stabilisation should also increase enzyme stability. In addition, the results of Risse et al. (1992a) showed that quaternary contacts play a significant role in the stabilisation of lpPOX wt. Formation of oligomer and cofactor binding are both common between GOD and pyruvate oxidase (see paragraph 3.2.2). Nevertheless, lpPOX behaves differently compared to GOD. In lpPOX, the binding of cofactors is not an indispensable requirement for monomer association. In pyruvate oxidase, the cofactors can also be released without dissociation of di- or tetramers. So one can conclude that in GOD, the release of cofactors is more effectively avoided by the above mentioned special mechanism than in lpPOX.

79 3 Protein Stability

3.4.2.2 Stability of the lpPOX Triple Mutant Developed by Schu- macher et al. (1990)

At acid or alkaline pH-values (below pH 5 or above pH 7) and also at high salt concentrations, lpPOX wt rapidly loses its activity. The reason is - as mentioned above - that the enzyme dissociates into the inactive monomers accompanied by the release of cofactors. With the intention of improving an enzymatic assay, Schumacher et al. (1990) created a more stable mutant of pyruvate oxidase. Because no structural data was available for the enzyme at that time, Schu- macher et al. used random mutagenesis and subsequent selection runs to generate mutants that work under conditions where the wild-type enzyme is inactivated (pH 8 and 0.2 mol/l NaCl). They found that the following mutations led to increased stability at higher pH-values and at higher salt concentrations: P178S, S188N and A458V as well as the combination of these three mutations in a triple mutant. The triple mutant of lpPOX showed increased stability in 0.1 mol/l potassium phosphate buffer containing 0.15 mol/l NaCl, at pH 7.5 up to an alkaline pH of 8.7, both at 25° and at 37°C (Schumacher et al. 1991). Risse et al. (1992b) observed that the midpoint of inactivation temperature increased from 42°C for the wild type enzyme to 51.5°C for the triple mutant. They also elucidated the stabilising mechanism of these point mutations and showed that the enhanced stability is based on the stabilisation of the active tetrameric quaternary structure of the holoenzyme. None of the above mentioned amino acid substitutions enhanced the stability of the tertiary structure of the dissociated apoenzyme. In 1994 Muller et al. elucidated the crystal structure of lpPOX wt and the above mentioned triple mutant. They showed that all three mutations are at or near subunit interfaces, indicating that they stabilise the quaternary structure. The stabilising effect of the A458V mutant dominates over the increments gained by the two other point mutations. This mutation strengthens the interface between the subunits αI and

αII subunits, where the ThDP molecules are buried. This also explains why the dissociation rate of the binary apoenzyme-ThDP complex is reduced for this mutant.

The S188N mutation affects the contact between αI and αIII subunits. The P178S mutation is at least located in a β-turn near the segment, which connects the CORE and FAD domains and which is part of the αI and αIII interface. The P178S and S188N mutants had no effect on ThDP binding, and the apparent increase in FAD binding may be attributed to improved subunit interactions.

80 3.4 Structure and Stability of lpPOX

3.4.2.3 The Achilles Heel of lpPOX

All the investigations regarding the stability of lpPOX wt and triple mutant demonstrate that the holotetramer formation limits the stability of the enzyme. With regard to lpPOX, protein inactivation steps have to be added to the common scheme of protein degradation in figure 3-1 page 53 (figure 3-9). active tetrameric release of lpPOX holoenzyme ThDP

unfolding of monomers

release of irreversible FAD protein degradation

dissociation of the tetrameric state

Figure 3-9: Inactivation cascade of lpPOX in addition to the common protein denaturating process The figure shows the whole inactivating cascade of lpPOX. On the left side the tetrameric holoenzyme is illustrated. FAD molecules are showed as orange circles and ThDP as blue circles. The protein moiety is drawn in black.

As shown in figure 3-9, the first step of lpPOX inactivation is the loss of FAD molecules as described in the minimum reaction scheme for tetramer-dimer-monomer transition in Risse et al. (1992a). The protein then dissociates to dimers and further to monomers accompanied by the loss of ThDP. The remaining monomeric apoenzyme undergoes the common unfolding and denaturating processes as described in paragraph 3.1.1 and illustrated in figure 3-1. The weakest point or so called „Achilles heel“ of lpPOX in solution is the dissociation of the tetrameric state accompanied by the release of cofactors. It is obvious that stabilising strategies for pyruvate oxidase have to begin at this weak point. All the more, especially because released cofactors can be washed out in the on-line biosensor. Therefore, the equilibrium showed on figure 3-9 is shifted more to the left side, to an inactivated enzyme. Factors that influence protein degradation at the monomer level are most likely less decisive for lpPOX stability in the amperometric biosensor. As mentioned previously, the exact limitating factor in the biosensor itself can not be evaluated absolutely reliably, but all the stabilising mutations found by Schumacher et al. (1990) favours the tetramer formation of the enzyme and therefore confirm the chance of further stabilisation on this level. In the next paragraph, the most important extrinsic factors, which can affect protein stability in the amperometric biosensor and which can also enforce the unwanted dissociation of

81 3 Protein Stability tetramers, are discussed. The most suitable strategies for a stabilisation of lpPOX in the biosensor are mentioned in paragraph 3.5.

3.4.3 Protein Destabilising Extrinsic Factors in the Amperometric Biosensor

The decisive factors that can reduce protein stability in the amperometric biosensor are the following; hydrophobic surfaces and the hydrophobic matrix of silicon oil, extremes of pH-range and low ionic strength of the sample solution, electric field and mediator molecules which favour redox reactions. A moderate temperature of about 25°C and high water activity are not optimal for the long-term stability of the protein, but these factors are not avoidable for an optimal enzyme catalysis. The rationale for neglecting the other factors listed on the table on page 54 during the application of lpPOX in the amperometric biosensor is outlined later on. There are plans to optimise the matrix in the biosensor in a subsequent project. The hydrophobic matrix of silicon oil should not cause problems in biosensors. Enzymes can also reach good catalytic activities also in organic solvents under optimal conditions and adequate saturation with water, as outlined in the paragraph about organic solvents in point 3.1.2. Saturation with water can easily be achieved in the biosensor via an exchange over the membrane with the sample solution. The silicon oil matrix has also been used and sucessfully implemented in many different amperometric biosensors with a variety of different enzymes, for example, those described by Fernandez et al. (1998), Rajendran et al. (1998), or by Korell and Spichiger (1994). The intrinsic protein instability revealed itself as the main problem during sensor development and protein production. A comparison with immobilised GOD in the sensor (see figure 2-9 page 49) shows the susceptibility of POX to inactivation. A change of the transducing technique and therefore a prevention of the electric field and mediator molecules is not advisable. This is because amperometry is the most promising transducing technology for phosphate sensors with regard to detection limits, dynamic response ranges, costs and technical requirements (see paragraph 2.4). The pH-range and ionic strength of the sample solution has to be controlled by the addition of a standard buffer. The corresponding cosubstrate, pyruvate or phosphate (depending on the wanted analyte), has to be mixed with the sample solution in a defined concentration. This prerequisite of defined cosubstrate concentration implies high demands on method development in the medical field, where it is intended to directly analyse body fluids like blood, plasma or urine. In such medical samples, certain amounts of both substrates (phosphate and pyruvate) ex-

82 3.4 Structure and Stability of lpPOX ist. Therefore, all possible interactions while buffer addition to the sample solution have to be considered during the measurement of these medical samples. Additionally, these methods have to be acurately validated in order to avoid unwanted influences of pre-existing cosubstrates.

Influence of Extreme pH-values on lpPOX Stability and Activity

As mentioned in paragraph 3.1.2 the ionisation state of acidic and basic amino acids can affect the equilibrium between native and unfolded protein. Charged amino acids in the active site are also highly influenced by pH changes. Their ionisation state can have an effect on the kcat and

Km values of an enzyme. With regard to lpPOX, the pH-value plays a crucial role for optimal activity. The ThDP cofactor in lpPOX has to be activated by the Glu59 of the adjacent subunit as mentioned in paragraph 2.2.4 and illustrated in figure 2-6 page 42. The interaction of Glu59 and the N1‘ in the pyrimidine ring of ThDP leads to an increased basicity of the 4‘ amino group and to fast deprotonation of C2-ThDP, which is the active centre during pyruvate oxidation (Kern et al. 1997; Friedmann and Neef 1998). Of course, in the active tetrameric state the amino acids in the active site and Glu59 especially are protected by the surrounding protein moiety. But as it is generally known the enzyme exists in an equilibrium between mono-, di- and tetrameric states. And the carboxyl-group of Glu59 in the monomeric state is indeed embedded in a cavity at the protein surface but exposed to solvent and therefore exposed to protonation at low pH-values. The wild type lpPOX showed optimal activity at pH-values around 5.7 (Sedewitz et al. 1984). But Risse, Möllering, Schumacher, Stempfer, Rudolph and Jaenicke developed a new triple mutant of lpPOX (P178S, S188N, A458V) with a higher tolerance to pH-values between 4.5 and 7.5, whereas optimal activity remains at a pH around 5.7 (Schumacher and Moellering 1989). At higher pH-values, the midpoint of inactivation temperature was shifted from 6.7 (wt- lpPOX) to 7.85 (triple mutant-lpPOX) (Risse et al. 1992b). The triple mutant with its higher pH tolerance therefore presents itself as a convenient enzyme for application in biosensors, that work in aqueous samples at pH-values around 7. It is also the most promising candidate for measurements in environmental analytics where aqueous sample solutions in pH-ranges from 6 to 9 have to be measured.

Further Extrinsic Factors

Chaotropic salts, redox agents, metal ions and detergents do not appear in sample solutions in

83 3 Protein Stability critical concentrations, nor in technical nor in medical applications. The enzyme paste inside the biosensor is perfectly protected against electromagnetic waves and the membrane between the sample solution and enzyme paste prevents the enzyme from protease attacks. There is a lack of an air-liquid interface in the sensor under operating conditions. Furthermore, a physical degasification module preceding the biosensor removes dissolved gases from the sample solution. Shear forces can be excluded because the enzyme is embedded and fixed in the paste and therefore not exposed to agitation. Destabilising high pressure is also prevented by intelligent sensor assembly. The pump is placed behind the sensor and draws the sample solution through the biosensor. (see Nagel 2004; paragraph 2.1.B.1)

3.5 Most Promising Stabilising Methods for lpPOX in the Biosensor

The lpPOX triple mutant (P178S, S188N, A458V) developed by Schumacher et al. (1991) was chosen as starting point for further investigations and stabilisation experiments because of its strong cofactor binding and its stability against higher pH-values and salt concentrations. After this selection, the stabilisation techniques summarised in figure 3-4 page 66 are checked for application in the biosensor setup.

3.5.1 Extrinsic Protein Stabilisation Techniques for lpPOX

As mentioned in paragraph 3.4.3, most extrinsic protein stabilisation techniques summarised in figure 3-4 have already been considered during the development of the biosensor setup (protection from electromagnetic waves, optimal pressure, non covalent immobilisation by a membrane). Or if not considered, they are currently applied to the maximal possible extent (optimal temperature, pH, buffer, salt concentration). A few techniques are not at all applicable in the amperometric biosensor (lyophilisation, protection from oxidation). The improvement of the enzyme matrix in the biosensor (optimal solvent, eventually covalent immobilisation) should be investigated in a parallel project and is therefore not a topic of this thesis. Chemical additives such as polyoles (glycerine and so forth) are inappropriate because they leach out of the sensor during long-term measurements.

84 3.5 Most Promising Stabilising Methods for lp-

Stabilisation by addition of other proteins e.g. bovine serum albumin (BSA) could be envisaged, but this procedure can only prevent protein aggregation and not the more crucial steps of tetramer dissociation and cofactor release. Because of the above mentioned reasons, further stabilisation of lpPOX based on extrinsic factors is not the most promising approach. Therefore, it was postponed for the benefit of the more favourable intrinsic stabilisation techniques.

3.5.2 Intrinsic Protein Stabilisation Techniques for lpPOX

The weak points of pyruvate oxidase are already discussed in paragraph 3.4.2.3. Intrinsic protein stabilisation techniques interfering exactly at the level of tetramer dissociation and cofactor release of lpPOX are the most promising steps on the way to stable phosphate/pyruvate biosensors.

3.5.2.1 Intrinsic Stabilisation by Change in the Amino Acid Se- quence

In principle, two methods are applicable to search for new mutants of the enzyme. Firstly, the random based evolutionary approach and secondly, the rational knowledge based method of structure guided site-directed mutagenesis (see also paragraph 3.2). In this project, the rational approach is the more qualified to stabilise of lpPOX for application in the amperometric biosensor because of the different reasons described in detail in paragraph 3.2; Firstly, the unphysiological environment of the biosensor makes it impossible to apply standard selection methods based on cells or phage display. Secondly, the weak points of the enzyme are well elucidated and several structural details are clarified by the crystal structure done by Muller et al. (1994). Changes in the amino acid sequence can be tested with the intention of strengthening the interactions between the monomeric subunits or with an aim to improve cofactor binding.

Improvement of Subunit Interactions and Mutants Investigated by Nagel (2004)

The starting template for further protein engineering was a threefold mutated lpPOX (P178S, S188N, A458V) provided by Roche Diagnostics (Penzberg, GER). Hence this triple mutant enzyme is called quasi-wt in this thesis.

85 3 Protein Stability

Nagel (2004) investigated a Val141Cys mutant of the lpPOX quasi wt, which should stabilise the tetrameric quaternary structure via disulphide bonds between the αI and αIV subunits as well as between αII and αIII. However, this mutant was not active without cofactors in the sample solution and was therefore not further investigated. Aside from that, this, it is rather difficult to predict the reduction potential and therefore stability of a certain disulphide bond in a protein. The reduction potential of the latter cysteine residues are dependent on the structure in which they are embedded and can vary over a wide range. Above all, a resistance to reduction of this putative stabilising disulphide bond in the electric field of the biosensor should have been demonstrated. A second mutant (Ala131Ile) investigated by Nagel (2004) stabilised lpPOX against high temperature inactivation caused by better contacts in the symmetry centre of the tetrameric enzyme. The midpoint of inactivation temperature increased about +1.1°C. An increase of the non-polar interaction area between the four monomers led to the abovementioned stabilisation. However, this mutant was not tested with regard to long term stability.

Conclusion

Aside from the fact that mutant A131I investigated by Nagel (2004) showed a small thermosta- bilisation during the stress test experiments, the undertaking to develop new interactions between the monomeric subunits is rather complex. Replaced amino acid residues at the monomeric interfaces should not destabilise the monomer itself. Secondly, they must interact better than the parent amino acid residue with the neighbouring residues of the adjacent subunit. Presumably a random based approach with iterative mutation and selection runs as applied by Schumacher et al. (1991) is the most appropriate method to improve the subunit interactions of lpPOX. However, due to the expected and abovementioned difficulties applying random based methods in combination with the unphysiological sensor matrix, this approach has been postponed.

Amplification of Cofactor Binding

Cofactor binding can be amplified by three different techniques; Firstly, direct interactions of amino acid residues in proximity to the cofactor molecules can be reinforced or newly arranged. The difficulty in this approach is the susceptibility of the active part of a cofactor. The introduction of new interactions between proteins and the active part of a cofactor, therefore, can lead to less active or totally inactivated holoenzymes. Because the number of amino acids which

86 3.5 Most Promising Stabilising Methods for lp- come into consideration is rather small, a rational driven stabilisation experiment can be performed based on this technique. A second approach is the reinforcement of tertiary structures, strengthening cohesion of the cofactor binding motifs and domains. An impressive example of this concept is discussed by means of GOD in paragraph 3.2.2. However, a transformation of POX in analogy to the GOD model enzyme would be very difficult with the nowadays currently available methods in protein engineering. The aim with pyruvate oxidase has to be limited to small adaptations. The problematic in this approach is the selection of the most promising amino acids among the whole sequence, as already mentioned in the previous paragraph about subunit interactions. A search for random mutagenesis would be the most promising method for a better cofactor immobilisation based on stabilisation of the tertiary structure. Because of the discussed difficulties in such an approach, this kind of cofactor stabilisation has also been postponed. The third point where cofactor stabilisation can be addressed is the rigidisation of protein backbone by Xxx->Pro and Gly->Xxx mutations respectively. Of course, a suitable selection mechanism for promising amino acids is also the key step in this approach. However, Nagel (2004) proposed such a selection system, which should be tested during this project on the basis of biochemical experiments.

3.5.2.2 Intrinsic Stabilisation by Biotechnological Modification of the Protein

Glycosylation

Since the lpPOX is an enzyme originally expressed in the procaryotic Lactobacillus plantarum and not in a eucaryotic host, no glycosylation could be expected. Indeed, the sequence of lpPOX shows one possible N-glycosylation site (sequence Asn-Xxx-Ser/Thr) at the N137-V138-T139 position. However, this site lays at the interface between the two CORE domains of monomers

αI and αIII (see figure 3-7) and therefore a hypothetical glycan at this position would hinder the assembly of tetramers. No O-glycosylation sites have been predicted. Possible stabilisation of lpPOX by glycosylation can therefore be excluded. Glycosylation after the engineering of new gycosylation sites was not further investigated during this thesis, because as other stabilisation techniques show much more promise.

Truncation of flexible ends

Truncation of flexible ends or large flexible loops may prevent proteolytic degradation or pro-

87 3 Protein Stability tein precipitation due to lower adsorption between protein molecules or between protein and surfaces (McHugh et al. 2004). Some extremozymes are stabilised based on this mechanisms in comparison to their mesophilic counterpart. An application of this promising technique presup- poses further structural analysis of the protein. This technique is basically only accessible by rational mutagenesis and should be retained during the project as a reserve strategy.

Addition of Stabilisation Tags

The addition of stabilisation tags should be investigated during this project. This modification of proteins can also be applied for a better protein solubilisation and stabilisation during the expression and purification.

3.5.2.3 Intrinsic Stabilisation by Chemical Modifications

Proteins can be chemically modified in several ways. These modifications can be used to optimise protein stability and the pharmacokinetic and immunogenetic properties of therapeutic proteins. A review written by Frokjaer and Otzen (2005) discusses the benefits of chemical modifications applied on therapeutic proteins and articles by DeSantis and Jones (1999) or Gov- ardhan (1999) summarise the in vitro applications of chemical modified proteins. Chemical crosslinking has emerged as an easy and inexpensive method to enhance thermal stability and increase tolerance to organic solvents. Both inter- and intramolecular crosslinking, for example, accomplished by glutaraldehyde or dextran dialdehydes can stabilise proteins (Kazan et al. 1997, Venkatesh and Sundaram 1998). The fact that the extent and the exact location of the modifications are often not determined complicates the process of crosslinking. By a combination of site-directed mutagenesis and specifical chemical modification of the introduced amino acid residue this disadvantage can be overcome (DeSantis et al. 1998). PEGylation is a process in which polyethylene glycol (PEG) molecules attached to the protein surface can enhance protein stability (Garcia et al. 1998). The attachment of PEG to proteins can also increase their solubility in organic solvents. In therapeutic proteins, PEGylation reduces the plasma clearance rate by reducing metabolic degradation. It also shields at least partially antigenic and immunogenetic epitopes and therefore minimises safety problems in the therapeutic field. Acylation is a chemical modification mainly used for in vivo stabilisation of therapeutic proteins and is less applied in protein stabilisation for technical applications. Chemical attach-

88 3.6 Conclusions and Hypothesis ment of fatty acids to exposed amino acid residues on the protein surface can, in some cases, increase the affinity of the protein to serum albumin enough to increase its circulation time in the blood. With regard to lpPOX stabilisation for the biosensor, only the chemical crosslinking of enzyme could be considered. The other methods are either targeted to solubilisation, which is not the most sensitive point of lpPOX or they are more focused on therapeutic relevant in vivo stabilisation. However, also crosslinking of pyruvate oxidase is successful in the sensor, a possible leaching of cofactors cannot be excluded by this method (Zarpata-Burci and Burstein 1987). A chemically driven covalent immobilisation of the cofactors in the enzyme analogue to the covalently bound FAD in vanillyl alcohole oxidase (Fraaije et al. 2000, 2003) was already discussed by Nagel (2004; p. 195 ff) and valued with only a low success rate.

3.6 Conclusions and Hypothesis

The random mutagenesis experiments of Schumacher et al. (1991) proved that it is possible to stabilise this enzyme. Considering all the structural details and weak points of lpPOX and also keeping in mind the harsh conditions in the biosensor, the most promising approach for enzyme stabilisation is the one by structure based rational protein design. The hypothesis is that the enzyme can be further stabilised in the most reliable way by increasing the interactions between the cofactors and the protein (see chapter 5). Stabilisation of the tertiary structure by new mutations based on the theory of defined unfolding and modelling studies should also bring out some new enzymes with a higher longterm stability (see chapter 6). The starting template for further protein engineering was, as mentioned previously, the threefold mutated lpPOX (P178S, S188N, A458V) with a higher tolerance to pH-values between 4.5 and 7.5 (Risse et al. 1992b). The goal was not only to further improve the stability of lpPOX but also to achieve this goal without loss of activity.

89 3 Protein Stability

90 4 Expression and Purification of lpPOX

big challenge during this project was the expression and purification of lpPOX in Aamounts of milligrams which allows for stability testing experiments of the mutated enzymes and therefore also a verification of the working hypothesis. Roche Diagnostics GmbH (Penzberg, GER) kindly provided lpPOX gene. However, neither efficient expression protocols nor reliable production instructions that work properly in real laboratory conditions could be found in scientific or patent literature (Schumacher and Möllering 1989, Risse et. al 1991, Schu- macher and Möllering 1992). This chapter describes a new expression and purification procedure that was developed in several steps to produce lpPOX in amounts of milligrams. When the phosphate sensor can be commercialised, amounts for other diagnostic enzymes of about up to 10 kg/year would be needed. Enzymes for industrial processes or enzymes used in the detergent industry, for example, are produced in amounts of several 100 tons per year worldwide. Al- though such large amounts of lpPOX will not be needed and the sensor is still in the development phase, the expression and purification protocol was optimised during the whole project with regard to a process that can easily be upscaled.

4.1 Introduction - The Challenge of Soluble Expression of lpPOX

As so often in real laboratory conditions, the expression protocols published for lpPOX expression did not work properly. This paragraph gives a short introduction into the problematic of soluble lpPOX expression and enumerates a few techniques that were tested on the way to the currently established protocol. The results of these preliminary solubilisation experiments are reported in the thesis of S. Nagel (Nagel 2004, p77ff) and are summarised here for the sake of completeness.

4.1.1 Strategy for lpPOX Expression

As mentioned in chapter 3, the template for genetic engineering was a threefold mutated lpPOX provided by Roche Diagnostics GmbH (Penzberg, GER). The DNA sequence of lpPOX was sent in Escherichia coli (E. coli) ED 82+ cells containing the pBP2008 plasmid which contain

91 4 Expression and Purification of lpPOX the lpPOX gene. An exact description and sequence verification of the plasmid containing an ampicillin resistant gene and the tac-promoter system for the control of lpPOX expression is given in Nagel (2004) pp. 105-112. The well known and investigated host organism Escherichia coli was chosen for the production of lpPOX due to several reasons; Firstly the promoter system with the tac-promoter was developed for the expression of heterologous proteins in E. coli, the best known and undemanding host organism. The promoter allows the induction of gene expression at a certain cell density by addition of Isopropyl-β-D-thiogalactoside (IPTG). The yield of pyruvate oxidase should be much higher applying this upgraded expression system in comparison to the natural expression in the original cells of Lactobacillus plantarum (Sedewitz et. al 1984). Secondly, in a well known host cell line, possible concomitants like proteases or other proteins, which can interfere during the purification, or which cause instability or degradation of recombinant proteins (like Lon and ompT proteases), are easier to control and avoid. Further- more the effectiveness of translation is characterised better in E. coli than in some uncommon natural host cells from Lactobacillus sp. or other organisms. Application of E. coli for heterologous protein production is one of the quickest and cheapest methods and E. coli has all the ma- chinery necessary for folding of procaryotic proteins. Thirdly, in the underlying patent application (Schumacher and Möllering 1990) a protocol of expression is based on E. coli ED cells too. The patent applicant Toyo Jozo Kabushiki Kaisha, a competitor of Roche Diagnostics, also describes the expression of pyruvate oxidase of Aero- coccus viridans in E. coli cells (Matsumura et al. 1988).

4.1.2 Insoluble POX Precipitated in Inclusion Bodies

Unfortunately, lpPOX could not be expressed in a soluble and active form in E. coli cells. Nei- ther in the E.coli ED82+ strain provided by Roche together with the pBP2008 plasmid nor in a few other tested E.coli strains (Nagel 2004 p77ff). Different E. coli BL21 cell lines (provided by Stratagene, Amsterdam, NED) from the most convenient and effective bacterial hosts for protein production available were also tested. However, in all these cells the protein was always separated in inclusion bodies and insoluble . A strategy to prevent cells from expressing heterologous proteins insoluble is to decrease the induction rate and the expression to a low level, where the host cells can fold the heterologous protein correctly (Kopetzki et al. 1989, Kopetzki and Schumacher 1989). Different expressing parameters were varied to obtain lower induction rates and therefore to obtain soluble

92 4.1 Introduction - The Challenge of Soluble Ex- lpPOX; Temperature was varied between 12°C and 37°C, the induction rate was varied applying different concentrations of IPTG (0.24 mM down to 0.01 mM), different expression times were tested from 24 hours down to 4 hours, further expression at different density levels (OD600 0.05 up to 0.8) and expression in M9 minimal medium were tested. However, no combination of expression parameters led to soluble, active lpPOX (Nagel 2004, p125-134).

4.1.3 In vitro Refolding of Unsoluble Protein

Nagel applied the in vitro refolding method to lpPOX (Risse 1992a, Nagel 2004 p84ff). How- ever, only low amounts of 100 up to 500 µg lpPOX could be refolded under consumption of four litres of dialysis buffer containing 20% glycerol. For stability experiments and measurements in the biosensor, milligrams of lpPOX are required. Therefore, a new expression strategy based on fusion proteins has been tested.

4.1.4 Strategy of Fusion Proteins

To overcome the problematic of insoluble protein in inclusion bodies, the lpPOX gene was cloned into two different plasmids (pET43.1b Nus-tag™ and pET32b Trx-tag™) which have been reported to enhance the solubility via expression of fusion proteins with a highly soluble fusion partner. The NusA protein N-terminal fused to the lpPOX consists of 491 amino acids. With the Nus-tag™, high amounts of soluble but inactive fusion protein were produced. Unfortunately, the protein lost solubility and precipitated during storage at -20°C or when the Nus-tag™ was cleaved off with thrombin. It is assumed that the pyruvate oxidase part of the fusion protein was not correctly folded and was held in solution only by its high soluble fusion partner, the Nus- tag™. The possibility that the fusion protein could not form active forms of POX due to sterical hindrance by the fusion partner was excluded by Nagel (2004, p92). He showed that it was possible to receive active fusion proteins via denaturation of inacitve protein and following in vitro refolding. The amounts of active enzyme obtained in these experiments were much too small for application in the biosensor or even for common biochemical characterisation. A second fusion protein with the thioredoxin tag (Trx-tag™) was tested; the best conditions for this fusion protein were induction with 0.24 mM IPTG at an OD600 of 0.6 and expression during 24 hours at 37°C. The thioredoxin tag protein consist of only 109 amino acids and therefore has a much lower risk of causing sterical problems during tetramer formation in com-

93 4 Expression and Purification of lpPOX parison with the large Nus-tag™ protein. The same lysis buffer as for the NusA-fusion protein was used. The lpPOX thioredoxin-fusion protein of was soluble in E. coli as was the Nus-tag™ fusion protein. After the first run through with the inactive fusion protein, active fusion protein was successfully produced based on the improved purification protocols (see section 4.3.2). Because of substantial loss of soluble protein after thrombin cleavage and in search of an easier and much more efficient production procedure for lpPOX, a new method was taken into consideration.

4.1.5 Strategy of Controlled Induction Rate

The need for a method which provides the new designed mutants in an active and soluble form, in acceptable yields, directly form E. coli without any refolding forced me to look for a new expression method. The method which brought the breakthrough in expression of soluble lpPOX was based on BL21-SI™-cells delivered by Invitrogen™ (USA). These cells are optimised for recombinant protein expression, have been patented by Gowrishankar et al. (1998) and are described in the article written by Bhandari and Gowrishankar (1997). They contain a stable integrated chromosomal insertion of the T7 RNA polymerase gene under control of the proU promoter. The addition of a certain amount of NaCl (e.g. 0.3 mol/l) induces expression of the T7 RNA polymerase and consequently also of genes cloned behind the T7 promoter (see figure 4-1). The salt inducible proU promoter generally remains inactive in concentrations of up to approximately 50 mmol/l salt. Higher concentrations induce gene expression in rates dependent on salt concentration in the medium. A titration experiment was done to find the ideal salt concentration where the highest protein expression of soluble lpPOX occurs. Different salt concentrations from 100 mmol/l up to 1000 mmol/l were tested for the most efficient induction of protein expression.

94 4.2 Materials and Methods

BL21-SI™ genome plasmid containing the gene of interest

a) without salt

proU T7 RNA no T7 RNA gene of no promoter polym. gene transcription promoter interest transcription

b) with salt

proU T7 RNA T7 RNA gene of promoter polym. gene promoter interest

transcription of transcription of T7 RNA polymerase protein of interest

Figure 4-1: Working scheme of salt inducible BL21SI™-cells from Invitrogen™ a) In medium without sodium chloride (LBON medium) the proU promoter is not activated. Therefore the T7 RNA polymerase is not transcribed and is absent form the cell. The gene of interest is also not transcribed. Under these conditions the cells can grow up to a certain level, where heterologous protein expression can be started by the addition of a certain amount of sodium chloride. b) A certain concentration of NaCl induces the expression of T7 RNA polymerase. This polymerase lacking in normal E. coli cells enables the overexpression of a T7 promoter in front of the gene of interest. The translation of the heterologous protein can therefore be controlled by the sodium chloride concentration in the medium.

4.2 Materials and Methods

4.2.1 Materials

4.2.1.1 Chemicals, Oligonucleotides and Enzymes

Potassium-dihydrogen-phosphate, dipotassium-hydrogen-phosphate, imidazole, sodium chloride, phenylmethylsulfonyl fluoride (PMSF), Triton-X 100®, ThDP, FAD, magnesium sulphate, sodium dodecyl sulphate (SDS) and Coommassie brilliant blue were purchased from Fluka (Buchs, SUI). Agarose (low melting point) was purchased from Gibco BRL (Paisley, GBR). Acrylamide solution (4K, 30%) and IPTG were purchased from AppliChem (Darmstadt, GER).

95 4 Expression and Purification of lpPOX

Protein markers for SDS-PAGE (low range and high range) and the Quick Start Bradford Pro- tein Assay for the determination of protein concentrations were purchased from BioRad (USA). The DNA ladder was from Invitrogen (USA). Ampicillin sodium salt was purchased from Fluka (Buchs, SUI) and sulphate from Serva (Heidelberg, GER). Synthetic oligonucleotides (primers) used for cloning, site-directed mutagenesis and sequencing were obtained from Microsynth (Balgach, SUI). The ABI PRISM DNA sequencing kit containing BigDye Terminator v3.0 Cycle Sequencing Ready Reagent with AmpliTaq DNA Polymerase FS as well as the Template Suppression Reagent were obtained from Applied Bio- systems (USA). Single nucleotides was purchased from Pharmacia Biotech (Uppsala, SWE). Pfu Turbo DNA polymerase and the 10x Pfu reaction buffer were bought from Stratagene (Amsterdam, NED). Desoxyribonuclease I (from bovine pancreas, crude, lyophilised, 90% protein) abbreviated as DNAse I, was purchased from Sigma-Aldrich (St. Louis, USA). Restriction endonucleases DpnI, BamHI and NcoI and their corresponding 10x buffers were obtained from New England Bio Labs (USA).

4.2.1.2 Plasmids and host cells

The starting material for the design of the constructs was a pBP2008-lpPOX clone kindly provided by Roche Diagnostics GmbH (Penzberg, GER). It contained the cDNA of lpPOX triple mutant P178S/S188N/A458V. In order to simplify purification, a His3-tag was inserted by Ro- che between the three starting amino acids MVM and the following amino acids KQTK... compared to the original lpPOX wild type (Risse et al. 1992b). Sequence verification of this quasi- wt was done and a overview of the multiple cloning sites was outlined by Nagel (2004) on p105. The pET32 plasmids for expression as a thioredoxin-tagged fusion protein and the pET28 vector for expression as His-POX were purchased from Novagen® (USA). For amplification of the E. coli XL2-Blue plasmids, cells provided by Stratagene (Am- sterdam, NED) were used. These cells are able to process unmethylated DNA which is generated in the mutagenesis procedure. E. coli DH5-alpha cells provided by Invitrogen (USA) were used also for plasmid amplification. For protein expression under a controlled induction rate, E. coli strain BL21-SI™ from Invitrogen™ (Carlsbad, USA) served as host cells. They contain a stable integrated chromosomal insertion of the T7 RNA polymerase gene under control of the proU promoter, which allows for a fine tuned protein expression rate by induction with variable amounts of sodium chloride as described above.

96 4.2 Materials and Methods

4.2.1.3 Buffers and solutions

The lysis buffer for protein extraction was a potassium phosphate buffer (200 mM; pH=6.0) containing imidazole 10 mM, Triton-X 100 0.1%, PMSF 0.1 mM and a small amount of DNAse

(EC 3.1.21.1). The purification buffers for the first purification step on the chelating NiSO4-column were potassium phosphate buffers (200mM, pH=6.0). Loading buffer A contained additional 10mM imidazole to prevent unspecific binding, and elution buffer B contained additional 500mM imidazole. Purification buffer C for the second purification steps was a potassium phosphate buffer (200 mM, pH=6.0). The 10x cofactor equilibration solution was a potassium phosphate buffer (200 mM, pH=6.0) containing additional ThDP 100 mM, FAD 10 mM and MgSO4 100 mM. Reaction solutions for the activity assay are prepared 24h before use. Reaction solution I contained horseradish peroxidase 25 U/ml and 4-amino-antipyrine (4-AAP) 75 mM in water. Reaction solution II contained sodium-3,5-dichloro-2-hydroxy-benzolsulfonate (DHS) 52.8 mM in water adjusted to pH=7.0. Reaction solution III was an aqueous magnesium sulphate solution (92.7 mM). However, in experiments, where cofactors are not added during activity measurement, reaction solution III was replaced by pure water. The substrate solution contained potassium phosphate 0.25 M, imidazole 250 mM and pyruvate 125 mM in water adjusted to pH=7.0. All chemicals for the reaction solutions and the enzyme horseradish peroxidase were purchased from Fluka (Buchs, SUI). The reaction solutions were stored for 24h in the refrigerator (at +2° up to +8°C).

4.2.1.4 Media

LB medium (Luria-Bertani medium) was prepared by mixing 10 g Bacto™ Trypton and 5 g Bacto™ Yeast Extract (both provided by Becton Dickinson & Co., USA) and 8 g sodium chloride (provided by Fluka, SUI) in 1000 ml purified water (Elix3 reverse osmosis equipment provided by Milipore). The media were sterilised by autoclaving 20 minutes at 121°C and stored at room temperature in the dark. LBON medium (Luria-Bertani medium with sodium chloride omitted) was prepared by mixing 10 g Bacto™ Trypton and 5 g Bacto™ Yeast Extract (both provided by Becton Dickin- son & Co., USA) in 1000 ml purified water. The pH-value was adjusted to 7.0 with NaOH. The medium was sterilised and stored in the same way as LB medium, at room temperature in the dark. If required, the LB and the LBON media were supplemented with appropriate antibiotics directly before use.

97 4 Expression and Purification of lpPOX

LB-agar plates or LBON-agar plates were produced by addition of 14% (m/m) Bacto™-Agar (Becton Dickinson & Co., USA) to hot LB- or LBON medium respectively and supplemented with appropriate antibiotics. Agar plates were stored between +2° and +8°C.

4.2.2 Methods

4.2.2.1 Cloning of thioredoxin-tagged lpPOX

The construction of thioredoxin-tagged lpPOX is described in Nagel 2004 (p119f). The pET32b plasmid containing the lpPOX-Trx fusion protein contains the following characteristic sections:

A N-terminal fused thioredoxin tag (11,5 kDa Novy et al. 1995), a spacer encoding for a His6- tag, a thrombin cleavage site and a S-tag, and the lpPOX quasi-wt protein (66 kDa calculated by Risse et al. 1992a) N-terminal supplemented with 3x His between the third and fourth amino acid of lpPOX (as described in 4.2.1.2). The pET32b vector also carries the T7lac promoter, T7 transcription terminator, lacI gene, pBR322 origin of replication, f1 origin for single-stranded plasmid production, and bla gene for ampicillin resistance.

4.2.2.2 Cloning of the His-POX

The pBP2008-lpPOX plasmids containing the lpPOX-quasi-wt-gene were extracted out of an overnight culture of E.coli cells provided by Roche and purified by a GenElute™ Plasmid Mini- prep Kit (Sigma-Aldrich, SUI). In a first step, the NcoI restriction site at base pair 791 was erased using a QuickChange™ site-directed mutagenesis kit provided by Stratagene (Amsterdam, NED) using PCR. The plasmid was amplified with mutagenic primer no. 1 and 2 (see table 4-1) and Pfu Turbo polymerase. PCR conditions were: 1x (95°C for 30 sec); 16x (95°C for 30 sec, 55°C for 1 min, 68°C for 6 min); 1x(68°C for 1 min). Afterwards, parental DNA strands were digested with 20 units of DpnI for 60 min at 37°C. The remaining circular, nicked dsDNA was transformed into competent XL2-Blue cells. Sucessfully transformed cells were selected overnight on ampicillin containing (0.1 mg/ml) LB-agar-plates and proliferated overnight in ampicillin containing (0.1 mg/ ml) LB-medium. The plasimids were extracted by a GenElute™ Plasmid Miniprep Kit (Sigma- Aldrich, SUI).

98 4.2 Materials and Methods

Table 4-1: Primers used for plasmid construction: Primer no. Sequence 1 5´-AAATTCCAGT CGATTTACCC TGGCAACAGA TTT-3´ 2 5´-AAATCTGTTG CCAGGGTAAA TCGACTGGAA TTT-3´ 3 5´-ACACAGGAAA CAGAATCCAT GGTTATGCAT CA-3´ 4 5´-TGATGCATAA CCATGGATTC TGTTTCCTGT GT-3´

In a second step, a new NcoI restriction site was inserted at base pair 264 of the pBP2008-lp- POX plasmid directly in front of the lpPOX coding sequence. The PCR and selection procedures were the same as those outlined previously using mutagenic primers no. 3 and 4 (see table 4-1). After digestion with NcoI and BamHI, the lpPOX insert was ligated with Ligase T4 (New England Bio Labs, USA) into the multiple cloning site of the pET28a vector (provided by No- vagen®, USA). These new plasmids containing the lpPOX gene controlled by a T7 promoter were transformed into competent DH5-alpha cells (Invitrogen, USA). Sucessfully transformed cells were selected overnight on LB-agar-plates containing kanamycin (0.05 mg/ml) and proliferated in an overnight culture in LB-medium containing kanamycin (0.05 mg/ml). Ligation was controlled through agarose gel electrophoresis where new plasmids with around 7370 bp indicated successful ligation (data not shown). The plasimids were extracted out of DH5-alpha™ by a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, SUI), taken up in water and transformed into BL21-SI™ cells for protein expression. Sucessfully transformed cells were selected overnight on LBON-agar-plates containing kanamycin (0.05 mg/ml) and proliferated in an overnight culture in LBON-medium containing kanamycin (0.05 mg/ml).

4.2.2.3 Transformation with calcium chloride method

Competent cells are produced using common methods such as those described by Nagel 2004, p113. About 100 ng of plasmid DNA was added to one aliquote (60 µl) of directly unfreezed competent cells and the mixture was held on ice for 20 minutes. After a heat shock of exactly 60 seconds at 42°C and subsequent cooling on ice for 120 seconds, 200 µl of the corresponding medium (LB or LBON) containing 10 mM CaSO4 and 10 mM KCl was added. The cells were incubated for 1h at 37°C on a shaker. Finally, the successfully transformed clones were selected overnight on LB-agar plates supplemented with the corresponding antibiotics.

99 4 Expression and Purification of lpPOX

4.2.2.4 Sequencing

The DNA sequencing kit containing BigDye Terminator v3.0 Cycle Sequencing Ready Reac- tion with AmpliTaq DNA polymerase as well as the template suppression reagent were bought from Abi Prism Applied Biosystems (USA). The final cloned plasmid was identified by restriction digestion on agarose gel and by automated sequencing using the above mentioned sequencing kit and the sequencing primers listed in table 4-2.

Table 4-2: Primers used for plasmid sequencing: Primer no. Sequence 5 (T7fw) 5´-TAATACGACT CACTATAGGG-3´ 6 (Sq4fw) 5´-GAACTACTGG GATGAACATG GAT-3´ 7 (Sq6fw) 5´-GGCATTGGAG CTCGTAAGGC TGG-3´ 8 (Sq2fw) 5´-ATACGCGTTA TTTCTTACAA ATTG-3´ 9 (SqErw) 5´-TAGGCTTCTT TCCCTAATCG CAA-3´

4.2.2.5 Expression

Overnight cultures were prepared as follows; 25 ml of LBON medium containing 50 µg/ml kanamycin were inoculated with the corresponding glycerol stock culture and then incubated overnight on a shaker (about 16 hours) at 37°C. The following day, one litre of LBON medium was inoculated with a fresh 25 ml overnight culture and incubated on a shaker during a few hours at

37°C up to an OD600 > 0.6. At this growth rate, the culture temperature was reduced to 30°C and expression was induced by addition of 550 - 600 mmol/l sodium chloride in solid form. Af- ter 24 hours of expression the cells were harvested by centrifugation (3000 g, 10 min, at 4°C). The resulting pellets were kept at -80°C until reconditioning.

4.2.2.6 Preparation of soluble cell fraction

The whole procedure was carried out on ice. The bacteria pellet was resuspended in 25 ml ice cold lysis buffer (200 mM phosphate, 10 mM imidazole, 0.1% Triton-X 100 (a non ionic detergent for membrane solubilisation), phenylmethylsulphonyl fluoride (PMSF; a serine protease inhibitor). One tip of a spatula (about 100 mg) of DNAse was added to avoid viscosity problems. For further preparation, the cells were disrupted during two cycles using a precooled

100 4.2 Materials and Methods

French press at 1500 bar inner pressure. Subsequently the lysate was clarified from insoluble cell fragments by centrifugation (10´000 g, 30 min, at 4°C), then filtered (0.45 µm) and either used immediately or stored frozen at -20°C.

4.2.2.7 Purification

The filtrated crude extract was applied to a 5 ml HiTrap chelating column charged with nickel ions (Amersham Biosciences, GBR). The purification procedure using the Ni-HighTrap and Su- perdex columns was carried out with a Äkta™ protein purifier system (Amersham Pharmacia Biotech). The column was washed with a minimum of 10 ml of buffer A to remove of unspecific bound proteins. The lpPOX was then eluted in a first experiment with a continuous gradient with elution buffer B (slope of imidazole increase = 6.1 mM imidazol per ml) and later on with a adjusted step gradient (slope of imidazole increase during the first 60 ml = 1.4 mM imidazol per ml; slope of imidazole increase during the next 25ml = 16.3 mM imidazol per ml). Elution profiles were monitored by UV absorbance at 280 nm. The lpPOX appeared at about 55 to 78 mM imidazole. Peak fractions were pooled and verified by SDS-PAGE and Coomassie staining. The pooled fractions were first adjusted with dilution buffer C to a concentration of 111 µg/ml and further complemented with cofactors to a concentration of 100 µg/ml by addition of equilibration buffer 10x. The equilibration with cofactors was accomplished during 30 minutes on ice. After that step, the samples were concentrated at 4°C to 2.5 ml by using Centriprep® centrifugal filters with a cut off = 30 kDa (Millipore, USA). LpPOX proteins were further purified by size exclusion chromatography using a Super- dex 200 FPLC column at a flow rate of 0.5 ml/min, using buffer C. The fractions were pooled and verified by SDS-PAGE and Coomassie staining. In order to optimise the rate of yield, the second purification step was repeated. The equil- ibrated and concentrated fractions of the first purification step were desalted, exchanged into a phosphate buffer of the desired pH-value and cleaned of low molecular weight material such as cofactors or imidazole on PD10 Sephadex™ desalting columns (Amersham Biosciences, GBR). The elution was done with 3.5 ml of dilution buffer C.

4.2.2.8 Determination of Protein Concentration

Protein concentrations of flavoproteins cannot be determined by applying the common method of near UV absorbance at 280 nm due to the interference to strongly absorbing cofactors. There-

101 4 Expression and Purification of lpPOX fore, the Bradford method for protein quantification was applied in this work (Kruger 1996, Bradford 1976). A Quick Start Bradford Protein Assay for the determination of protein concentrations was supplied by BioRad (USA). The method was calibrated against a purified standard of lpPOX determined by a spectrophotmetric method independent of flavin-cofactors described in Risse et. al (1992a) or in Pajot (1975). Protein quantification was accomplished by mixing 10 µl of protein sample, 790 µl of water and 200 µl of BioRad Bradford reagent. After incubation for 10 minutes at room temperature, the absorption was measured at 595 nm on an Uvikon spectrophotometer. The 2 concentration was calculated using an extinction coefficient ε595 = 0.0533 (cm /µg).

4.2.2.9 Activity Assay

The principle of this acivity assay, which measures the catalytic activity of pyruvate oxidase, is based on the product of the enzyme reaction (eq. 1-1), namely hydrogen peroxide. In order to monitor the ongoing produced amount of hydrogen peroxide in situ, a second enzyme reaction was coupled to the lpPOX reaction. Horseradish peroxidase (HRP) (EC. 1.11.1.7) deoxidises hydrogen peroxide via oxidation of an appropriate electron donor (see eq. 2-1). In the activity assay, this reaction is coupled to the oxidative connection of 4-amino-antipyrine (4-AAP) and 3,5-dichloro-2-hyroxy-benzolsulfone acid (DHS) to a chinoneimine dye. (see figure figure 4-2).

HO O N H C O S OH 3 N O Peroxidase H O 2 H2O HCl 2 2 N H C O Cl H C N 3 N 3

chinoneimine Cl DHS H3C NH2 Cl dye 4-AAP O S O O OH

Figure 4-2: Second reaction of the activity assay. HRP oxidises 4-amino-antipyrine (4-AAP) and 3,5-dichloro- 2-hyroxy-benzolsulfone acid (DHS) to a chinoneimine dye under deoxidation of hydrogen peroxide. (Vasudevan and Li 1996, Allain et al. 1974). HRP is a widely used (indicator enzyme) label in the development of enzyme linked immuno sorbent assays (ELI- SAs) and in other analytical technologies, such as the biosensors field (Lindgren et al. 2000, Dong and Chen 2002). The HRP is a haem-containing glycoprotein of a molecular weight of 40 kDa, extensively known and investigated (Keilin and Hartree 1951). Its functions in armoracia rusticana (horseradish) are the removal of H2O2, due to environmental stress, the oxidation of toxic reductants and a few other metabolic catalyses. The most abundant HRP isoenzyme consists of 353 amino acids and a minimum of six carbohydrate prosthetic groups that are joined to the protein chain through N-glycosidic links (Clarke and Shannon 1976).

102 4.3 Results

Four different reaction solutions (see paragraph 4.2.1.3) were prepared 24h before processing the activity assay. The reaction mix was prepared immediately before activity measurement (reaction solution I, 40 µl; reaction solution II, 56 µl; reaction solution III, 20 µl and substrate solution 80 µl). The prepared lpPOX solution (4 µl) is added to this 196 µl reaction mix, and the enzymatic catalysis was monitored for 10 minutes at 25°C by measuring the absorption at 517 nm. The activity assay was performed three times with differently prepared pyruvate oxidase solutions and the specific activity was calculated according to eq. 4-1

------∆A 1 U tmin[]V []ml ------specActivity ------= ------× ------K - × mg mg ml V []ml c ------(Eq. 4-1) ε ------E ml µmol

∆Α = Difference in Absorbtion []; t = time [min]; VK = volume assay [ml]; VE = volume enzyme solution [ml]; ε = micromolar extinction coefficient at 517 nm = 13,68 [ml * µmol-1]; c = enzyme concentration [mg * ml-1]

4.3 Results

4.3.1 Expression Vector

The lpPOX gene with additional coding for a 3x His-tag between amino acid no 3 and 4 was cloned sucessfully into the BamH I and Nco I sites of the pET28 plasmid (see figure 4-3). This plasmid carries a gene for kanamycine resistance, which was used for the selection of successfully transformed host cells. It also contains a T7-promoter directly in front of the lpPOX gene.

The initial existing His6-tag, the thrombin cleavage site and the T7-Tag were removed from the pET28a plasmid. Hence the first start codon after the T7-promoter codes directly for the first Met of lpPOX. The pET28a vector also carries the T7lac promoter, T7 transcription terminator, lacI gene, pBR322 origin of replication, f1 origin for single-stranded plasmid production, and kan gene for kanamycin resistance.

Finally, two different constructs were expressed. On one hand the Trx-His6-His3-POX construct on vector pET32b described by Nagel (2004) called Trx-POX. And on the other hand, the His3-POX construct on vector pET28a (see figure 4-3) called His-POX.

103 4 Expression and Purification of lpPOX

lpPOX

MCS MCS T7 promoter f1 origin pET28a- lpPOX 7370 bp lacI

kan

ori

Figure 4-3: Expression vector. pET28a lpPOX construct for overexpression of lpPOX in salt inducible E. coli BL-SI cells. black = MCS (multiple cloning site); white = genes on the pET28a plasmid: kan (kanamycin resistance gene); grey = insert (lpPOX gene, hatched = uncoding insert).

The verification by seqencing confirmed the exact lpPOX gene inserted in the pET28a plasmid with only one silent mutation P173P whereas the codon has changed from CCC to CCA.

4.3.2 Protein Expression

The pyruvate oxidase was expressed as described in section 4.2.2 under induction of different concentrations of NaCl in salt inducible BL-SI™ cells. The titration for optimal induction rate showed that NaCl concentrations beneath 500 mM or above 750 mM are inapropriate for protein expression (data not shown). First step purification by Ni affinity chromatography showed that induction with NaCl concentrations of 500 or 550 mM (for Trx-POX, see figure 4-4) or 600 mM (His-POX, see figure 4-5) led to the most efficient protein production and therefore were favoured.

104 4.3 Results

200 kDa - 116 kDa - 97 kDa - 66 kDa -

45 kDa -

31 kDa -

front - M 1 2 3 4 5 6 7 c.e.c.e. ppt c.e.ppt c.e. ppt 100 200200 500500 750 750 NaCl conc. (mmol/l)

Figure 4-4: 10% SDS-PAGE of the titration over different NaCl concentrations for inducing the SI-cells. Proteins are visualised by Coomassie blue staining. The molecular weight of standard protein markers are indicated (lane M); c.e: crude extracts; ppt.: pellets; lane 1: crude extract at an induction with 100 mM NaCl. Lanes 2, 4 and 6 show the crude extracts containing the soluble heterologous and homologous proteins out of the host cells at induction with 200, 500 and 750 mM NaCl respectively. Lanes 3, 5 and 7 show the corresponding pellets consisting of all insoluble components of the host cells. The highest rate of soluble to insoluble lpPOX is produced at an induction rate of 500 mM NaCl (see lanes 4 and 5, bands at 85.5 kDa). At lower induction rates no overexpression can be observed. At higher NaCl concentration the ratio of soluble to insoluble Trx-POX was unfavourable.

105 4 Expression and Purification of lpPOX

a) b)

200 kDa- -200 kDa 116 kDa- 97 kDa- -116 kDa -97 kDa 66 kDa- -66 kDa 45 kDa- -45 kDa

31 kDa- -31 kDa front- -front M 1 2 3 4 5 6 7 8 M NaCl conc. ppt. c.e. ppt. c.e. ppt. c.e. ppt. c.e. (mmol/l) 300300 400400 500 500 600 600

Figure 4-5: 10% SDS-PAGE of the titration over different NaCl concentrations for inducing the SI-cells expressing His-POX performed on two independent gels a and b. Proteins are visualised by Coomassie blue staining. The molecular weight of standard protein markers are indicated (lane M); Lanes 1, 3, 5 and 7 show crude extracts (c.e.), containing the soluble heterologous and homologous proteins out of the host cells. Lanes 2, 4, 6 and 8 show pellets (ppt.) consisting of all the insoluble components of the host cells. Large amounts of soluble His- POX is produced at an induction rate of 600 mM NaCl (lane 8, protein of 66 kDa marked with an arrow). At lower induction rates, below 500 mM NaCl, no overexpression was observed. At higher expression rates the ratio of soluble to insoluble His-POX did not turn out well (data not shown).

4.3.3 Protein Purification

Ni Affinity Chromatography

The Trx-POX fusion protein containing a His6- and a His3-tag was purified by Ni affinity chromatography as described in section 4.3.3. The polyacrylamide gel electrophoresis (SDS PAGE) performed with different fractions after the first purification with a metal chelating NiSO4-column shows the soluble expressed Trx-POX fusion protein at 85.5kDa (see figure 4-6, lane 3 to 6). The outcome of different runs of protein expression and the first purification steps were on average about 5.9 mg total protein for the Trx-tagged lpPOX induced with 550 mM NaCl (n=3).

The achieved purity after one-step purification with a chelating NiSO4-column was estimated at about 95% (see fig figure 4-6). The thrombin digestion used to cut the Trx-tag from the lpPOX was successful. However, a separation of His-POX by size exclusion chromatography was not possible. The purification

106 4.3 Results of acceptable amounts of cleaved protein, in particular, appeared to be impossible (data not shown). The protein containing only the 3x His-tag was also purified on a Ni affinity column as mentioned above (see figure 4-7). The outcome of different runs of protein expression and the first purification steps were on average about 6.7 mg total His-POX expressed at 600 mM NaCl (n=3). The yield of purified lpPOX after the first purification step amounted from 2.1 to 9.8 mg per litre culture medium.

200 kDa -

116 kDa - 97 kDa - 66 kDa -

45 kDa -

31 kDa -

front - M 1 2 345 6

Figure 4-6: 10% SDS-PAGE of soluble Trx-POX fusion protein after first purification step Proteins are visualised by Coomassie blue staining. The molecular weight of standard protein markers are indicated (Lane M), Lane 1 shows the preliminary rinse with lots of unbound proteins. Lane 2 shows the first fractions during the gradient at about 7-16% of buffer B (corresponds to about 50 mM imidazole). Lane 3-6 show the collected fractions from the column at about 100 mM imidazole. Large bands of Trx-POX-fusion protein monomers at 85.5 kDa and small bands of Trx-POX-fusion protein dimers at about 170 kDa are noticeable.

107 4 Expression and Purification of lpPOX

a) b)

200 kDa - 200 kDa - 116 kDa - 97 kDa - 116 kDa - 97 kDa - 66 kDa - 66 kDa - 45 kDa -

45 kDa -

31 kDa - 31 kDa - front - front - M 1 2 3 4 5 6 M 7 8 9 10 11

Figure 4-7: First purification step (affinity chromatography) of His-POX after induction with 500 mM NaCl (a) and with 600 mM NaCl (b): SDS-PAGE using polyacrylamid-gel 10%, Coomassie blue staining. The molecular weight of standard protein markers are indicated (lane M); lpPOX (65 kDa) was bound to the Ni-column as indicated by the difference between lane 1 and 2 (crude extract and flow-through after the Ni-column) or between lane 8 and 9 (crude extract after filtration and flow-through after the Ni-column). After the forerun (lane 3) and elution of unspecific bound proteins during an increasing imidazole gradient (lane 4, 5 and 10) lpPOX can be eluted completely (lane 6 and 11) at a certain concentration of imidazole.

Equilibration with Cofactors and Desalting

The second purification step was done after equilibration with cofactors by size exclusion chromatography on a Superdex column for the Trx-fusion protein and for the His-POX (section 4.2.2.). The achieved purity was estimated at over 95% (see figure 4-8). The achieved specific activities after equilibration with cofactors and the second purification step were re- spectable; 3.69 U/mg for the fusion protein* and 1.47 U/mg for His-POX (*Specific activity for the fusion protein was calculated taking into account that lpPOX is only 78% of the mass of the whole fusion protein). Unfortunately, the recovery after this chromatographic purification was only about 6% for the Trx-tagged fusion protein and about 13.6% for His-POX. Only a few hun- dred micrograms of active protein was obtained. A second purification step was required for the removal of unbound cofactors resulting from equilibration with cofactors and for the removal of incorrectly folded protein (note: in the Ni-affinity chromatography also incorrectly or completely unfolded lpPOX has been purified.)

108 4.3 Results

By applying PD10 columns for the second purification step (section 4.2.2.), purity was also estimated at over 95%. The achieved specific activities were good (3.36 U/mg). The recovery after this second purification procedure was remarkable 97%, resulting not only in micrograms, but in milligrams of pure active protein (see figure 4-9).

a) b)

220 kDa - 220 kDa - 116 kDa - 116 kDa - 97 kDa - 97 kDa - 66 kDa - 66 kDa -

45 kDa - 45 kDa - 31 kDa - 31 kDa - 22 kDa - front - front - M 1 2 3 4 M 5 6 7 activity - + + +/- - activity + + + +

Figure 4-8: Second purification step (size exclusion chromatography) of Trx-POX fusion protein (a) and His-POX (b): SDS-PAGE using polyacrylamid-gel 10%, Coomassie blue staining. The molecular weight of standard protein markers are indicated (lane M); lanes 1 to 4 show different fractions from the Superdex column, while the fraction of fusion protein in lane 2 is highly active and the fraction in lane 4 rather inactive. Lane 5 shows the protein solution after equilibration with cofactors which was loaded in the size exclusion column. Lane 6 and 7 are different fractions containing active His-POX eluted from the Superdex column. LpPOX appears rather pure on both gels (> 95%) and is present in sufficient amounts.

97 kDa - 66 kDa - 45 kDa -

31 kDa -

21 kDa -

14 kDa - front - M 1 2 3 4 5 6 activity + + + + + +

Figure 4-9: Second purification step (PD10 column) of His-POX: SDS-PAGE using polyacrylamid-gel 10%, Coomassie blue staining. The molecular weight of standard protein markers are indicated (lane M); lane 1 to 6 show different fractions from the PD10 column (each 0,5 ml). LpPOX (65 kDa) appears pure (> 95%), active and is present in sufficient amounts in a total volume of 2,5 ml.

109 4 Expression and Purification of lpPOX

4.4 Discussion

4.4.1 Expression System

The suitability of the choosen expression system (E. coli) after implementing the right induction rate was confirmed. Indeed, the implementation of finely adjusted induction rates was crucial for expression of soluble and active protein in acceptable amounts. The undemanding host organism and the easily controllable induction system have been aproved by numerous runs of expression cycles. In this respect, the reports written by Schumacher and Möllering (1990), Risse et al. (1991) and Wille et al. (2003), which describe lpPOX expression in E. coli, can be confirmed, even though the purification processes mentioned in these references are rather different. The expression of Trx-fusion protein was not essential to the expression of soluble and active lpPOX. Two theories argue against the strategy of fusion proteins; firstly, two fractions of soluble fusion proteins separable using size exclusion chromatography are produced. One of them was highly active and the other, inacitve (see gel (a) on figure 4-8). With regard to His- POX, there was only one fraction of highly active protein visible after elution from PD10 columns (see gel (b) on figure 4-8). The second argument against the fusion protein strategy was the insufficient separation of active pyruvate oxidase after cleavage of the added tag by thrombin. It can therefore be concluded, that His-POX is most effectively expressed in E. coli BL-21 cells under a controlled induction rate. The published expression protocols for lpPOX based on E. coli (Schumacher and Möller- ing 1989, Risse et. al 1991, Schumacher and Möllering 1992) did not include any detailed data on the capacity of the expression systems. Schumacher et al. (1990) reported that nearly 50% of total soluble protein expressed by E. coli were pyruvate oxidase. Wille et al. (2003) described a rather time consuming expression and purification protocol based on E. coli cells, including a step with cofactor equilibration and dialysis, but no data about the yield were reported. In their original publication, Sedewitz et al. (1984) described a yield of 16 mg active lpPOX purified out of 10 litres Lactobacillus plantarum culture. Even though there was no direct comparison possible to other expression protocols, the developed procedure can be estimated as comparable to other protein expression protocols based on E. coli.

110 4.4 Discussion

4.4.2 Equilibration with Cofactors and Purification

After the development of appropriate and accurate expression protocols, different purification strategies were tested. The aim was to reach the target for yield and purity with the minimum number of steps and the simplest possible purification protocol. The intention was always to optimise purification towards a fast and simple process, which can be scaled up without too many modifications. By implementing the abovementioned methods for the determination of protein concentration and specific activity, fast and reliable analytical assays were selected to follow the progress of purification and to assess its effectiveness. The common three step purification process consisting of capture phase, intermediate purification to remove most of the bulk impurities and polishing phase has been well established (Amersham Pharmacia Biotech 1999). An additional step of cofactor equilibration as described in a similar form by Risse et al. (1992a) has been aproved. Because of the elevated loss of protein during the second step of purification (gel filtration), purification was further improved by the introduction of a step gradient elution procedure on the HiTrap™ chelating NiSO4-column. This new purification procedure led to a higher resolution between the protein fractions. In this way, the second purification step by size exclusion chromatography on a superdex column was necessary to achieve higher specific activity but low protein yields can be omitted. The purity of our protein was estimated at about 95% (see figure 4-6), although the gel depicted the presence of other weak bands. The high purity of about 95% is sufficient for the following physico-chemical characterisations or would be sufficient even for x-ray crystallog- raphy experiments. Only therapeutical use or in vivo studies require extremely high pure protein samples of more than 99%. The purity of lpPOX produced and purified in our laboratory as described was in the same range as the protein sold by Roche; >90%. The new strategy allowed much faster production of highly active lpPOX and generated higher yields with less time consumption and lower costs. This improvement is very important for any future scale up, needed for biosensor application, because it allows a reduction of time and materials.

111 4 Expression and Purification of lpPOX

112 5 Stabilisation of the Quaternary Structure and Cofactor Sites

fter the development of an appropriate expression and purification protocol for producing AlpPOX in sufficient amounts, stabilisation techniques could be experimentally tested in the laboratory. The first stabilisation experiments with lpPOX containing A131I or V141C mutations compared to the triple-mutant (P178S; S188D; A425V), failed (Nagel 2004). These mutants should stabilise the tetrameric state by increasing the interactions between the monomers. Another promising mutant regarding tetrameric stabilisation was tested in this chapter. As explained in section 3.5.2.1 the preferred approach of lpPOX stabilisation is an improvement of cofactor binding at the cofactor binding site. This chapter summarises the experiments that were made in the field of cofactor binding and tetrameric stabilisation.

5.1 Introduction

5.1.1 Stabilisation at the Tetrameric Interface

Because two stabilising mutations in the quasi-wt produced by Roche (P178S and S188A) increased the interactions between the different subunits in the tetrameric state (Muller et al. 1994), further investigations were done in this direction. Structure-based analysis pointed out several regions of the enzyme where possible inter- ventions could be performed. One region was at the subunit interface in which water molecules have been found, namely from Pro178 to Asp198 (loop connecting the CORE and the FAD domains) and from Lys317 to Asp325 (region in the FAD domain next to the loop described previously). The amino acid substitution in this region will allow an increase in the complementarity of the interface. Between αII and αIV or αI and αIII respectively, mutation of Leu323 to Gln allows the formation of a new favourable contact point between Gln323-ND2 and Thr191´-O of the second subunit (see figure 5-1).

113 5 Stabilisation of the Quaternary Structure and Cofactor Sites

a) b)

Leu323 Gln323

2.78 Å

Thr191´ Thr191´

Figure 5-1: Proposed mutation L323Q for better subunit interaction in lpPOX; a) shows the neighbouring site of quasi-wt and b) the proposed mutant of quasi-wt. On both pictures the ThDP domain (aa no 323) of the first monomer is shown in yellow and the loop of the second monomer in purple. The interacting amino acids are coloured in white. The new hydrogen bond in mutant L323Q is marked as dotted white line (2.78 Å) between Gln- ND2 and Thr191´-O.

5.1.2 Effects of Cofactors on Protein Stability

The first step of lpPOX inactivation is the loss of FAD molecules as outlined in paragraph 3.4.2.3. During the dissociation of the tetrameric state ThDP is also released. The weakest point or so called „Achilles heel“ of lpPOX is the dissociation of the tetrameric state accompanied by the release of cofactors as mentioned. The first temperature-stress-experiments with the triple-mutant lpPOX with or without the cofactors FAD, ThDP and Mg2+ showed that an absence of cofactors is associated with a decrease of tolerated temperature (Nagel 2004, p. 125ff). This leads to a further working hypothesis, namely that the enzyme can be further stabilised by increasing the interactions between the cofactors and the protein. Cofactors are important ligands fastening the chemical reactions within the active site of enzymes. The role of cofactors in protein folding or protein stability is scarcely investigated. In principle, cofactor binding can influence correct folding of native proteins acting as a nucleation site for 3D-structure formation. Such cofactors could bind to an unfolded polypeptide, reduce the conformational freedom and thereby speed up the folding process. For example, apo-cyto- chromeC only folds upon incorporation of its heme cofactor (Fisher et al. 1973). In other cases, ligands do not interact with unstructured protein states but become incorporated

114 5.1 Introduction during the final stages of protein folding and therefore influence the protein 3D-structure. In this way, the tertiary structure of a protein can depend on binding of cofactors. For instance, Steensma et al. (1998) put forward that unfolding of Azotobacter vinelandii holoflavodoxin only occurs after release of the FMN cofactor. There are other enzymes where cofactors stabilise the correct multimeric assembly of subunits and therefore react as stabilisers on the level of quaternary structure. For example, inactive monomeric apo-lipoamide dehydrogenase (EC 1.8.1.4) forms active dimers after incorporation of FAD (Berkel et al. 1991). Another example is the dihydroorotate dehydrogenase (EC 1.3.99.11) of Lactobacillus lactis (llDHOD) where the FAD cofactor stabilises the tetrameric structure of the native enzyme (Jensen et al. 1999). In that study, the stabilisation effect of FAD had been shown by electrophoresis under nondenaturing conditions. While the apoenzyme without FAD was torn apart into different small subunits, the apoenzyme in the presence of FAD showed clear bands indicating the precence of a single species, namely the tetrameric holoenzyme. Another example, where a dependence of ThDP binding and oligomer formation was described is acetohydroxyacid synthase (EC 2.2.1.6), homologous to pyruvate oxidase and other members of the ThDP-dependent enzyme family (Bar-Ilan et al. 2001, Kim et al. 2004). Yet another example is one of the already introduced mutations (A458V); the quasi-wt stabilises lp- POX by means of increased binding of ThDP. In a first step, a systematic search of sites with less favourable interactions between the four subunits of lpPOX as well as between subunits and cofactors based on published structures was carried out by calculations and visual inspection.

5.1.3 Selection of Promising Mutations Concerning Cofactor Binding

Based on the conclusion of paragraph 3.4.2.1 indicating that insufficient cofactor binding, especially that of FAD, affects lpPOX stability, different mutations in the FAD and ThDP binding sites were selected for enzyme stabilisation. At the FAD binding site, mutating Ile221 into a Thr (I221T) will generate new hydrogen bonds between Thr221-OG and the oxygen (AO1) of the alpha-phosphate of FAD (see figure 5-2).

115 5 Stabilisation of the Quaternary Structure and Cofactor Sites

a) b)

Ile221 Thr221 2.84 Å

FAD FAD

Figure 5-2: Proposed I221T mutation for better FAD binding in lpPOX; a) shows the FAD binding site of quasi-wt and b) the proposed mutant of quasi-wt. On both pictures FAD is shown in white and the protein in yellow. At the FAD binding site the Ile221->Thr mutation is assumed to generate new hydrogen bonds (2.84 Å) between Thr221-OG and the oxygen (AO1) of the alpha-phosphate of FAD.

Another important point for a possible intervention to further stabilise the enzyme is the presence of water molecules within a cavity of the protein in close contact with ThDP. At this site, the introduction of a Lys substituting Ala373 will allow the replacement of a water-mediated hydrogen-bond involving the oxygen from the β-phosphate of ThDP by a direct hydrogen bond with the amine (NZ) of Lys373.

5.2 Materials and Methods

5.2.1 Chemicals and Enzymes

Thrombin (EC 3.4.21.5) from bovine plasma was bought from Sigma-Aldrich (St. Louis, USA). Urea and ethanol were from Hänseler (Herisau, SUI) whereas Tris buffer and sodium acetate trihydrate were from Fluka (Buchs, SUI). All other materials were purchased as described in section 4.2.1.1.

116 5.2 Materials and Methods

5.2.2 Methods

5.2.2.1 Mutagenesis of Proposed Stabilising Mutants

The pET32b plasmids containing the lpPOX-wt-gene combined with the Trx-tag were extracted out of an overnight culture of E.coli DH5alpha cells produced as described in section 4.2.2.1 and purified by a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, SUI). I221T, A373K and L323Q mutations were introduced into the lpPOX genome using a QuickChange™ site-directed mutagenesis kit provided by Stratagene (Amsterdam, NED) using PCR. The plasmid was individually amplified with mutagenic primers no. 10 to 15 (see table 5-1) and Pfu Turbo polymerase. PCR conditions were: 1x(95°C for 30sec); 16x(95°C for 30sec, 55°C for 1min, 68°C for 7min 45sec); 1x(68°C for 1min). PCR results were verified on agarose gels. Following this, parental DNA strands were digested with 20 units of DpnI for 60min at 37°C. The remaining circular, nicked dsDNA was transformed into competent XL2- Blue cells using the described calcium chloride method (see paragraph 4.2.2.3) using 50ng of plasmid and a heat shock time of exactly 120 seconds at 42°C. Sucessfully transformed cells were selected overnight on ampicillin containing (0,05 mg/ml) LB-agar-plates and allowed to grow overnight in ampicillin containing (0,05 mg/ml) LB-medium.

Table 5-1: Primers used for mutagenesis: mutated codons are underlined Primer no. Sequence 10 5´-CCGGTGAATA AATGATACCG TGACCTCGAG CATTCCGACC (I221Tfwd) ATTTC-3´ 11 5´-GAAATGGTCG GAATGCTCGA GGTCACGGTA TCATTTATTC (I221Trev) ACCGG-3´ 12 5´-CGTCCTTCCC GGAAATGTTT TTATAGTTCA CGATGCACGC (A373Kfwd) C-3´ 13 5´-GGCGTGCATC GTGAACTATA AAAACATTTC CGGGAAGGAC (A373Krev) G-3´ 14 5´-GTATTTTGTC TATAACGCCA TGTTCGACTA CGTGTTTTCT (L323Qfwd) GCG-3´ 15 5´-CGCAGAAAAC ACGTAGTCGA ACATGGCGTT ATAGACAAAA (L323Qrev) TAC-3´

5.2.2.2 Sequencing of Different Mutants

Different clones, resulting from ampicillin selection, were verified by automated sequencing using the abovementioned sequencing kit (see 4.2.2.4) and the sequencing primers listed in

117 5 Stabilisation of the Quaternary Structure and Cofactor Sites table 4-2. For the PCR reaction, 500 ng plasmid DNA, 2 pmol primer and 4 µl big dye reagent were pipetted into a PCR-tube. Bidest. water was added to an end volume of 10 µl. The PCR conditions were: 1x(96°C for 60sec); 30x(96°C for 10sec, 50°C for 5sec, 60°C for 4min); 1x(60°C for 5min). DNA precipitation was done by adding 15 µl bidest. water and 1 µl sodium acetate 3 M pH=5,2 to the processed PCR reaction mix. Then the mixture was transferred into an Eppendorf tube and 45 µl cooled ethanol 100% (-20°C) was added, mixed and centrifuged at 4°C, 14000 rpm for 10 minutes. The supernatant was discarded and the DNA remaining in the tube was washed twice with 45 µl cooled ethanol 70% (-20°C) and centrifuged using the abovementioned procedure. The purified DNA was dissolved in TSR buffer from the sequencing kit, further processed following the sequencing protocol and analysed on a ABI PRISM 310 genetic analyser (Applied Biosystems).

5.2.2.3 Transformation into Expressing Cells

The verified plasmids were extracted out of XL2-blue™ cells by a GenElute™ Plasmid Mini- prep Kit (Sigma-Aldrich, SUI) taken up in water and transformed into BL21-SI™ cells for protein expression (see paragraph 4.2.2.3). Sucessfully transformed cells were selected at 37°C overnight on LBON-agar-plates containing ampicillin (0,05 mg/ml). Selected cells were grown in an overnight culture on LBON-medium containing ampicillin (0,05 mg/ml) at 37°C.

5.2.2.4 Expression and Purification

Expression and purification were performed as described in paragraphs 4.2.2.5ff, based on the three step purification protocol with Ni-affinity chromatography, equilibration with cofactors and size exclusion chromatography. The purification protocol was not developed to the final stage at this time of the project.

5.2.2.5 Stability Testing

Mutants which showed acceptable high activity (similar to the quasi-wt) in a first activity assay (see 4.2.2.9) were tested for stability in a temperature-stress test developed in our laboratory. By measuring the remaining activity of the enzyme over time at different temperatures, it is possible to estimate the thermodynamic stability of the protein. Samples containing lpPOX protein 100 mg/ml in potassium phosphate buffer (200 mM, pH=7) were incubated for 10 minutes at different temperatures between 25 and 75°C. Temper- ature stress tests were performed in thermoshakers and were stopped by cooling the samples for

118 5.3 Results

5 minutes on ice. The specific activity assay was performed afterwards at 25°C as described in section 4.2.2. where remaining activity was measured under standard conditions.

Influence of the Thioredoxin-tag on lpPOX Activity

Thioredoxin, as a low molecular weight oxidoreductase, may potentially influence the activity assay in either a positive or negative way. To disprove this hypothesis, pure thioredoxin origi- nated from E. coli (a gift from Andreas Limacher ETH Zürich) was added to the activity assay in the corresponding amount of 0,086 µg per assay. The assay was performed with that amount of thioredoxin, and again a second time with pure lpPOX protein supplemented with same amount of thioredoxin. The tests were done in triplicate.

5.3 Results

5.3.1 Mutagenesis and Transformation

The three mutations mentioned above were successfully introduced at DNA level into the expression plasmid. Transformation into XL2-blue™ cells was successful in all mutants, namely L323Q, A373K and I221T. Mutations were confirmed by sequencing (see figure 5-3). In order to verify the lpPOX gene, two clones of each mutant were sequenced over the whole lpPOX insert. This sequencing of six complete lpPOX genes showed one additional silent mutation in one of the A373K mutants. Sequencing confirmed CCC instead of CCT according to the codon coding for Pro386. Nevertheless, the chosen strategy revealed itself to be very powerful and can also be used for further mutation steps.

119 5 Stabilisation of the Quaternary Structure and Cofactor Sites

a) Mutation L323Q b) Mutation I221T c) Mutation A373K orig. seq. mut. seq.

Figure 5-3: Assessment of mutagenesis by sequencing; First lines show the interesting base sequences of the Roche lpPOX quasi-wt on DNA-level. The second line indicates the corresponding sequences of the mutated plasmid coding for lpPOX. The codons that define the amino acids which have been mutated are pointed out by black frames. a) CTT is encoding for leucine, whereas ATT is defining glutamine; b) ATT is encoding for isoleucine, whereas ACT is defining threonine; c) GCA is encoding for alanine, whereas AAA is defining lysine.

5.3.2 Expression

After sequencing, the mutant plasmids were transformed into competent BL21-SI™ cells as described in 4.2.2.3 and expressed. Mutant I221T could be expressed in a soluble form according to the protocol outlined in 4.2.2.5 using 500 mM NaCl for induction. Several clones had to be tested and tailor made expressions had to be performed for mutants L323Q and A373K as they did not show any expression using different subclones resulting from the first sequenced clone in the XL2-blue cells. The selection of another clone of mutant L323Q allowed the expression of soluble lpPOX fusion protein at an induction rate of 500 mM NaCl. The second clone of mutant A373K showed overexpression at an induction rate of 500 mM NaCl too. However, the whole lpPOX protein was insoluble and appeared in inclusion bodies. Neither testing of different subclones, different induction rates (from 200 up to 750 mM NaCl), different expression durations nor different resuspending buffers (Tris and phosphate) allowed expression of a soluble, active A373K mutant.

5.3.3 Purification

Protein purification was performed as outlined in paragraph 4.3.3 using affinity chromatography followed by size exclusion chromatography. The size exclusion purification was necessary to increase specific activity. The reason is that the lpPOX active species can be separated from

120 5.3 Results the inactive species during size exclusion chromatography (see figure 5-4).

a) b) 200 kDa- 116 kDa- 97 kDa- 66 kDa-

45 kDa-

31 kDa-

20 kDa- front- M 1 2 3 4 + ++ ± - activity

Figure 5-4: Size exclusion chromatography of L323Q; a) Chromatogram with two peaks resulting from different protein fractions. Blue line: absorption at 280nm, brown line; conductivity. Peak at 10 ml = active lpPOX species; peak at 12ml = inactive species. b) SDS-polyacrylamide gel with 10% polyacrylamide. M: broad range marker (Bio Rad). Lane 2-5 shows a protein of 86kDa. 1: fraction 9 (active), 2: fraction 10 (active), 3: fraction 11 (weak active), 4: fraction 12 (inactive). A similar picture was obtained with the I221T lpPOX.

Soluble expressed protein from the I221T and L323Q mutants was successfully purified using the abovementioned method. The yields of soluble pure protein from the I221T and L373Q mutants were 495 µg and 280 µg protein per litre culture medium, respectively. Despite the numerous experiments for optimising the expression and purification of mutant A373K, it was not possible to obtain soluble active A373K lpPOX. Urea denaturation (8 M) followed by renaturation using dialysis with potassium phosphate buffer (200 mM, pH=6) did not yield enough protein for further analysis. Therefore, activity and stability tests for mutant A373K could not be performed. Thrombin digestion to cleave the Trx-tag was successful for both mutants I221T and L323Q. However, the purification of the mutants after thrombin digestion was not successful because it caused a substantial loss of soluble protein (compare with 4.3.3) (data not shown). This is why the Trx-fusion protein, with seemingly unchanged stability, was used for assessing stability and activity.

121 5 Stabilisation of the Quaternary Structure and Cofactor Sites

5.3.4 Trx Does not Influence the Activity of lpPOX

The addition of thioredoxin to the blank or to a sample containing pure lpPOX only marginally influenced activity. The results were 4.144 U/mg or 4.074 U/mg for the test containing wt lp- POX with or without Trx respectively. Whereas the blanks showed an almost undetectable rest activity 0.0056 U/mg (with Trx) and 0.0039 U/mg (without Trx).

5.3.5 Specific Activity of Mutant Proteins

Specific activities were measured based on the test mentioned in paragraph 4.2.2.9. The results are summarised in table 5-2.

Table 5-2: Specific activity of original triple mutant and further mutated pyruvate oxidases: ¹ Purification Purification by Ni-affinity- Protein was renaturated by Ni-affin- chromatography and size with urea 8M, refolded by ity chroma- exclusion chromatography dialysis and purified by Ni- tography affinity-chromatography Specific Specific Specific Specific Specific activity activity activity activity activity (lpPOX) (fusion pro- (lpPOX¹) (fusion pro- (lpPOX¹) tein) tein) Roche wt 4.07 up to 3.01 U/mg (product specifica- 9.32 U/mg tions: 3.89 U/mg, measuring conditions unknown) Trx-wt 2.9 U/mg 3.69 U/mg 2.03 up to 2.59 up to 4.12 U/mg 5.24 U/mg Trx-mutant I221T 9.57 U/mg 12.19 U/mg 0.82 U/mg 1.04 U/mg Trx-mutant L323Q 2.46 U/mg 3.13 U/mg 0.94 U/mg 1.20 U/mg Trx-mutant A373K ² ² 0.78 U/mg 0.99 U/mg 1) Specific activity calculated for the active component, taking into consideration that lpPOX is only 78.5% of the Trx-fusion protein; 2) amounts of active protein were too low for an exact detection of specific activity

The specific activities of Roche wt and our Trx-wt are comparable. The values for purified and renatured Trx-wt and Roche wt are within the same range. The activity resulting from mutant renaturation experiments were considerably lower than that resulting from standard purification protocols (factor 3 up to 10). I221T and L323Q enzymes showed activity equal or better than that of the wt, whereas no active protein was produced with A373K based on standard purification protocols.

122 5.3 Results

5.3.6 Stability Testing Using Heat Inactivation

The remaining activity after heat inactivation was measured and plotted against inactivation temperatures in figure 5-5 and figure 5-6. Temperature stress tests were done in triplicate and means are shown in combination with standard deviations. Heat inactivation experiments with cofactors added to the sample solution generally showed higher thermal stability than the experiments performed without cofactors. This affirms our new hypothesis; that the enzyme can be further stabilised by increasing the interactions between cofactors and protein. Secondly, the results show the difference in thermal stability between lpPOX with and without the Trx-tag. In both experiments (with or without cofactors) stand alone lpPOX produced by Roche showed significantly higher thermostability than all types of fusion-proteins (p<0,05). In figure 5-5 and figure 5-6 it can be seen that the L323Q mutant shows higher thermostability than the comparable wt in terms of a Trx-fusion protein. For better comparison and statistical analysis, the results of the heat inactivation experiments were further processed to determine the midpoint of inactivation temperature (Td).

140

120 )

100

I 60

40 Activity after Heat Inactivation (% Inactivation Heat after Activity 20

0 40 45 50 55 60 65 70 Inactivation Temperature (°C) Figure 5-5: Heat inactivation curves of Trx-fusion lpPOX quasi-wt and mutants with cofactors added to the sample solution during heat inactivation; Activity after heat inactivation in per cent related to the activity at 25°C is plotted against inactivation temperature. The inactivation experiments were done in triplicate and means are shown as () Trx-lpPOX quasi-wt, (z) mutant L323Q, () mutant I221T, () Roche quasi-wt

123 5 Stabilisation of the Quaternary Structure and Cofactor Sites

120

100 )

Activity after Heat Inactivation (% Inactivation Heat after Activity 20

0 25 30 35 40 45 50 55 60 Inactivation Temperature (°C)

Figure 5-6: Heat inactivation curves of Trx-lpPOX quasi-wt and mutants without cofactors in the sample solution during heat inactivation; Activity after heat inactivation in per cent related to the activity at 25°C is plotted against inactivation temperature. The inactivation experiments were done in triplicate and means are shown as () Trx-lpPOX quasi-wt, (z) mutant L323Q, () mutant I221T, () Roche quasi-wt.

Processing results for Td Determination and Statistical Analysis

The midpoint of inactivation temperature (Td) is used as a parameter of thermodynamic stability. Td describes the temperature at which half the enzyme is inactive after a given time of incubation. In other words, at Td [proteinactive] = [enzymeinactive]. At rising temperatures, proteins tend to lose activity in cooperative ways resulting in sigmoidal curves when the fraction of active protein is recorded against the denaturating factor.

In order to determie of Td, the individual activity values resulting from the three heat inactivation runs were separately fitted with Microcal™ Origin® 6.0 software (Northampton, USA) using sigmoidal fitting functions based on the Boltzmann equation eq. 5-1.

()A – A y = ------1 ------2 - + A ()xx– ⁄ ()dx 2 Eq. 5-1 1 + e 0

A1 = init value; A2 = final value; x0 = midpoint of inactivation (Td); dx = time constant (also called reciprocal slope factor)

124 5.3 Results

One exemplary fitting graph (Roche lpPOX) is shown in figure 5-7 for clarification.

. 120

100

Activity after Heat Inactivation (%) Activity after 20

0 25 30 35 40 45 50 55 60 65 Inactivation Temperature (°C)

Figure 5-7: Calculation of Td; Roche quasi-wt without cofactors in the sample solution during heat inactivation. Black, grey and white are the inactivation values for three runs of stress tests. Fitting curves were calculated as described in the text based on eq. 5-1.

The resulting Td values are listed in table 5-3 including standard deviations. Based on the three resulting Td values, statistical analysis using a F-test for comparison of variance and t-test for comparison of mean values were performed. The results are summarised in figure 5-8.

Table 5-3: Stability of different mutated pyruvate oxidases: Midpoint of deactivation by temperature (incubation for 10 minutes) in 0.2M potassium phosphate buffer, pH=7, 25°C, cofactors added for the activity assay;) Roche-wt Trx-wt Trx-mutant Trx-mutant L323Q with I221T a) with cofactors 59.55°C 53.54°C 54.56°C 49.80°C during the heat inacti- ±0.56°C ±0.41°C ±2.59°C ±3.78°C vation b) without cofactors 46.8°C 39.68°C 41.06°C 37.67°C during heat inactivation ±0.39°C ±0.34°C ±0.86°C ±0.72°C

125 5 Stabilisation of the Quaternary Structure and Cofactor Sites

65 a) b) 60 59.6 55 54.6 53.5 50 49.8

Td (°C) 45 46.8 Ile221 *

40 41.1 39.7 35 37.7

30 FAD FAD Roche quasi wt Trx-lpPOX quasi wt mutant L323Q mutant I221T

Figure 5-8: Stability of original triple mutant and further mutated pyruvate oxidases; Td values of Roche lpPOX wt Trx-lpPOX as internal standard and two mutants. Grey bars: heat inactivation with added cofactors in the sample solution; white bars: heat inactivation without additional cofactors in the sample solution. * Asterisk indicates significant difference (p <0.05).

All six samples based on Trx-fusion proteins (figure 5-8 b) are significantly less stable against heat inactivation than the samples containing Roche lpPOX quasi-wt (figure 5-8 a) (p<0.05). Compared with Trx-lpPOX quasi-wt, only mutant L323Q without cofactors shows significantly higher stability against heat inactivation (p<0.05; indicated by asterisk). The Td value is 1.4°C higher than that of the wt fusion protein.

5.4 Discussion

We have been able to produce Trx-lpPOX using our protocol and the salt inducible BL21-SI E. coli strains with the same specific activity as the lpPOX, sold by Roche. From this it can be concluded that the addition of the Trx-tag does not influence the activity of the enzyme. However, the Trx-tag reduces the thermal stability of lpPOX. Because the intended thrombin cleavage did not work as expected, the first mutants were analysed as fusion proteins and the production protocol of pure lpPOX was developed further as described in chapter 4 and implemented in chapter 6.

5.4.1 Quaternary Stabilisation

Mutant L323Q led to an expected stabilisation. It shows significantly better thermostability than

126 5.4 Discussion the comparable Trx-wt tested without cofactors during heat inactivation. The proposed improvement of inter subunit contacts may be responsible for the increased Td of +1.4°C at pH=7.0 and at a protein concentration of 100 µg/ml. The resulting difference in Td of +1.4°C is comparable to the increasing Td of +1.1°C for the lpPOX mutant A131I which also stabilised the tetrameric state of the enzyme (Nagel 2004). These stabilisations at a quaternary structure level confirmed the hypothesis of possible stabilisation by improving the interactions between the POX monomers and indirectly suggested lpPOX inactivation cascade (see figure 3-9). These results also conincide with the underlying model put forward by Risse et al. (1992a) where one of the early steps in POX inactivation is the disintegration of the tetrameric state. The accomplished stabilisation cannot be compared to the one obtained by covalent crosslinkining of other tetrameric proteins (+16°C) as reported by Wurth et al. (2001) or (+15°C) by Bjork et al. (2003) for other proteins. These improved thermal stabilities were measured by far-UV-circular dichroism and therefore do not show loss of activity but do show unfolding of alpha helices. The values are simply reported here to give an impression of which stabilisations could be possible using protein engineering. The possibility of stabilising lpPOX covalently on the quaternary level was attempted by Nagel (2004) with the V141C mutant but did not succeed. Therefore, further stabilisation strategies are put forward and tested in chapter 6.

5.4.2 Cofactor Mutants

At the ThDP site mutation Ala373->Lys tends to result in incorrectly folded insoluble proteins. The fact that active protein could be produced by urea denaturating and slow refolding demonstrated that the exchange is possible in that position at the beginning of helix 19 even though an uncharged amino acid has been replaced by a charged one. The difficulties encountered during production of the mutant protein showed that replacement by a fundamentally different amino acid close to the active site might by hazardous. Due to persistent problems during production of the A373K mutant, the investigations with this suggested mutation were discontinued. Fur- thermore, a comparison with the POX of Oceanobacillus iheyensis (figure 3-6) led to a new suggested stabilising mutant, A373P, reported in chapter 6. Mutant I221T showed similar activity and stability as the wt (comparison between the Trx-fusion proteins). The expected stabilisation by generating a new hydrogen bond between Thr221-OG and the oxygen (AO1) of the alpha-phosphate of FAD could not be confirmed. Al-

127 5 Stabilisation of the Quaternary Structure and Cofactor Sites though this mutation could be found in the POX of Oceanobacillus iheyensis (see figure 3-6), the theoretical effect of better cofactor binding did not lead to measurable improvement of thermal stabilisation. Rational stabilisation by mutations near the cofactor binding sites is a big challenge as shown by these examples. Each enzyme contains an uniquely optimised cofactor binding site, allowing exact control of the specific redox potential, for example of FAD, that is required in a given redox system. Hydrogen bonds, Pi-stacking and donor atom-Pi effects between the apoprotein and prosthetic group influence the redox (reduction-) potential of bound cofactor. This unstable balance between interactions and redox potential and the fact that many hydrogen bonds between apoprotein and cofactor functions through backbone carbonyls or amides, makes it difficult to improve the cofactor binding by rational mutagenesis. A few theoretical considerations on how to stabilise the enzyme in a following step on the cofactor site are summarised in the following paragraph, in order to give an outlook on what could be done further in this field. Investigations concerning cofactor-stabilisation were stopped here in order to follow promising common stabilisation concepts (see chapter 6).

5.4.3 Perspective on Further Cofactor Stabilisation

The binding of ThDP to the apoprotein could be improved by an adjustment of the ThDP binding site. A comparison of the lpPOX ThDP binding motif with one of the other enzymes binding ThDP (iHawkins et al. 1989) suggests that T473N would be a promising mutant. In this respect T473N would complete the established ThDP fingerprint (iHawkins et al. 1989), which runs in lpPOX from the conserved GDG motif at amino acid 446 to a possible NN motif at amino acids 473/474 (see figure 3-6). Another indication for this mutant is the fact that oiPOX already contains an asparagine at that position. In most flavoproteins the flavin cofactor is generally non-covalently bound and can be reversibly removed especially since FAD is water soluble (Hefti et al. 2003). In a small number of cases, FAD is covalently attached to the apoprotein via histidyl-, tyrosyl- or cystidyl-bonds. (Robinson et al. 1994; Robinson and Lemire 1996; Decker and Brandsch 1997). A covalent attachment of FAD to lpPOX would be a promising approach for further stabilisation.

128 6 Entropic Stabilisation

irst experiments with mutants that should stabilise the active tetrameric form and mutants F that should improve cofactor binding showed only small or negligible effects in stabilisation. As outlined in paragraph 3.5.2.1, one promising approach for lpPOX stabilisation is a rigidisation of the protein backbone by Xaa->Pro and Gly->Xaa mutations respectively. This chapter highlights the practical work and the results which were derived in the field of lpPOX entropic stabilisation based on theoretical calculations published elsewhere (Nagel 2004).

6.1 Introduction

In order to decrease the conformational entropy of a protein in the unfolded state, existing amino acids (aa) have to be replaced by aa with more restricted rotational degrees of freedom. This should reduce the degree of rotational freedom in the unfolded enzyme and thus cause a lower loss of entropy during protein folding and stabilises the native state. (see figure 3-9). The rigidisation leads to two stabilising effects; Firstly a rigidisation of the protein backbone leads to a shift of the equilibrium between unfolded and folded protein towards the folded species (figure 3-9 on the right). On the other hand the rigidisation can also indirectly stabilise the protein. Limiting the overall protein flexibility can lead to the less favourable separation of bound cofactors (figure 3-9 on the left). Two well-known approaches for backbone rigidisation can be applied. One of these is the replacement of selected aa by proline (Pro). Another approach for decreasing the rotational degrees of freedom is the replacement of glycine (Gly) residues by different amino acids with side chains.

6.1.1 Entropic Stabilisation by Proline

Unfolded polypeptide chains can adopt different rotameric positions around the backbone dihedral angles ω, ψ and φ. Whereas ω is restricted to 180° (trans) or 0° (cis) respectively due to the partial double bound character of the peptide bond (see figure 3-3), the polypeptide side chains can adopt different rotameric positions around χ, the side chains angles. In the native state of a protein, almost all φ and ψ angles are restricted to a defined position, as are the majority of the χ angles. This loss of degree of conformational entropy must be overcome by interactions in

129 6 Entropic Stabilisation order to fold a stable protein. If the conformational entropy of the unfolded peptide chain can be reduced, the decrease of entropy during protein folding will be lower and at constant enthalpic forces the equilibrium will shifted from the unfolded state towards the folded state. The pyrrolidine ring of proline restricts the rotational degrees of freedom of the protein backbone and side chain. Due to the bond between the δ-carbon atom of the proline side chain and the amide nitrogen, the φ backbone dihedral angle is locked at approximately -75° and the χ angles are also restricted (see figure 6-1). Therefore, proline has exceptional conformational rigidity compared to other aa and loses less conformational entropy upon folding. This may account for its higher prevalence in proteins of thermophilic organisms (Watanabe et al. 1994 and 1996, Scandurra et al. 1998). If inserted in regular secondary structure elements such as beta sheets and alpha helices, proline is known to disrupt them. However, proline is commonly found as the first residue of an alpha helix and also in the edge strands of beta sheets. Proline is also commonly found in turns, which may account for the peculiar fact that proline is the only aa possessing an aliphatic side chain, which is usually solvent-exposed. Because proline lacks a hydrogen on the amide group, it can not act as a hydrogen bond donor, only as a hydrogen bond acceptor. .

Cδ Cγ

ω φ Cβ

Cα ψ

Figure 6-1: Proline in a peptide chain Dihedral angles ω, ψ and φ are shown by arrows. Due to the partial double bound between the carbonyl C-atom and the amide nitrogen atom ω is restricted to 180° (trans) or 0° (cis) re- specitvely.

130 6.2 Methods

6.1.2 Entropic stabilisation by Glycine Replacement

Glycine has higher conformational backbone flexibility than all other amino acids due to the lack of a side chain. In other words, the backbone of a glycine residue in solution has greater conformational entropy than other amino acids i.e. alanine. Therefore, the stability of a protein should be increased by the careful replacement of glycines with other amino acids such as alanine (Matthews et al. 1987). Of course, threonine, valine and isoleucine branched side chains restrict backbone conformation more than alanine with a non-branched β-carbon atom. How- ever, during the replacement of glycine by a branched amino acid, all other reinduced interactions and repulsions of these new side chains must be checked carefully. A further challenge is the replacement of glycines in alpha helices. Glycines normally dis- favours alpha helix formation but sometimes occur in alpha helices due to helix bending. Be- cause of its specialised structural properties in protein architecture, this amino acid is often conserved, through evolution, for example, in the G-D-G binding motif of ThDP binding enzymes (iHawkins et al. 1989) or in the G-x-G-x-x-G binding motif of dinucleotide binding proteins (Wirenga et al. 1985). This has to be considered when designing glycine replacements.

6.2 Methods

6.2.1 Selection of Stabilising Xaa-Pro Mutations

In order to decrease the conformational entropy of lpPOX in the unfolded state, amino acids with more restricted rotational degrees of freedom have to be carefully chosen, inserted into the enzyme aa-sequence and tested in the laboratory. Because of the abovementioned restrictions resulting out form the structural peculiarities of proline and glycine, the suggested mutations have to be verified carefully. Furthermore, it has to be considered that amino acids with strong interactions within the protein structure cannot be replaced. If they were replaced, an undesira- ble loss of stability due to a loss of enthalpic contributions could happen. The four step selection procedure for new stabilising proline mutants was established by Nagel (2004) and is summarised here only for clarity. In a first step an assessment of the φ and ψ dihedral angles of the aa that has to be replaced with accepted dihedral angles of proline was performed. Positions within alpha helices or beta sheets were not selected, because of the structural breaking properties of proline. In a second step the φ and ψ dihedral angles of the proceed- ing amino acid were also evaluated. In step three the remaining mutants were tested for sterical

131 6 Entropic Stabilisation hindrance based on the Van der Waals radii of the nearly insented proline. Finally, the mutations were inspected to see whether they led to a loss of stabilising interactions upon replacement by proline. The procedure was also verified successfully by Nagel through the application of the procedure for T4 lysozyme and oligo-1,6-glucosidase of Bacillus cereus (EC 3.2.1.10). The theoretical calculated values were compared to experimental data on the basis of numerous published mutations of these two enzymes. The results of that proline mutant selection procedure were ten promising Xaa-Pro mutants. On further consideration, two of these ten mutants were excluded because of their proximity to cofactor binding sites and the resulting risk of losing enzyme activity during replacement by proline. Thus, the suggested mutations were; G112P, A143P, L193P, V199P, A252P, A373P, S559P and A564P. The location of these mutations is shown in figure 6-2.

Figure 6-2: Promising mutations for entropic stabilisation of lpPOX One monomer of lpPOX is shown; alpha helices are marked in red, beta sheets are marked in blue, cofactors are showed in stick and ball mode.

132 6.2 Methods

6.2.2 Selection of Stabilising Gly-Xaa Mutations

As with the suggested proline mutants, the Gly-Xaa mutations were also selected by a multi step procedure developed by Nagel (2004). Due to the lack of published mutations, no verification of the method was applied. Only one suggested mutation G508A resulted out of this procedure, because all other possible sites were buried in the protein without any space for an additional side chain (Nagel 2004). The location of that mutation is shown in figure 6-2. Two doubtful mutations next to the cofactor binding sites (G25N and G590N) discussed in Nagel (2004) p. 181ff were also produced in order to verify this rigorous selection.

6.2.3 Mutagenesis of Proposed Stabilising Mutants

The pET28 plasmids containing the lpPOX-wt-gene were extracted out of an overnight culture of E.coli DH5alpha cells produced as described in section 4.2.2.1 and purified by a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, SUI). All eleven mutations were introduced into the lpPOX genome using the same method as described in paragraph 5.3.1 and pairwise mutagenic primers no. 16 to 37 (see table 6-1). The remaining circular, nicked dsDNA was transformed into competent XL2-Blue cells by means of the described calcium chloride method (see paragraph 4.2.2.3) using 50 ng of plasmid and a heat shock time of exactly 60 seconds at 42°C. Sucessfully transformed cells were selected overnight on kanamycin containing (0.05 mg/ml) LB-agar-plates, selected clones were expanded overnight in kanamycin containing (0.05 mg/ml) LB-medium. The double mutant L193P_S559P was built using the same protocols and primers as for the single mutants, simply by introducing one mutation after another. The double mutant was created to verify the hypothesis that the stabilising effects of proline mutations are cumulative. For this approach, the most promising stabilising mutants out of the first selection round were chosen and combined (see figure 6-4). By selecting the combination, we have considered that mutations located far from each other may be more proof of an additive effect of stabilisation, than a double mutant with two proline mutations side by side.

133 6 Entropic Stabilisation

Table 6-1: Primers used for mutagenesis:

Primer no. Sequence 16 (G112Pfwd) 5'-CGTATCCATG TTCATCCCAG TAGTTGGAAA TTGACCAATA AGTGC-3' 17 (G112Prev) 5'-GCACTTATTG GTCAATTTCC AACTACTGGG ATGAACATGG ATACG-3' 18 (A143Pfwd) 5´-CATGTGGCAA CGTGGCAGGA TTGACGGCTG TTAC-3´ 19 (A143Prev) 5´-GTAACAGCCG TCAATCCTGC CACGTTGCCA CATG-3´ 20 (L193Pfwd) 5´-CGTCGGGTTC TGGTAATGGC GGCGTTTGAT AATTATTAGC G-3´ 21 (L193Prev) 5´-CGCTAATAAT TATCAAACGC CGCCATTACC AGAACCCGAC G-3´ 22 (V199Pfwd) 5´-CTCGTCACTG CTTGAGGGTC GGGTTCTGGT AATAACG-3´ 23 (V199Prev) 5´-CGTTATTACC AGAACCCGAC CCTCAAGCAG TGACGAG-3´ 24 (A252Pfwd) 5'-GGCTGGATAA CGATCCGGGA CAATACCCTT AGCTG-3' 25 (A252Prev) 5'-CAGCTAAGGG TATTGTCCCG GATCGTTATC CAGCC-3' 26 (A373Pfwd) 5´-CGCACGTAGC ACTTGATATG GTTGTAAAGG CCCTTCC-3´ 27 (A373Prev) 5´-GGAAGGGCCT TTACAACCAT ATCAAGTGCT ACGTGCG-3´ 28 (S559Pfwd) 5´-GCCGAACTCA TTGCCGGATC TAAACGAAGC TTTTC-3´ 29 (S559Prev) 5´-GAAAAGCTTC GTTTAGATCC GGCAATGAGT TCGGC-3´ 30 (A564Pfwd) 5´-GCTTCAATATCAGCTGGCGAACTCATTGCCGAATC-3´ 31 (A564Prev) 5´-GATTCGGCAATGAGTTCGCCAGCTGATATTGAAGC-3´ 32 (G25Nfwd) 5´-CCTCCAGGAA TACCATACAA ATGATCTACG TTCCAAGCTT CTAAAAC-3´ 33 (G25Nrev) 5´-GTTTTAGAAG CTTGGAACGT AGATCATTTG TATGGTATTC CTGGAGG-3´ 34 (G508Afwd) 5´-GCTTGCATGT GCACGGCATC GGCAATCTTA C-3´ 35 (G508Arev) 5´-GTAAGATTGC CGATGCCGTG CACATGCAAG C-3´ 36 (G590Nfwd) 5´-CCACCCTGTC CAATTTGATG TTGCAAATCA TCTAAGTTAA ATTGTTT-3´ 37 (G590Nrev) 5´-AAACAATTTA ACTTAGATGA TTTGCAACAT CAAATTGGAC AGGGTGG-3´

6.2.3.1 Sequencing and Transformation into Expressing Cells

Different clones, resulting from the kanamycin selection, were verified by automated sequencing using the previously mentioned sequencing procedure (see 4.2.2.4) and the sequencing primers listed in table 4-2. The verified plasmids were extracted out of XL2-blue™ cells by a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, SUI) taken up in water and transformed into BL21-SI™ cells for protein expression (see paragraph 4.2.2.3). Sucessfully transformed cells were selected overnight on LBON-agar-plates containing kanamycin (0.05 mg/ml) at 37°C and expanded at 37°C in an overnight culture in LBON-medium containing kanamycin (0.05 mg/ml).

134 6.2 Methods

6.2.4 Expression and Purification of the Mutants

After transformation into competent BL21-SI™ cells, lpPOX was expressed on the basis of the pET28 vector, in this way we were able to express the lpPOX with only the 3xHis-Tag™. With regard to the nine promising single mutants, there was sufficient expression according the protocol outlined in 4.2.2.5 using 550 mM NaCl for induction. The mutanted enzymes were successfully expressed, purified at pH=6.0 (near the optimal pH=5.7) at 4°C and tested for activity and thermal stability. Protein purification was performed as outlined in paragraph 4.3.3 by using affinity chromatography in a first step and then size exclusion chromatography in a second step. Size exclusion purification was revealed to be as necessary to increase the specific activity but during this second purification step, 74 to 96% of the protein was lost. In order to overcome this dramatic loss of protein and to have sufficient amounts of protein for biophysical characterisation, the purification protocol was improved, as mentioned in chapter 4, by the introduction of a step gradient on the HiTrap™ chelating NiSO4-column. In this way, the second purification step by size exclusion chromatography could now be omitted (see paragraph 4.2.2.7) and was replaced by a PD10 column purification step using potassium phosphate buffers (0.2M, pH=6 or 7). The rationale for choosing these two pH values for the biochemical and biophysical characterisation is that pH=6 corresponds to an optimum stability for lpPOX (Sedewitz et al. 1984) and pH=7 simulates the conditions in the biosensor, which should work at a neutral pH.

6.2.5 Thermal Stability monitored by far-UV-CD

Thermal unfolding experiments of selected mutants were monitored by measuring the circular dichroism (CD) signal at 222nm of samples containing 100 µg/ml lpPOX in a temperature range of 20 to 85°C with a heating rate of 40°C/h (cell d = 0.5cm). All CD measurements were ac- quired on a JASCO J720 spectropolarimeter attached to a Neslab111 water bath circulator. The measurements were done in triplicate in potassium phosphate buffer 0.2 mM, adjusted to a defined pH. To verify that the Tm values were not dependent on protein concentration, three different samples of wt protein at different concentrations, e.g. 10, 100 and 1000 µg/ml, were thermally unfolded. For evaluation of the CD unfolding profiles, the buffer profile was removed from the protein CD profile. The exact melting points (Tm) were calculated using Microcal™ Origin® 6.0 software (Northampton, USA). A non linear curve fit as described by Wurth et al. (2001) based

135 6 Entropic Stabilisation on eq. 6-1 was performed for this purpose.

()Tm× ()– m + y – y Eq. 6-1 Y = ------N U------N ------U - ++m × T y T U U ⎛⎞⎛⎞⎛⎞1 – ⎛⎞------⎝⎠⎝⎠ ⎜⎟⎜⎟Tm ⎜⎟1 + exp⎜⎟–∆H × ------⎜⎟⎜⎟RT ⎝⎠⎝⎠

Y is the molar ellipticity per residue [Θ]MRW at 222nm, measured and calculated out of the CD denaturating curve; T is the temperature (K) recorded by the CD spectrophotometer; Tm is the melting temperature (K) at which half of the material exists in the native state and the other half in the denatured state, calculated by non linear curve fit; ∆H is the enthalpy change between the native and the denatured state, calculated by non linear curve fit; yN and yU are the signals contributed by the native (N) and the unfolded (U) state, calculated out of the denaturating curve; mN and mU are the slopes of the baselines, calculated out of the CD denaturating curve.

6.2.6 Kinetics of Protein Inactivation and Longterm Stability Experiments

There are two methods available for testing protein formulations for stability or for shelf life: 1) longterm stability tests 2) stress tests with enhanced degradation The longterm stability tests allow for the most precise predictions on real shelf life because kinetic and thermodynamic aspects of destabilisation are included in equal shares. The disadvantages of longterm stability tests are their long duration and the high demands on analytical methods to indicate only small amounts of degradation products. Stress tests and also microcalorimetrical assays at higher temperatures allow for the acceleration of stability tests but the disadvantages are the unnatural conditions of degradation (high temperatures, high concentrations of chaotropic salts,...) and the risk of unilateral consideration of the thermodynamic aspects of destabilisation. Thus preliminary experiments have to show with regard to accelerated temperatures the same mechanisms and kinetics are prone to degradation as at moderate temperatures. After proving this, accelerated stability tests can be used for fast prediction of stabilising effects but can never replace the longterm stability test, especially in the field of protein formulations or biopharmaceuticals. Longterm stability experiments were performed in Eppendorf tubes containing potassium phosphate buffer (0.2M; pH=7 or 6) with the protein in a concentration of 100 µg/ml. The samples were stored under controlled temperatures of 25°C at all times and were sealed with para- film against loss of water. The remaining protein activity was tested at different points of time

136 6.2 Methods under application of the activity assay described in chapter 4. For a discussion of kinetic factors in protein stabilisation, a few general considerations about the kinetics of protein inactivation are summarised in the following subchapter.

6.2.7 Kinetics of Protein Inactivation

With regard to irreversible unfolding and the general protein inactivation process illustrated by figure 3-1 or at figure 3-9, the rate of the overall process is termed kobs. This simplified description of kinetics is based on the fact that kinetics characterises an empirically ascertained tempo- ral sequence of events but not the exact mechanism of a reaction. The determination of the appropriate order of reaction was done by the graphical method described by Grimm et Gothier (1999) p547f. Based on that analysis it can be assumed that the inactivation of pyruvate oxidase most probably works in the way of first order kinetics. The ve- locity equation of a first order reaction is given in eq. 6-2:

dA[ ] v = –------Eq. 6-2 t dt

Integration of eq. 6-2 leads to eq. 6-3:

Eq. 6-3 ln[]At = ln[]A0 – kt

By converting the natural logarithm into the decadic logarithm one arrives at eq. 6-4.

kt log[]A = log[]A – ------Eq. 6-4 t 0 2, 303

Considering the inactivation of lpPOX as a reaction of first order kinetic, the half life of a protein t1/2 is given by the following equation 6-5:

ln2 Eq. 6-5 t12⁄ = ------kobs kobs at a given temperature is used frequently as an operational measure of kinetic stability.

137 6 Entropic Stabilisation

6.3 Results

6.3.1 Mutagenesis and Transformation

All eleven mutations mentioned previously (including the two doubtful mutations G25N and G590N) were successfully introduced at DNA level into the expression plasmid and transformed into XL2-blue™ expressing cells. The double mutant L193P_S559P was also introduced into the lpPOX gene in a second site-directed mutagenesis run. Mutations were confirmed by sequencing.

6.3.2 Expression and Purification of the Mutants

All nine promising single mutants were successfully expressed, purified at a pH=6.0 (near the optimal pH=5.7) at 4°C and tested for activity and thermal stability. Protein yields resulting from one litre of culture medium are listed in table 6-2. Most of the mutants were producible in the same range as lpPOX wt (around 1 mg per litre culture) despite the loss of protein during size exclusion based purification. Only mutants A143P and A373P resulted in small amounts of active enzyme in the range of one order of magnitude less than the wt and the other mutants. The purity of the recieved proteins was estimated to be >95% based on SDS-PAGE. The two mutants G25N and G590N could not be expressed in a soluble form according to the previously mentioned protocol and therefore were not further investigated. In contract to size-exclusion, the use of the PD10 columns as second step showed only a small loss of protein. This new strategy allowed us to produce highly active lpPOX in a much faster and efficient way. The total yields achieved were as follows 1.01 mg/l (L193P), 0.851 mg/ l (V199P), 1.52 mg/l (S559P), 1.54 mg/l (L193P_S559P) and 1.14 up to 9.52 mg/l (wt). The achieved purity with this new purification protocol (chelating NiSO4-column, equilibration with cofactors and removing of unbound cofactors by a PD10 desalting column) is high enough (at least > 90%) for the biochemical and biophysical characterisation of our mutant proteins and for an application in the sensor.

6.3.3 Specific Activity of Mutant Proteins

Specific activity was measured based on the test mentioned in paragraph 4.2.2.9. The results are summarised in table 6-2. In general most of the mutants showed activity in the same order of

138 6.3 Results magnitude as lpPOXwt. Mutants A143P, V199P and S559P had a remarkably higher specific activity than the other mutants. Mutants G112P and A373P showed clearly lower activity than the wt and the other mutants. It must be noted that these values and also the yields of produced protein fluctuate noticeably between different production runs within one order of magnitude.

Table 6-2: Yield and specific activity of wt and mutant pyruvate oxidases: ¹ Yield in mg per litre culture Specific activity (U/mg) purified by Ni-affinity, cofactor measured at pH=7.0 equil. and size excl. chromatogr. wt 1.17 U/mg ±0.039 G112P 0.756 mg 0.257 U/mg ±0.117 A143P 0.028 mg 1.917 U/mg ±0.251 L193P 4.025 mg 0.93 U/mg ±0.102 V199P 4.590 mg 2.08 U/mg ±0.094 A252P 0.524 mg 0.905 U/mg ±0.137 A373P 0.020 mg 0.396 U/mg ±0.067 S559P 1.475 mg 2.24 U/mg ±0.353 A564P 2.067 mg 0.875 U/mg ±0.042 G508A 2.762 mg 1.212 U/mg ±0.099

The specific activities (U/mg) of three selected mutant enzymes and of the double mutant were measured in phosphate buffer (KH2PO4 0.2M) at pH 6.0 and at pH 7.0 (see figure 6-3). The specific activity of the mutant enzymes are in the same order of magnitude as the wild type enzyme. However, the V199P, S559P and L193P_S559P mutants showed a statistically significant (p <0.05) increase in specific activity at pH=7.0 and V199P also at pH=6.0 compared to wt (figure 6-3).

139 6 Entropic Stabilisation

4 )

2 Specific Activity (U/mg Activity Specific 1 9 4

0 3.36 1.17 2.15 0.92 4.23 2.08 1.46 2.2 2.26 2.02 wt L193P V199P S559P L193P_S559P

Figure 6-3: Specific Activities of Selected Mutant Enzymes; measured at different pH values, pH 6.0 (blue) and pH 7.0 (purple); specific activity is given in (Umg).

6.3.4 Stability Testing Using Heat Inactivation

The remaining activity after heat inactivation was measured, processed and Td values were calculated as mentioned in paragraph 5.3.6 for comparison and statistical analysis. Temperature stress tests were done in triplicate and means are shown in combination with standard deviations. The results are listed in table 6-3 and illustrated in figure 6-4.

Table 6-3: Stability of different mutated pyruvate oxidases: Midpoint of deactivation by temperature (incubation for 10 minutes) in 0.2M potassium phosphate buffer, pH=7, 25°C, cofactors added for the activity assay;) Td ∆Td compared to wt Roche wt 45.83°C ±0.34°C -0.21°C wt 46.04°C ±1.57°C G112P 41.50°C ±0.22°C -4.54°C A143P 44.84°C ±0.17°C -1.20°C L193P 50.16°C ±0.22°C +4.12°C V199P 48.70°C ±0.10°C +2.66°C A252P 43.94°C ±2.26°C -2.10°C A373P 44.64°C ±0.24°C -1.40°C S559P 48.92°C ±0.05°C +2.88°C A564P 47.26°C ±1.19°C +1.22°C G508A 46.97°C ±0.81°C +0.93°C

140 6.3 Results

* 50 50.2 * * 48.7 48.9 a) b) 47.3 47.0 45 45.8 46.0 44.8 44.6 * 43.9 Td (°C) 40 41.5

35 Ile221 *

30 wt wt G112P A143P L193P V199P A252P A373P S559P A564P G508A RocheFAD FAD

Figure 6-4: Stability of wt and mutants of pyruvate oxidase; Values of Td from Roche wt, and wt expressed in our laboratory as standards and nine mutants. Heat inactivation without additional cofactors in the sample solution. * significant different Td compared to wt (p<0.05).

The wt delivered by Roche and the wt produced in our laboratory showed a comparable Td of about 46°C. Three mutants showed significantly higher Td values and therefore higher thermal stability than the wt (L193P +4.12°C; V199P +2.66°C and S559P +2.88°C) when tested without adding cofactors during heat inactivation. G112P had a significantly lower Td value (-4.54°C) than the wt and is therefore less stable against heat inactivation. All the other mutants did not have significantly differing midpoints of inactivation temperatures compared to the wt.

6.3.5 Biophysical Characterisation of Selected Mu- tants

The three proline mutants (L193P, V199P, S559S) and the double mutant (L193P_S559P) where submitted to further experiments for a clearer understanding of the stabilisation mechanism. The unfolding of the secondary structure due to temperature was measured by CD in order to determine the melting temperature (Tm) which indicates the temperature where half of the secondary structure is lost. The heat inactivation (Td) curves are displayed in the same graph for a comparison of the degradation of different protein structures.

141 6 Entropic Stabilisation

120 0 Tm L193P; 322.99 K Tm wt; 324.02 K -1 100 -2 ) 80 -3

60 -4

-5

Relative Activity (% Activity Relative 40 ]MRW x10³ (deg cm² dmol-1 cm² (deg x10³ ]MRW Θ

-6 [ 20 Td L193P; 323.28 K -7 Td wt; 318.70 K 0 -8 FAD FAD 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 Temperature (K)

Figure 6-5: Thermal denaturation of L193P The graph shows the fitted data (n=3) from thermal denaturation experiments measured by CD at a wavelength of 222nm (solid lines) and by thermal inactivation (dotted lines). The corresponding curves from wt are coloured in red and the one from L193P coloured in yellow. The differences of Td and Tm between mutant and wt are shown by vertical dotted lines.

120 0 Tm V199P; 323.96 K Tm wt; 324.02 K -1 100 1

-2 -

) -3 80 2dmol ^ -4

60 -5 3(degcm ^ -6

Relative Activity (% Activity Relative 40 -7 ]MRW x10 Θ -8 [ 20 Td V199P; 321.18 K -9 FAD Td wt;FAD 318.70 K 0 -10 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 Temperature (K)

Figure 6-6: Thermal denaturation of V199P The graph shows the fitted data (n=3) from thermal denaturation experiments measured by CD at a wavelength of 222nm (solid lines) and by thermal inactivation (dotted lines). The corresponding curves from wt are coloured in red and the one from L199P coloured in green. The differences of Td and Tm between mutant and wt are shown by vertical dotted lines.

142 6.3 Results

. 120 1 Tm S559P; 323.42 K Tm wt; 324.02 K 0 100 -1 1

) 80 -2

-3 60 -4

Relative Activity (% Activity Relative 40 -5 ]MRW x10^3 (deg cm^2 dmol- cm^2 (deg x10^3 ]MRW Θ

-6 [ 20 Td S559P; 321.73 K -7 Td wt; 318.70 K FAD FAD 0 -8 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 Temperature (K)

Figure 6-7: Thermal denaturation of S559P The graph shows the fitted data (n=3) from thermal denaturation experiments measured by CD at a wavelength of 222nm (solid lines) and by thermal inactivation (dotted lines). The corresponding curves from wt are coloured in red and the one from S559P coloured in purple. The differences of Td and Tm between mutant and wt are shown by vertical dotted lines.

120 1 Tm wt; 324.02 K

Tm L193P_S559P; 0 1 100 325.80 K -1 ) -2 80 -3 60 -4 -5 40 -6 Relative Activity (% Activity Relative Td L193P_S559P; -7 dmol- cm^2 (deg x10^3 ]MRW

20 Θ

318.22 K [ Td wt; 318.70 K -8 0 -9 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 FAD FAD Temperature (K)

Figure 6-8: Thermal denaturation of double mutant L193P_S559P The graph shows the fitted data (n=3) from thermal denaturation experiments measured by CD at a wavelength of 222nm (solid lines) and by thermal inactivation (dotted lines). The corresponding curves from wt are coloured in red and the one from L193P_S559P coloured in grey. The differences of Td and Tm between mutant and wt are shown by vertical dotted lines.

143 6 Entropic Stabilisation

The comparison of Tm and Td for the three stabilising proline mutants shows that a better stabilisation due to a higher Td does not have a mandatory strong effect on the melting temperature of the secondary structure (Tm values are more or less the same for the mutants and for the wt).

Interestingly, the increase of Td values for L193P and S559P are not cumulative in view of the double mutant. On the contrary, the Td value of the double mutant is lower than each one of the two single mutants. A second interesting fact is the increased Tm of the double mutant (see figure 6-8) of about +1.78 K compared to wt.

6.3.6 Longterm Stability Data

Longterm stability experiments were performed in potassium buffer (0.2M) at different pH-values in the absence of added cofactors in the sample solution in order to simulate the conditions in a biosensor. It has been showen that lpPOX and all different mutants are more stable at pH=6 than at pH=7. Mutant S559P is the only protein species which achieved the primary objective of remaining activity of a minimum 50% after two weeks. This achievement was only accomplished at a pH=6.0. At pH=7.0 only mutant V199P was more stable than the wt (see figure 6-9).

144 6.3 Results

a) b)

120 120

100 100

80 80

60 60 Relative Activity(%) 40 Relative Activity (%) 40

20 20

0 0 0102030FAD 0102030FAD Days Days

c) d)

2.5 2.5

2 2

1.5 1.5

1 1 Log Relative Activity Log Relative Activity 0.5 0.5

0 0 01020300102030 Days Days

Figure 6-9: Longterm Stability The graphs show the relative activity remaining after storage at 25°C in potassium buffer (0.2 M) at pH=6.0 (a) and (c) or at pH=7.0 (b) and (d). The longterm stability experiments were done without added cofactors in the sample solution during storage. The relative activity is outlined in normal (a and b) and in logarithmic scale (c and d). The corresponding curves from wt are marked in red, mutant L193P in yellow, V199P in purple, S559P in blue and the double mutant L193P_S559P in green.

145 6 Entropic Stabilisation

6.4 Discussion

6.4.1 Stability of Single Xaa->Pro Mutants

Four mutants (L193P, V199P, S559P and A564P) out of eight showed a better thermostability with ∆Td from +1.2°C to +4.1°C. The measured ∆Td values of these four promising lpPOX mutants were comparable to stabilizing effects investigated in other enzymes stabilized by application of the same backbone rigidisation strategy. Watanabe et. al. (2000) reported an increase of Td between +1.3°C to +5.4°C for each proline residue introduced in oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus.

The four remaining proline mutants (G112P, A143P, A252P and A373P) showed lower Td values than wt lpPOX (∆Td from -4.5°C up to -1.4°C). The significant destabilisation measured for mutant G112P (∆Td -4.5°C) and also the destabilisation of mutant A373P (∆Td -1.40°C) was rather unexpected due to the fact that pyruvate oxidase from Oceanobacillus iheyensis (extremophile organism, see figure 3-6) had proline residues at these two positions. This and also the moderate destabilisation effects of mutants A143P and A252P can be explained by two reasons or a combination of the two. Firstly, it could be that lpPOX needs some backbone flexibility at these positions at higher temperatures to remain active. The second explanation is that the more voluminous side chains from proline encounter structural hindrance during folding and unfolding. The fact that mutants A143P and A373P are only expressed at low amounts also shows that lpPOX does not prefere proline residues at these positions. A comparison of surface accessibility pointed out that all four destabilising proline mutants are deeply buried (Ooi term: 0.66 to 1.1; mean: 0.81) and the four stabilising proline mutants are obviously more solvent exposed (Ooi term: 0.2 to 0.4, mean: 0.29). The corresponding “Ooi“-values have been calculated by Nagel (2004) p173ff and represent the count of the number of other Cα atoms within a radius of 14 Å of the given residue‘s own Cα atom. The Ooi-number was converted into to a scale from 0 (solvent exposed) to 1 (buried). For solvent exposed Xaa->Pro mutants, a higher flexibility of the neighbouring amino acids allows for better adaptation of the whole mutation site.

146 6.4 Discussion

6.4.2 Discussion of CD Experiments

∆Tm values ranging from -1.03° K to +1.78° K were observed for the four selected mutants (L193P, V199P, S559P and L193P_S559P) compared to the wt protein. A comparison with data from Gaseidnes et. al. (2003) and Matthews et. al. (1987) showed similar effects of thermal stabilization by introducing Xaa->Pro or Gly->Xaa mutations. The chitinase double mutant G188A_A234P of Serratia marcescens showed an increase of the melting Temperature (∆Tm, measured by CD) of +4.2°C but also had additivity effects for applied mutations (Gaseidnes et al. 2003). Matthews et. al. (1987) described a stabilisation effect concerning ∆Tm from +0.9°C after introducing Gly->Xaa and up to +2.1°C if an additional Xaa->Pro or Gly->Xaa mutation was introduced in the bacteriophage T4 lysozyme respectively.

The comparison of Tm and Td for the three stabilising proline mutants and for the double mutant showed, that a better stabilisation due to a higher Td does not have a mandatory strong effect on the melting temperature of the secondary structure (Tm values are more or less the same for the single mutants and for the wt). Therefore one can conclude that the three proline mutants (L193P, V199P and S559P) stabilise the active lpPOX more at the tertiary structure level or on the level of preventing movement of the three domains against each other. This supports the lpPOX inactivation model put forward in figure 3-9.

Tm values of the double mutant are discussed in the following paragraph.

6.4.3 Stability of Double Mutant (L193P_S559P)

Interestingly, the increase of Td values of L193P and of S559P are not cumulative with regard to the double mutant. On the contrary, the Td value of the double mutant is lower than each value of the two single mutants. A second interesting observation is that the Tm of the double mutant (see figure 6-8) increases about +1.78 K compared to the wt. The latter can be explained by a rigidisation of the whole monomer and therefore leads to a better protection of the secondary structure from thermal inactivation. The absence of additivity with respect to ∆Td values is not easy to explain but may result from a high rigiditisation of the enzyme, which negatively influences protein refolding. Further experiments with different double mutants are needed to clarify this unexpected result.

147 6 Entropic Stabilisation

6.4.4 Glycine to Alanine Mutations

The increased ∆Td of +0.93°C of the G508A mutant (α-helix, solvent exposed) compared to lp- POX wt was in the same range as generally reported in literature for Gly -> Ala mutants. Mat- thews et al. (1987) found a ∆Td of +0.9°C in the bacteriophage T4 lysozyme mutant G77A (α- helix, solvent exposed). Nicholson et al. (1992) measured a ∆Td of +0.8°C for the same protein with mutant G113A (loop, solvent exposed). Marguerite et al. (1992) investigated Bacillus subtilis neutral protease and found a ∆Td of +2.2°C for mutant G147A (α-helix, buried) and ∆Td of +1.1°C for mutant G189A (loop, buried). Veltman et al. (1996) established in Bacillus stearo- thermophilus a thermolysine-like protease (TLP) mutant (G141A; α-helix, buried) with a similar ∆Td of +0.7°C and another more thermostable mutant (G58A; β-strand, solvent exposed) with ∆Td +3.9°C. Ganter and Pluckthun (1990) found that the G316A mutation (α-helix, buried) of chicken glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) induces an excellent thermal stabilisation of ∆Td +7°C. The replacement of Gly by Ala can be a successful stabilisation strategy but general stabilisation effects can neither be predicted nor calculated, either from the secondary structure or from the location of the mutation site (buried or solvent exposed). Interestingly the main effect of stabilising Gly -> Ala mutants is also controversially discussed. Whereas in the literature mentioned above the effect of Gly -> Ala stabilisation was explained by rigidisation of the protein backbone, Scott et al. (2007) found that unfolded alanine- and glycine-containing peptides are approximately isoenergetic. They hypothesised that the higher conformational freedom of glycine sequences in the unfolded state is compensated by hydrophobic and van der Waals' stabilisation of the alanine side chain in the helical state. Fur- thermore, the helix-stabilising propensity of Ala relative to Gly thus mainly results from more favourable interactions of Ala in the folded helical structure and not from reducing entropic effects in the unfolded state. Two promising lpPOX mutants G25N and G590N (at the surface of the protein) could not be expressed in a soluble form. These two amino acids appear in lpPOX with excellent dihedral angles in the so called left-handed helical region in the Ramachandran plot, where only glycine and asparagine residues and no other aa exist. It can be concluded that the glycines in these positions are fundamental for correct protein folding and can not easily be replaced by asparagines. Stabilisation by replacing these glycines by other aa is impossible, because any other aa than asparagine cannot adopt the required dihedral angles. The stabilisation of proteins by replacing glycines with dihedral angles in the left-handed Ramachandran plot does not seem to be a prom-

148 6.4 Discussion ising approach for protein stabilisation.

6.4.5 Long-term Stability Experiments

As it could have been expected from preliminary stress test data, the shelf lifes were shorter at pH=7 than pH=6 near the enzyme optimal pH. The single exception to this common trend was the double mutant with almost the same slope on the graphs (see figure 6-9) and therefore with a comparable k-value at pH 6 and 7. At both pH-values, two out of four selected mutants were inactivated at a similar rate to the wt-protein, one was inactivated faster and one more slowly than the wt. Interestingly, at pH=6 mutant S559P was more stable and the double mutant L193P_S559P was less stable than the wt. At pH=7 V199P was more stable and S559P less stable than the wt. Mutant S559P is the only enzyme species which can fulfil the aim of this project. The aim of this project was to develop and produce a stabilised lpPOX in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor would be less than 50%. As reported by McAteer et al. (1999), shelf stability and the experimental protocols used to predict the shelf-life of biosensors or enzymes are generally well guarded company secrets and little attempt has been made to standardise such methods. A comparison of reference shelf lives is therefore rather difficult. Nevertheless, a list of pyruvate sensors based on POX not originating from Lactobacillus plantarum was described in chapter 2.2.4. Arai et al. (1999), Gajovic et al. (2000), Revzin et al. (2002) and Situmorang et al. (2002) did not measure the operational stability of sensors but the storage stability. The latter varies between less than 50 and about 95% residual activity after 7 days. Therefore, the requested shelf life of 50% remaining activity after two weeks of measurement is certainly an ambitious aim but a real prerequisite.

6.4.6 Outlook

It has been demonstrated that single Xaa-Pro mutations can stabilise lpPOX against temperatures degradation. The approach of stabilising a protein by backbone rigidisation was successful. However, a cumulative effect of more then one proline mutation in the same enzyme on functional stabilisation could not be confirmed thus far. Further experiments and other combined mutants are needed to answer the question about whether lpPOX can be stabilised in a cumulative way by adding more than one proline mutation to the enzyme. A comparison of stress tests and longterm stability experiments showed that not all stabil-

149 6 Entropic Stabilisation ising mutations at high temperatures (L193P, V199P and S559P; thermodynamic stabilisation) also led to an extended kinetic stability resulting in an extended shelf life. The assumption that a thermodynamic stabilisation of the protein at high temperatures would also lead to kinetic stabilisation at moderate temperatures could not be confirmed. Therefore the inactivation does not proceed with the mechanism at high and at moderate temperatures. An extrapolation of shelf life duration from stress tests at high temperatures can not be done to estimate shelf lives at moderate temperatures. Therefore, in future, all promising stabilising mutants have to be tested with long term tests at room temperature. Long term stability experiments have shown that at least one mutant out of four selected mutants also had an extended shelf life in an environment similar to that of the sensor, namely only phosphate buffer (without cofactors, sodium chloride or other stabilizers). The remarkable extended shelf life of mutant S559P has to be confirmed in the biosensor setup.

150 7 Final Discussion and Outlook

he results and findings of the previous chapters are discussed here as a general overview. T At the end a brief outlook provides suggestions for follow up projects aimed at engineering a stable pyruvate oxidase to be used for a phosphate or pyruvate sensor.

7.1 Methods of Producing lpPOX

The developed expression protocol allows now for the production of pure lpPOX in a fast, straightforward way. The problems raised such as producing low amounts, insoluble or inactive protein are circumvented. Yield, purity and activity of the protein comply with the requirements for an efficient protein production process. During development of the production procedure the implementation of a finely adjusted induction rate was the crucial step for the expression of soluble and active protein in acceptable amounts. The new production procedure could be scaled up without too many modifications. In this respect, the reports written by Schumacher and Möl- lering (1990), Risse et al. (1991) and Wille et al. (2003), which describe lpPOX expression in E. coli, can be confirmed, although the purification processes mentioned in these reports are rather different and were difficult to reproduce. LpPOX can be seen as a rather delicate enzyme. The introduction of certain single mutations, for example, A373K (at the ThDP-binding site), G25N or G590N (at the surface of the protein) already results in the impossibility of expressing the enzyme in a soluble form. The expression and purification of mutants A143P and A373P resulted in small amounts of active enzyme in the range of one order of magnitude less than for the wt and the other mutants. One of the three selected mutations (A373K) at the cofactor binding sites did not allow for expression of soluble protein and mutation A373P was only expressible in small amounts. Taking these results together, one can suggest, that mutations near the cofactor binding sites bear a high risk of making protein folding impossible. This is especially the case if a neutral amino acid is replaced by a basic one.

151 7 Final Discussion and Outlook

7.2 Stabilising Concepts

7.2.1 Quaternary structure Stabilisation

Mutant L323Q led to a significant stabilisation of ∆Td +1,4°C compared to the wt. This improvement is comparable to the increasing ∆Td of +1,1°C for the lpPOX mutant A131I, which also stabilised the tetrameric state of the enzyme (Nagel 2004). These results at a quaternary structure level confirmed the hypothesis of possible stabilisation by improving the interactions between the POX monomers. The point mutations in the gene of the lpPOX quasi-wt P178S, S188N and A458V do not affect the secondary and tertiary structures. Risse et al. (1992b) showed that both improved lig- and binding and subunit interactions contribute to the observed thermal stabilisation. So a quaternary stabilisation strategy is the most promising stabilisation approach, especially with regard to lpPOX. Although the first experiments done with a possible mutant that have formed disulfid bridges between two monomers failed (Nagel 2004), the strategy of connecting monomers covalently could be developed further. The effect of covalent stabilisation of proteins by introducing disulfid bonds are described as very important and linked with a high increase in thermal stability. Mansfeld et al. (1997) reported on the introduction of disulfid bonds between protein domains and measured an increase of Td of +16.7°C for mutants of thermolysine-like protease. Wurth et al. (2001) connected whole monomers of a multimeric enzyme and showed an increase of the Tm determined on circular dichroism of +16°C for herpes simplex virus type 1 thymidine kinase.

7.2.2 Stabilisation at the Cofactor Binding Site

Mutation Ala373->Lys at the ThDP binding site tends to result in incorrectly folded insoluble protein. The difficulties during production of the mutant protein showed that a replacement by a fundamentally different amino acid (neutral versus basic) close to the active site might be hazardous and cannot be recommended as a strategy for other protein stabilising projects. Mutant I221T showed comparable stability to the wt. The expected stabilisation by generating a new hydrogen bond between Thr221 and FAD could not be confirmed. Although this mutation could be found in the POX of Oceanobacillus iheyensis (see figure 3-6) the theoretical effect of better cofactor binding did not lead to measurable improvement of thermal stabilisa-

152 7.3 Outlook tion. Although a few further strategies stabilising the enzyme at the cofactor site are discussed in paragraph 5.4.2 the existing results can be summarised as unsuccessful and I would recommend focussing on other stabilising strategies. Rational stabilisation by mutations near the cofactor binding sites is a big challenge as it has been shown by this example.

7.2.3 Stabilisation by Backbone Rigidification

Different rigidisation mutants led to more thermostable enzymes, but the higher thermostability is not connected to longer shelf life of the enzyme, as it has been shown in chapter 6. The reason for this is different deactivation mechanisms at high and at room temperature. S559P is the first mutant that fulfils the requirements defined in the research plan of losing less than 50% of activity at pH=6. Thus the aim of this project which was to develop and produce a stabilised lpPOX in a way that the decrease in POX-activity during two weeks of continuous measurement with the biosensor would be less than 50% is partially fulfilled. Nevertheless, at the moment no protein could be engineered, which fulfilled the project objective of less than 50% loss of activity at pH =7. The goal was not only to further improve the stability of lpPOX but also to achieve that goal without loss of activity. This secondary objective is also fulfilled with the similar activity of mutant S559P compared to the wt (see figure 6-3). The stability of the enzyme was improved and the enzyme activity was at least conserved. Therefore, one can conclude that protein engineering by rational protein design was a successful approach for stabilising lpPOX. However, so far, it is not known whether these results can be translated into a more stable biosensor. Stability and activity tests are planned for the first stabilised lpPOX enzymes in a biosensor measuring pyruvate or phosphate. The suggested procedure developed by Nagel (2004) for searching and selecting new proline mutation sites was feasible to a certain extend and could be applied to other proteins that have to be stabilised after implementing one confinement. The “Ooi“ term should be limited to a value of maximal 0,6 (see paragraph 6.4.1).

7.3 Outlook

In the next step the kinetic stabilisation of the two quaternary stabilising mutants A131I and L323Q had to be tested in longterm stability assays. In successive steps, all mutations (including

153 7 Final Discussion and Outlook

S559P) which showed a better shelf life than the wt had to be inserted into one or different multiple-mutant enzymes and activity and stability of that protein had to be investigated. A further step in rational stabilisation of lpPOX should involve searching for the primary unfolding region as described by Köditz et al. (2004). When the weak sites of the protein, where movements for cofactor loss are originate, are known, one can focus protein engineering to that region. If the analysis of rational designed mutants in the biosensor are not satisfactory, the use of structure-guided random mutagenesis or DNA-shuffling can be considered in order to achieve the desired stabilisation of lpPOX. However, the unsolved problem of simulating the environment in the paste is not easy to avoid. In principle, other phosphate measuring techniques must also be considered in the case, if lpPOX or an other enzymatic cascade for phosphate does not achieve the proposed aims. In the phosphate sensors research field, great efforts have been made to design artificial, stable recognition molecules. Further inputs about this topic have been given within chapter 3.

154 7.3 Outlook

155 156 Bibliography

Ahern, T. J., and Kilbanov, A. M. (1985). The Mechanism of Irreversible Enzyme Inactivation at 100 degrees C. Science 228, 1280-1284.

Akhtar, M., S., Ahmad, A., and Bhakuni, V. (2002). Divalent Cation Induced Changes in Struc- tural Properties of the Dimeric Enzyme Glucose Oxidase. Biochemistry 41(22), 7142 - 7149.

Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., and Fu, P.C. (1974) Enzymatic determination of total serum-cholesterol. Clinical Chemistry, 20(4), 470-475.

Ambühl, H. (1982). Phosphate und Gewässerschutz: Untersuchungen zur Eutrophierung der Alpenrandseen. Seifen-Öle-Fette-Wachse 108(15), 453-459.

Amersham Pharmacia Biotech AB; Protein Purification Handbook (1999) Uppsala, Sweden

Antonisse, M.M.G., Snellink-Ruel, B.H.M., Engbersen, J.F.J., and Reinhoudt, D.N. (1998) H2O4-selective CHEMFETs with uranyl salophene receptors. Sensors and Actuators B: Chemical, 47(1-3), 9-12.

Arai, G., Noma, T., Habu, H., and Yasumori, I. (1999). Pyruvate sensor based on pyruvate oxidase immobilized in a poly(mercapto-p-benzoquinone) film. Journal Of Electroanalytical Chemistry 464(2), 143-148.

Athes, V., Guerra, P., and Combes, D. (1999). Effect of soluble additives on enzyme thermo- and/or baro-deactivation. Journal of Molecular Catalysis B: Enzymatic 7(1-4), 1-9.

Bar-Ilan, A., Balan, V., Tittman, K., Golbik, R., Vyazmensky, M., Hubner, G., Barak, Z., and Chipman, D.M. (2001) Binding and activation of thiamin diphosphate in acetohydroxyacid synthase. Biochemistry, 40(39), 11946-11954.

Becker, N., Oroudjev, E., Mutz, S., Cleveland, J. P., Hansma, P. K., Hayashi, C. Y., Makarov, D. E., and Hansma, H. G. (2003). Molecular nanosprings in spider capture-silk threads. Nature materials 2.

Benson, D. E., Conrad, D. W., de Lorimier, R. M., Trammell, S. A., and Hellinga, H. W. (2001). Design of Bioelectronic Interfaces by Exploiting Hinge-Bending Motions in Proteins. Science 293(5535), 1641-1644.

Bergmann, W., Rudolph, R., and Spohn, U. (1999). A bienzyme modified carbon paste electrode for amperometric detection of pyruvate. Analytica Chimica Acta 394(2-3), 233-241.

Berkel, W.J.H., Benen, J.A.E., and Snoek, M.C. (1991) On the FAD-induced dimerization of apo-lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluore- scens. Kinetics of reconstitution. European Journal of Biochemistry, 197(3), 769-779.

157 Bhandari, P., and Gowrishankar, J. (1997). An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer. Journal of Bacteriology 179(13), 4403-4406.

Bjork, A., Dalhus, B., Mantzilas, D., Eijsink, V.G., and Sirevag, R. (2003) Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface. J Mol Biol., 334(4), 811-821.

Böhm, H. J., Klebe, G., and Kubinyi, H. (1996). Protein Ligand Wechselwirkungen. In "Wirk- stoffdesign" (H. J. Böhm, ed.), pp. 95-111. Spektrum Akad. Verl., Heidelberg.

Boonyaratanakornkit, B. B., Park, C. B., and Clark, D. S. (2002). Pressure effects on intra- and intermolecular interactions within proteins. Biochimica et Biophysica Acta (BBA) - Pro- tein Structure and Molecular Enzymology 1595(1-2), 235-249.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254.

Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994). Direct, Real-Time Measure- ment of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Ap- plication to Actomyosin Subfragment 1 ATPase. Biochemistry 33, 8262-8271.

Brune, M., Hunter, J. L., Howell, S. A., Martin, S. R., Hazlett, T. L., Corrie, J. E. T., and Webb, M. R. (1998). Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37, 10370-10380.

Bryan, P. N. (2000). Protein engineering of subtilisin. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1543(2), 203-222.

Campanella, L., Cordatore, M., Mazzei, F., Tomassetti, M., and Volpe, G. (1992). Phosphate determination in foodstuffs using a plant tissue electrode. Food Chemistry 44(4), 291- 297.

Carey, C. M., and Riggan, W. B. J. (1994). Cyclic polyamine ionophore for use in a dibasic phosphate-selective electrode. Anal Chem. 66(21), 3587-91.

Chaniotakis, N. A., Jurkschat, K., and Ruhlemann, A. (1993). Potentiometric phosphate selective electrode based on a multidendate--tin (IV) carrier. Analytica Chimica Acta 282(2), 345-352.

Chen, Z., De Marco, R., and Alexander, P. W. (1997). Flow-injection Potentiometric Detection of Phosphates Using a Metallic Cobalt Wire Ion-selective Electrode. Analytical Commu- nications 34(3), 93 - 95.

Clark, L. C. J., and Lyons, C. (1962). Electrode systems for continuous monitoring in cardio- vascular surgery. Ann N Y Acad Sci. 102, 29-45.

Clarke, J., and Shannon, L. M. (1976). The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C. Biochim Biophys Acta. 427(2), 428-42.

158 Conrath, N., Grundig, B., Hüwel, S., and Cammann, K. (1995). A novel enzyme sensor for the determination of inorganic phosphate. Analytica Chimica Acta 309(1-3), 47-52. d’ Urso, E. M., and Coulet, P. R. (1990). Phosphate-sensitive enzyme electrode: a potential sensor for environment control. Anal.Chim.Acta 239(1), 1-5. d’ Urso, E. M., and Coulet, P. R. (1993). Effect of enzyme ratio and enzyme loading on the performance of a bienzymatic electrochemical phosphate biosensor. Analytica Chimica Acta 281(3), 535-542.

Danson, M. J., and Hough, D. W. (1998). Structure, function and stability of enzymes from the Archaea. Trends in Microbiology 6(8), 307-314.

De Lorimier, R. M., Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M., Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., and Hellinga, H. W. (2002). Construction of a fluorescent biosensor family. Protein Sci 11(11), 2655-2675.

Decker, K., and Brandsch, R. (1997) Determining covalent flavinylationMethods in Enzymolo- gy, p. 413-423. Academic Press.

Demuth, C., Zerbe, O., Rognan, D., Soll, R., Beck-Sickinger, A., Folkers, G., and Spichiger, U. E. (2001). A rationally designed oligopeptide shows significant conf changes upon binding to sulphate ions. Biosensors & Bioelectronics 16(9-12), 783-789.

DeSantis, G., and Jones, J. B. (1999). Chemical modification of enzymes for enhanced functionality. Current Opinion in Biotechnology 10(4), 324-330.

DeSantis, G., Berglund, P., Stabile, M., R., Gold, M., and Jones, J., B. (1998). Site-directed mutagenesis combined with chemical modification. Biochemistry. 37(17), 5968-73.

Directive No. 95/2/EC on Food Additives other than Colours and Sweeteners, decreed by the European Parliament and Council, (status 20. Feb 1995).

Dong, S., and Chen, X. (2002). Some new aspects in biosensors. Reviews in Molecular Biotech- nology 82(4), 303-323.

Durrschmidt, P., Mansfeld, J., and Ulbrich-Hofmann, R. (2005). An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding. FEBS J 272(6), 1523-1534.

Dwyer, M. A., and Hellinga, H. W. (2004). Periplasmic binding proteins: a versatile superfami- ly for protein engineering. Current Opinion in Structural Biology 14(4), 495-504.

Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad, B., van den Burg, B., and Vriend, G. (2004). Rational engineering of enzyme stability. J Biotechnol. 113(1-3), 105-120.

Eijsink, V. G., Gaseidnes, S., Borchert, T. V., and van den Burg, B. (2005). Directed evolution of enzyme stability. Biomol Eng. 22(1-3), 21-30.

159 Elstner, E., Schleifer, K., H., Götz, F., Sedewitz, B., Röder, A., Möllering, H., and Seidel, H. (1987). Pyruvate-oxidase which does not require FAD TPP or divalent metal ions Boe- hringer Mannheim GmbH; Germany.

Engblom, S. O. (1998). Determination of inorganic phosphate in a soil extract using a cobalt electrode. Plant and Soil 206(2), 173-179.

Engblom, S. O. (1998). The phosphate sensor. Biosensors-Bioelectron. 13(9), 981-94.

Fedor, M. J., and Williamson, J. R. (2005). The Catalytic Diversity of RNAs. Nature Reviews Molecular Cell BiologyNat Rev Mol Cell Biol 6(5), 399-412.

Fernandez, J. J., Lopez, J. R., Correig, X., and Katakis, I. (1998). Reagentless carbon paste phosphate biosensors: preliminary studies. Sensors and Actuators B: Chemical 47(1-3), 13-20.

Fibbioli, M., Berger, M., Schmidtchen, F. P., and Pretsch, E. (2000). Polymeric Membrane Electrodes for Monohydrogen Phosphate and Sulfate. Anal. Chem. 72(1), 156-160.

Fischer, G., Bang, H., and Mech, C. (1984). Determination of enzymatic catalysis for the cis- trans-isomerization of peptide binding in proline-containing peptides. Biomed Biochim Acta. 43(10), 1101-11.

Fisher, W.R., Taniuchi, H., and Anfinsen, C.B. (1973) On the Role of Heme in the Formation of the Structure of Cytochrome c. J. Biol. Chem., 248(9), 3188-3195.

Fraaije, M. W., van den Heuvel, R. H. H., Mattevi, A., and van Berkel, W. J. H. (2003). Cova- lent flavinylation enhances the oxidative power of vanillyl-alcohol oxidase. Journal of Molecular Catalysis B-Enzymatic 21(1-2), 43-46.

Fraaije, M. W., van den Heuvel, R. H. H., van Berkel, W. J. H., and Mattevi, A. (2000). Struc- tural analysis of flavinylation in vanillyl-alcohol oxidase. Journal of Biological Chemistry 275(49), 38654-38658.

Friedemann, R., and Neef, H. (1998). Theoretical Studies on the Electronic and Energetic Pro- perties of the Aminopyrimidine Part of Thiamin Phosphate. Biochimica et Biophysica Acta 1385, 245-250.

Frokjaer, S., and Otzen, D. E. (2005). Protein drug stability: a formulation challenge. Nat Rev Drug Discov 4(4), 298-306.

Gajovic, N., Binyamin, G., Warsinke, A., Scheller, F. W., and Heller, A. (2000). Operation of a miniature redox hydrogel-based pyruvate sensor in undiluted deoxygenated calf serum. Analytical Chemistry 72(13), 2963-2968.

Gajovic, N., Habermuller, K., Warsinke, A., Schuhmann, W., and Scheller, F. W. (1999). A pyruvate oxidase electrode based on an electrochemically deposited redox polymer. Electro- analysis 11(18), 1377-1383.

160 Gajovic, N., Warsinke, A., and Scheller, F. W. (1997). Comparison of two enzyme sequences for a novel L-malate biosensor. J.Chem.Technol.Biotechnol. 68(1), 31-36.

Ganjali, M. R., Mizani, F., Emami, M., Niasari, M. S., Shamsipur, M., Yousefi, M., and Javan- bakht, M. (2003). Novel Liquid Membrane Electrode for Selective Determination of Mo- nohydrogenphosphate. Electroanalysis 15(2), 139 - 144.

Ganter, C., and Pluckthun, A. (1990) Glycine to alanine substitutions in helices of glyceraldehyde- 3-phosphate dehydrogenase - effects on stability. Biochemistry, 29(40), 9395- 9402.

Garcia, D., Ortéga, F., and Marty, J., L. (1998). Kinetics of thermal inactivation of horseradish peroxidase: stabilizing effect of methoxypoly(ethylene glycol). Biotechnol. Appl. Bio- chem. 27, 49-54.

Gaseidnes, S., Synstad, B., Jia, X., Kjellesvik, H., Vriend, G., and Eijsink, V.G.H. (2003) Sta- bilization of a Chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro Mutations. Protein Engineering, 16(11), 841-846.

Gaseidnes, S., Synstad, B., Nielsen, J. E., and Eijsink, V. G. H. (2003). Rational engineering of the stability and the catalytic performance of enzymes. Journal of Molecular Catalysis B- Enzymatic 21(1-2), 3-8.

Gavalas, V. G., and Chaniotakis, N. A. (2001). Phosphate biosensor based on polyelectrolyte- stabilized pyruvate oxidase. Analytica Chimica Acta 427(2), 271-277.

Gerard, M., Ramanathan, K., Chaubey, A., and Malhotra, B. D. (1999). Immobilization of lactate dehydrogenase on electrochemically prepared polyaniline films. Electroanalysis 11(6), 450-452.

Goddette, D. W., Christianson, T., Ladin, B. F., Lau, M., Mielenz, J. R., Paech, C., Reynolds, R. B., Yang, S. S., and Wilson, C. R. (1993). Strategy and Implementation of a System for Protein Engineering. Journal of Biotechnology 28(1), 41-54.

Goff, T. L., Braven, J., Ebdon, L., and Scholefield, D. (2004). Phosphate-selective electrodes containing immobilised ionophores. Analytica Chimica Acta 510(2), 175-182.

Götz, F., and Sedewitz, B. (1991). Physiological role of pyruvate oxidase in the aerobic metabolism in Lactobacillus plantarum. In "in Biochemistry and Physiology of Thiamine-Di- phosphate Enzymes" , pp. 286-293. Verlag VCH Weinheim, Basel.

Gouda, M. D., Singh, S. A., Rao, A. G. A., Thakur, M. S., and Karanth, N. G. (2003). Thermal Inactivation of Glucose Oxidase: Mechanism and Stabilization Using Additives. J. Biol. Chem. 278(27), 24324-24333.

Govardhan, C. P. (1999). Crosslinking of enzymes for improved stability and performance. Current Opinion in Biotechnology 10(4), 331-335.

161 Gowrishankar, J., Poonam, B., and Kaveti, R. (1998). Process for producing polypeptides pat. no. US-5830690.

Graziano, G., Catanzano, F., Riccio, A., and Barone, G. (1997). A reassessment of the molecular origin of cold denaturation. J Biochem (Tokyo) 122(2), 395-401.

Griffiths, D., and Hall, G. (1993). Biosensors - what real progress is being made? Trends Bio- technol 11(4), 122-130.

Grimm, W., and Gothier, D. (1999) Stabilität. In K.-D. Herzfeldt, and J. Kreuter, Eds. Grund- lagen der Arzneiformenlehre - Galenik 2. Springer Verlag, Berlin.

Guilbault, G. G., and Nanjo, M. (1975). A phosphate-selective electrode based on immobilized alkaline phosphatase and glucose oxidase. Anal Chim Acta 78(1), 69-80.

Gupta, V. K., Ludwig, R., and Agarwal, S. (2005). Anion recognition through modified calixarenes: a highly selective sensor for monohydrogen phosphate. Analytica Chimica Acta 538(1-2), 213-218.

Halle, B., Denisov, V. P., Modig, K., and Davidovic, M. (2005). Protein Conformational Tran- sitions as Seen from the Solvent. In "Protein Folding Handbook" (J. Buchner and T. Kief- haber, eds.), Part I, pp. 201-246. Wiley-VCH Verlag GmbH & Co., Weinheim.

Hartley, G. (1936). Aqueous solutions of paraffin-chain salts; Hermann and Cie; Paris as quoted in "The Hydrophobic Effect - Formation of Micelles and Biological Membranes" John Wiley & Sons, New York.

Haupt, K., and Mosbach, K. (2000). Molecularly Imprinted Polymers and Their Use in Biomi- metic Sensors. Chem. Rev. 100(7), 2495-2504.

Hawkins, C.F., Borges, A., and Perham, R.N. (1989) A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Letters, 255(1), 77-82.

Hayashi, S., and Nakamura, S. (1981). Multiple forms of glucose oxidase with different carbohydrate compositions. Biochimica et Biophysica Acta (BBA) - Enzymology 657(1), 40- 51.

He, X. Y., and Rechnitz, G. A. (1995). Plant Tissue-Based Fiberoptic Pyruvate Sensor. Analy- tica Chimica Acta 316(1), 57-63.

Hecht, H. J., Kalisz, H. M., Hendle, J., Schmid, R. D., and Schomburg, D. (1993). Crystal-Struc- ture of Glucose-Oxidase from Aspergillus-Niger Refined at 2 .3 Angstrom Resolution. Journal of Molecular Biology 229(1), 153-172.

Hefti, M.H., Vervoort, J., and van Berkel, W.J.H. (2003) Deflavination and reconstitution of flavoproteins: Tackling fold and function. Eur J Biochem, 270(21), 4227-4242.

Hei, D. J., and Clark, D. S. (1994). Pressure Stabilization of Proteins from Extreme Thermophi- les. Appl Environ Microbiol. 60(3), 932-939.

162 Henson, A. F., Mitchell, J. R., and Mussellwhite, P. R. (1970). The surface coagulation of proteins during shaking. Journal of Colloid and Interface Science 32(1), 162-165.

Hirshberg, M., Henrick, K., Haire, L. L., Vasisht, N., Brune, M., Corrie, J. E. T., and Webb, M. R. (1998). Crystal Structure of PBP Labeled with a Coumarin Fluorophore, a Probe for Pi. Biochemistry 37(29), 10381 -10385.

Hossain, M. T., Suzuki, K., Yamamoto, T., Imamura, S., Sekiguchi, T., and Takanaka, A. (2002). Crystal Structure of Pyruvate Oxidase Complexed with FAD and TPP, from Ae- rococcus viridans. pdb database 1V5F.

Hough, D. W., and Danson, M. J. (1999). Extremozymes. Current Opinion in Chemical Biology 3(1), 39-46.

Hüwel, S., Haalck, L., Conrath, N., and Spener, F. (1997). Maltose phosphorylase from Lacto- bacillus brevis: purification, characterization, and application in a biosensor for orthophosphate. Enzyme Microb Technol. 21(6), 413-20.

Ikebukuro, K., Nishida, R., Yamamoto, H., Arikawa, Y., Nakamura, H., Suzuki, M., Kubo, I., Takeuchi, T., and Karube, I. (1996). A novel biosensor system for the determination of phosphate. Journal of Biotechnology 48(1-2), 67-72.

Ikebukuro, K., Wakamura, H., Karube, I., Kubo, I., Inagawa, M., Sugawara, T., Arikawa, Y., Suzuki, M., and Takeuchi, T. (1996). Phosphate sensing system using pyruvate oxidase and chemiluminescence detection. Biosensors & Bioelectronics 11(10), 959-965.

Institute for Clinical Chemistry, University Hospital Zurich. (1998). Vademecum IKC. Vonder- schmitt, D. J.

IUPAC. (1999). Electrochemical Biosensors: Recommended Definitions and Classification. Pure Appl. Chem. 71(12), 2333-2348.

Jensen, K. F., et al. (1999). Roles of Three Prosthetic Groups in the Tetrameric Dihydroorotate Dehydrogenase B from Lactobacillus lactis. In "Flavins and Flavoproteins" (S. Ghisla, et al., eds.), pp. 599-602. Ag. f. Sc. Publ., Berlin.

Jones, M. N., Manley, P., and Wilkinson, A. (1982). The dissociation of glucose oxidase by sodium n-dodecyl sulphate. Biochem J. 203(1), 285-291.

Kalisz, H. M., Hecht, H.-J., Schomburg, D., and Schmid, R. D. (1991). Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger. Biochimica et Biophysica Acta, 1080(2), 138-142.

163 Karube, I., and Nomura, Y. (2000). Enzyme sensors for environmental analysis. Journal of Mo- lecular Catalysis B-Enzymatic 10(1-3), 177-181.

Kazan, D., Ertan, H., and Erarslan, A. (1997). Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers. Appl Microbiol Biotechnol. 48(2), 191-7.

Keilin, D., and Hartree, E. F. (1951). Purification of horse-radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. J Biochem (Tokyo) 49(1), 88- 104.

Keller, H. (1991). "Klinisch-chemische Labordiagnostik für die Praxis." second ed. Georg Thie- me Verlag, Stuttgart.

Kern, D., Kern, G., Neef, H., Tittmann, K., KillenbergJabs, M., Wikner, C., Schneider, G., and Hubner, G. (1997). How thiamine diphosphate is activated in enzymes. Science 275(5296), 67-70.

Kim, J., Beak, D.-G., Kim, Y.-T., Choi, J.-D., and Yoon, M.-Y. (2004) Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding. Biochem. J., 384(1), 59-68.

Klibanov, A. M. (1997). Why are enzymes less active in organic solvents than in water? Trends in Biotechnology 15(3), 97-101.

Köditz, J., Ulbrich-Hofman, R., and Arnold, U. (2004) Probing the unfolding region of ribonuclease A by site-directed mutagenesis. Eur. J. Biochem., 271, 4147–4156.

Kopetzki, E., and Schumacher, G. (1988). Verfahren zur Herstellung von Proteinen in löslicher Form, No. 0 300 425 A2, Boehringer Mannheim GmbH, Deutschland.

Kopetzki, E., Schumacher, G., and Buckel, P. (1989). Control of Formation of Active Soluble or Inactive Insoluble Bakers-Yeast Alpha-Glucosidase-Pi in Escherichia-Coli by Induc- tion and Growth-Conditions. Molecular & General Genetics 216(1), 149-155.

Korell, U., and Spichiger, U. E. (1994). Kinetic-Studies on Membraneless Amperometric Bio- sensors Prepared from Xanthine-Oxidase, Organic Conducting Salt, and Silicone Oil. Electroanalysis 6(4), 305-315.

Kruger, N.J. (1996) The Bradford Method for Protein Quantitation. In J.M. Walker, Ed. The Protein Protocols Handbook, p. 15-20. Humana Press Inc., Totowa.

Kubo, I., Inagawa, M., Sugawara, T., Arikawa, Y., and Karube, I. (1991). Phosphate Sensor Composed from Immobilized Pyruvate Oxidase and an Oxygen-Electrode. Analytical Letters 24(10), 1711-1727.

Kulys, J., Wang, L., and Daugvilaite, N. (1992). Amperometric methylene green-mediated pyruvate electrode based on pyruvate oxidase entrapped in carbon paste. ANALYTICA CHIMICA ACTA 265(1), 15-20.

164 Kunz, W., Henle, J., and Ninham, B. W. (2004). Zur Lehre von der Wirkung der Salze (about the science of the effect of salts): Franz Hofmeister’s historical papers. Current Opinion in Colloid & Interface Science 9(1-2), 19-37.

Kutuzov, M. A., and Andreeva, A. V. (2001). Noncompetitive Inhibition of Plant Protein Ser/ Thr Phosphatase PP7 by Phosphate. Biochemical and Biophysical Research Communica- tions 283(1), 93-96.

Kwan, R. C. H., Leung, H. F., Hon, P. Y. T., Barford, J. P., and Renneberg, R. (2005). A screen- printed biosensor using pyruvate oxidase for rapid determination of phosphate in synthetic wastewater. Applied Microbiology and Biotechnology 66(4), 377-383.

Kwan, R. C. H., Leung, H. F., Hon, P. Y. T., Cheung, H. C. F., Hirota, K., and Renneberg, R. (2005). Amperometric biosensor for determining human salivary phosphate. Analytical Biochemistry 343(2), 263-267.

Lehninger, A. L., Nelson, D. L., and Cox, M. M. (1993). Secondary structures of proteins. second ed. In "Principles of Biochemistry", pp. 187. Worth Publishers, Inc., New York.

Lehninger, A. L., Nelson, D. L., and Cox, M. M. (1993). Three dimensional structures of proteins. second ed. In "Principles of Biochemistry" Worth Publishers, Inc., New York.

Lilley, D. M. J. (2003). The origins of RNA catalysis in ribozymes. Trends in Biochemical Sci- ences 28(9), 495-501.

Lindgren, A. et al. (2000). Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer. Biosensors and Bioelectronics 15(9-10), 491-497.

Liu, D., Chen, W.-C., Yang, R.-H., Shen, G.-L., and Yu, R.-Q. (1997). Polymeric membrane phosphate sensitive electrode based on binuclear organotin compound. Analytica Chimi- ca Acta 338(3), 209-214.

Liu, J., Masuda, Y., and Sekido, E. (1990). Response properties of an ion-selective polymeric membrane P electrode prepared with cobalt phthalocyanines. Journal of Electroanalytical Chemistry 291(1-2), 67-79.

Lu, J., Nogi, Y., and Takami, H. (2001). Oceanobacillus iheyensis gen. nov., sp. nov., a deep- sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol Lett. 205(2), 291-7.

Luecke, H., and Quiocho, F. A. (1990). High specificity of a phosphate transport protein determined by hydrogen bonds. 347(6291), 402-406.

Maa, Y. F., and Hsu, C. C. (1997). Effect of primary emulsions on microsphere size and protein- loading in the double emulsion process. J Microencapsul. 14(2), 225-41.

Maillard, L. C. (1912). Action des acides amines sur les sucres: formation des melanoidines par voie methodique. Seances Acad Sci 154, 66-68.

165 Mak, W. C., Chan, C., Barford, J., and Renneberg, R. (2003). Biosensor for rapid phosphate monitoring in a sequencing batch reactor (SBR) system. Biosensors and Bioelectronics 19(3), 233-237.

Male, K. B., and Luong, J. H. T. (1991). An FIA biosensor system for the determination of phosphate. Biosensors and Bioelectronics 6(7), 581-587.

Mansfeld, J., Vriend, G., Dijkstra, B., W., Veltman, O., R., Van den Burg, B., Venema, G., Ul- brich-Hofmann, R., and Eijsink, V.G.H. (1997) Extreme stabilisation of a thermolysine- like protease. the journal of biological chemistry, 272(17), 11152-11156.

Marcus, R. A., and Sutin, N. (1985). Electron transfers in chemistry and biology. Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics 811(3), 265-322.

Margarit, I., Campagnoli, S., Frigerio, F., Grandi, G., Defilippis, V., and Fontana, A. (1992) Cu- mulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease. Protein Engineering, 5(6), 543-550.

Mascini, M., and Mazzei, F. (1987). Amperometric sensor for pyruvate with immobilized pyruvate oxidase. Analytica Chimica Acta 192, 9-16.

Matsumura, E., Imamura, S., Sagai, H., Misaki, H., and Nogata, K. (1988). Pyruvate oxidase, its preparation and use; pat. no: EP-0274425-A3.

Matthews, B.W., Nicholson, H., and Becktel, W.J. (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Nat. Acad. Sci. of the United States of America, 84(19), 6663-6667.

Maurer, P., and Hohenester, E. (1997). Structural and functional aspects of calcium binding in extracellular matrix proteins. Matrix Biol. 15(8-9), 569-80.

Mayr, L. M., Landt, O., Hahn, U., and Schmid, F. X. (1993). Stability and Folding Kinetics of Ribonuclease T1 are Strongly Altered by the Replacement of Cis-proline 39 with Alanine. Journal of Molecular Biology 231(3), 897-912.

McAteer, K., Simpson, C.E., Gibson, T.D., Gueguen, S., Boujtita, M., and El Murr, N. (1999) Proposed model for shelf-life prediction of stabilised commercial enzyme-based systems and biosensors. Journal of Molecular Catalysis B: Enzymatic, 7(1-4), 47-56.

McHugh, C. A., Tammariello, R. F., Millard, C. B., and Carra, J. H. (2004). Improved stability of a protein vaccine through elimination of a partially unfolded state. Protein Sci 13(10), 2736-2743.

Mizutani, F., Yabuki, S., Sato, Y., Sawaguchi, T., and Iijima, S. (2000). Amp determination of pyr, Pi and urea using enzyme electrodes based on POX-containing poly(vinyl alcohol)/ polyion complex-bilayer membrane. Electrochimica Acta 45(18), 2945-2952.

Mousty, C., Cosnier, S., Shan, D., and Mu, S. L. (2001). Trienzymatic biosensor for the determination of inorganic phosphate. Analytica Chimica Acta 443(1), 1-8.

166 Mozhaev, V. V., Lange, R., Kudryashova, E. V., and Balny, C. (1996). Application of high hydrostatic pressure for increasing activity and stability of enzymes. Biotechnology and Bioengineering 52(2), 320-331.

Mozhaev, V. V., Lange, R., Kudryashova, E. V., and Balny, C. (1996). Application of high hydrostatic pressure for increasing activity and stability of enzymes. Biotechnology and Bioengineering 52(2), 320-331.

Mueller, J. (2000). Entwicklung eines modularen Durchflusssystems auf der Basis von ampe- rometrisch-enzymatischen Biosensoren unter Verwendung von TTF-TCNQ als Mediator. Ph. D. thesis. Swiss Federal Institute of Technology Zurich.

Muller, Y. A., and Schulz, G. E. (1993). Structure of the thiamine-and flavin-dependent enzyme pyruvate oxidase. SCIENCE 259(5097), 965-968.

Muller, Y. A., Schumacher, G., Rudolph, R., and Schulz, G. E. (1994). The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum. Journal of Molecular Biology 237(3), 315-335.

Nagel, S. (2004) Towards a Stable Enzymatic Phosphate and Pyruvate Sensor. Institut für Phar- mazeutische Wissenschaften, p. 206. Swiss Federal Institute of Technology Zurich, Zu- rich.

Nakamura, H., Ikebukuro, K., McNiven, S., Karube, I., Yamamoto, H., Hayashi, K., Suzuki, M., and Kubo, I. (1997). A chemiluminescent FIA biosensor for phosphate ion monitoring using pyruvate oxidase. Biosensors and Bioelectronics 12(9-10), 959-966.

Nakamura, H., Tanaka, H., Hasegawa, M., Masuda, Y., Arikawa, Y., Nomura, Y., Ikebukuro, K., and Karube, I. (1999). An automatic FIA system for determining Pi-ion in river water using POX G (from Aerococcus viridans). Talanta 50(4), 799-807.

Nakamura, S., and Hayashi, S. (1974). A role of the carbohydrate moiety of glucose oxidase: Kinetic evidence for protection of the enzyme from thermal inactivation in the presence of sodium dodecyl sulfate. FEBS Letters 41(2), 327-330.

Nicholson, H., Tronrud, D.E., Becktel, W.J., and Matthews, B.W. (1992) Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme. Biopolymers, 32(11), 1431-1441.

Novy, R., Berg, J., K., Y., and Mierendorf, R. (1995) pET TRX Fusion System for Increased Solubility of Proteins Expressed in E. coli. inNovations - Newsletter of Novagen, Inc.(3), 7-9.

Pajot, P. (1976) Fluorescence of Proteins in 6M Guanidie Hydrochloride. European Journal of Biochemistry, 63, p. 263-269.

Parellada, J., Narvaez, A., Lopez, M. A., Dominguez, E., Fernandez, J. J., Pavlov, V., and Ka- takis, I. (1998). Amperometric immunosensors and enzyme electrodes for environmental applications. Analytica Chimica Acta 362(1), 47-57.

167 Pazur, J. H., and Kleppe, K. (1964). The Oxidation of Glucose and Related Compounds by Glu- cose Oxidase from Aspergillus niger. Biochemistry 3(4), 578-583.

Perutz , M. F., and Raidt, H. (1975). Stereochemical basis of heat stability in bacterial ferredo- xins and in haemoglobin A2. Nature 255, 256 - 259.

Petrucelli, G. C., Kawachi, E. Y., Kubota, L. T., and Bertran, C. A. (1996). Hydroxyapatite-based electrode: a new sensor for phosphate. Analytical Communications 33(7), 227 - 229.

Rahman, M. A., Park, D.-S., Chang, S.-C., McNeil, C. J., and Shim, Y.-B. (2005). The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determi- nations. Biosensors and Bioelectronics In Press, Corrected Proof.

Rajendran, V., Csoregi, E., Okamoto, Y., and Gorton, L. (1998). Amperometric peroxide sensor based on horseradish peroxidase and toluidine blue O-acrylamide polymer in carbon paste. Analytica Chimica Acta 373(2-3), 241-251.

Rebecca L. Rich, David G. Myszka (2005). Survey of the year 2003 commercial optical biosensor literature. Journal of Molecular Recognition 18(1), 1-39.

Revzin, A. F., Sirkar, K., Simonian, A., and Pishko, M. V. (2002). Glucose, lactate, and pyruvate biosensor arrays based on redox polymer/oxidoreductase nanocomposite thin-films. Sensors and Actuators B-Chemical 81(2-3), 359-368.

Rieger, P. H. (1994). Electrochemistry Chapter 4. II ed. In "Electrochemistry" Chapman and Hall, New York.

Risse, B., Schumacher, G., and Jaenicke, R. (1991) Characterization of pyruvate oxidase from Lactobacillus plantarum: Stability and reconstitution of the apoenzyme. BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 372(9), 737-738.

Risse, B., Stempfer, G., Rudolph, R., Moellinger, H., and Jaenicke, R. (1992a) Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum. Protein Science, 1(12), 1699-1709.

Risse, B., Stempfer, G., Rudolph, R., Schumacher, G., and Jaenicke, R. (1992b) Characteriza- tion of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum, Protein Science, 1(12), 1710-1718.

Robinson, K., Rothery, R., Weiner, J., and Lemire, B. (1994) The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria. Eur J Biochem, 222(3), 983-990.

Robinson, K.M., and Lemire, B.D. (1996) Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein Requires Import into Mitochondria, Presequence Removal, and Folding. J. Biol. Chem., 271(8), 4055-4060.

Rubingh, D. N. (1997). Protein engineering from a bioindustrial point of view. Current Opinion in Biotechnology 8(4), 417-422.

168 Russell, R. J. M., Gerike, U., Danson, M. J., Hough, D. W., and Taylor, G. L. (1998). Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 6(3), 351-361.

Ruzicka, J., and Marshall, G. D. (1990). Sequential injection: a new concept for chemical sensors, process analysis and laboratory assays. Analytica Chimica Acta 237, 329-343.

SAEFL. (2002). Fish from Natural or Overfertilized Waters. in Environment Switzerland 2002. p221-226. Bern: Swiss Agency for the Environment, Forests and Landscape.

SAEFL. (2002). Nothing Works Without Water. in Environment Switzerland 2002. p33-46. Bern: Swiss Agency for the Environment, Forests and Landscape.

Salins, L. L. E., Deo, S. K., and Daunert, S. (2004). Phosphate binding protein as the biorecogni- tion element in a biosensor for phosphate. Sensors and Actuators B: Chemical 97(1), 81- 89.

Scandurra, R., Consalvi, V., Chiaraluce, R., Politi, L., and Engel, P.C. (1998) Protein thermostability in extremophiles. Biochimie, 80(11), 933-941.

Scheller, F. W., Lisdat, F., and Wollenberger, U. (2005). Application of Electrically Contacted Enzymes for Biosensors. 1 ed. In "Bioelectronics - From Theory to Applications" (I. Will- ner and E. Katz, eds.), pp. 99-126. Wiley-VCH, Weinheim.

Schroers, A., Krämer, R., and Wohlrab, H. (1997). The Reversible Antiport-Uniport Conversi- on of the Phosphate Carrier from Yeast Mitochondria Depends on the Presence of a Single Cysteine. Journal of Biological Chemistry 272(16), 10558–10564.

Schumacher, G. and H. Moellering (1989). Pyruvatoxidase-Mutanten;. Patent No: EP 0365836, Boehringer Mannheim GmbH (D).

Schumacher, G., and Moellering, H. (1989). Pyruvatoxidase-Mutanten; Boehringer Mannheim GmbH (D), Patent No: EP 0365836.

Schumacher, G., and Möllering, H. (1990) Pyruvatoxidase Mutanten;. Boehringer Mannheim GmbH (Germany), pat. no: DE-3833601-A1.

Schumacher, G., and Möllering, H. (1990). Pyruvatoxidase Mutanten; Boehringer Mannheim GmbH (Germany), pat. no: DE-3833601-A1.

Schumacher, G., and Möllering, H. (1992) Pyruvate oxidase mutants, DNA expressing pyruvate oxidase and methods of the use thereof. Boehringer Mannheim GmbH; Germany, Pat No US-5153138.

Schumacher, G., Moellering, H., Geuss, U., Fischer, S., Tischer, W., Deneke, U., and Kresse, G. B. (1991). Properties and application of recombinant lpPOX and a mutant with enhanced stability. Int. Meeting on the function of thiamine diphosphate enzymes.

169 Schumacher, G., Möllering, H., Geuss, U., Fischer, S., Tischer, W., Deneke, U., and Kresse, G.B. (1990) Prop. and Appl. of Recomb. lpPOX and a Mutant. In H. Biswanger, Ed. Int. Meeting on the Function of ThDP Enzymes, 453, p. 300-308, Blaubeuren.

Schweizerische Gewässerschutzverordnung, SR 814.201 (status July 2005).

Schweizerische Lebensmittelverordnung, SR 817.02 (status 12. Juli 2005).

Scott, K., Alonso, D., Sato, S., and Daggett, V. (2007) Conformational entropy of alanine versus glycine in protein denatured states. Proc Natl Acad Sci, 104((8)), 2661–2666.

Sedewitz, B. (1983). Die Pyruvat-Oxidase aus Lactobacillus plantarum und ihre Bedeutung im aeroben Stoffwechsel. Ph. D. thesis. Technische Universität München.

Sedewitz, B., Moellering, H., Kresse, G. B., and Comer, M. (1986). Isolation and purification of pyruvate-oxidase from Lactobacillus plantarum.

Sedewitz, B., Schleifer, K.H., and Götz, F. (1984) Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum. J Bacteriol, 160(1), 273-278.

Situmorang, M., Gooding, J. J., Hibbert, D. B., and Barnett, D. (2002). The development of a pyruvate biosensor using electrodeposited polytyramine. Electroanalysis 14(1), 17-21.

Spichiger, U. E. (1998). Potentiometric Chemical Sensors and Biological Applications. In "Chemical Sensors and Biosensors for Medical and Biological Applications", p. 199. Wi- ley-VCH, Weinheim.

SR 817.021.23, Verordnung des EDI über Fremd- und Inhaltsstoffe in Lebensmitteln, decreed by the Swiss Federal Department of Home Affairs (status 22. Februar 2005).

Steensma, E., Nijman, M., Bollen, Y., De-Jager, P.A., Van-Den-Berg, W., Van-Ddongen, W., and Van-Mierlo, C. (1998) Local stability of the sec structure of Azotobacter vinelandii holoflavodoxin II as probed by H-exchange, Protein Sci, 7(2), 306-317.

Sun, M. M. C., Caillot, R., Mak, G., Robb, F. T., and Clark, D. S. (2001). Mechanism of pressure-induced thermostabilization of proteins: Studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis. Protein Sci 10(9), 1750-1757.

Swoboda, B. E. P. (1969). The mechanism of binding of flavin-adenine dinucleotide to the apoenzyme of glucose oxidase and evidence for the involvement of multiple bonds. Biochimica et Biophysica Acta (BBA) - Protein Structure 175(2), 380-387.

Takami, H., Takaki, Y., and Uchiyama, I. (2002). Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments. Nucl. Acids Res. 30(18), 3927-3935.

Takeda, E., Taketani, Y., Sawada, N., Sato, T., and Yamamoto, H. (2004). The regulation and function of phosphate in the human body. Biofactors 21(1-4), 345-55.

170 Tittmann, K. (2000). Untersuchungen zu Katalysemechanismen von Flavin- und Thiamindi- phosphat-abhängigen Enzymen. Dissertation Universität Halle-Wittenberg.

Tittmann, K., Golbik, R., Ghisla, S., and Huebner, G. (2000). Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum. Biochemistry-. [print] Sep- tember 5, 2000; 39(35), 10747-10754.

Tittmann, K., Proske, D., Spinka, M., Ghisla, S., Rudolph, R., Huebner, G., and Kern, G. (1998). Activation of ThDP and FAD in the phosphate-dependent pyruvate oxidase from Lactobacillus plantarum. Journal of Biological Chemistry 273(21), 12929-12934.

Tuena de Gómez-Puyou, M., and Gómez-Puyou, A. (1998). Enzymes in Low Water Systems. Critical Reviews in Biochemistry and Molecular Biology 33(1), 53-89.

Van den Burg, B., and Eijsink, V. G. H. (2002). Selection of mutations for increased protein stability. Current Opinion in Biotechnology 13(4), 333-337.

Vanbelle, C., Halgand, F., Cedervall, T., Thulin, E., Akerfeldt, K. S., Laprevote, O., and Linse, S. (2005). Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding. Protein Sci 14(4), 968-979.

Vasudevan, P.T., and Li, L.O. (1996) Kinetics of phenol oxidation by peroxidase. Applied Bio- chemistry and Biotechnology, 60(3), 203-215.

Veltman, O.R., Vriend, G., Middelhoven, P.J., van den Burg, B., Venema, G., and Eijsink, V.G.H. (1996) Analysis of structural determinants of the stability of thermolysinlike proteases. Protein Eng., 9(12), 1181-1189.

Venkatesh, R., and Sundaram, P., V. (1998). Modulation of stability properties of bovine tryp- sin after in vitro structural changes with a variety of chemical modifiers. Protein Eng. 11(8), 691-8.

Walsh, G. (2005). Biopharmaceuticals: recent approvals and likely directions. Trends in Bio- technology 23(11), 553-558.

Walsh, G. Biopharmaceuticals: recent approvals and likely directions. Trends in Biotechnology In Press, Corrected Proof.

Wang, Z., Luecke, H., Yao, N., and Quiocho, F. A. (1997). A low energy short hydrogen bond in very high resolution structures of protein receptor-phosphate complexes. Nat Struct Biol 4(7), 519-522.

Warriner, K., Higson, S., and Vadgama, P. (1997). A LDH amperometric pyruvate electrode exploiting direct detection of NAD(+) at a poly(3- methylthiophene): poly(phenol red) modified platinum surface. Materials Science & Engineering C, 5(2), 91-99.

Watanabe, K., Fujita, Y., Usami, M., Takimoto, A., and Suzuki, Y. (2000) Thermodynamic analysis of Bacillus cereus oligo-1,6-glucosidase and its cumulatively proline-introduced mutant proteins by DSC. Journal of Molecular Catalysis B, 10(1-3), 257-262.

171 Watanabe, K., Kitamura, K., and Suzuki, Y. (1996) Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans. Appl Environ Microbiol., 62(6), 2066–2073.

Watanabe, K., Masuda, T., Ohashi, H., Mihara, H., and Suzuki, Y. (1994) Multiple proline substitutions cumulatively thermostabilize Bacillus cereus oligo-1,6-glucosidase. European Journal of Biochemistry, 226(2), 277-283.

Wierenga, R.K., De Maeyer, M.C., and Hol, W.G.J. (1985) Interaction of Pyrophosphate Moie- ties with a-Helixes in Dinucleotide Binding Proteins. Biochemistry, 24, 1346-1357.

Wille, G., Ritter, M., Friedemann, R., Mantele, W., and Hubner, G. (2003) Redox-Triggered FTIR Difference Spectra of FAD in Aqueous Solution and Bound to Flavoproteins. Bio- chemistry, 42(50), 14814-14821.

Willner, I., and Katz, E. (2000). Integration of Layered Redox Proteins and Conductive Sup- ports for Bioelectronic Applications. Angewandte Chemie International Edition 39(7), 1180-1218.

Wollenberger, U., and Scheller, F. W. (1993). Enzyme activation for activator and enzyme activity measurement. Biosensors and Bioelectronics 8(6), 291-297.

Wollenberger, U., Schubert, F., and Scheller, F. W. (1992). Biosensor for sensitive phosphate detection. Sensors and Actuators B: Chemical 7(1-3), 412-415.

Wroblewski, W., Wojciechowski, K., Dybko, A., Brzozka, Z., Egberink, R. J. M., Snellink- Ruel, B. H. M., and Reinhoudt, D. N. (2000). Uranyl salophenes as ionophores for phosphate-selective electrodes. Sensors and Actuators B: Chemical 68(1-3), 313-318.

Wroblewski, W., Wojciechowski, K., Dybko, A., Brzozka, Z., Egberink, R. J. M., Snellink- Ruel, B. H. M., and Reinhoudt, D. N. (2001). Durability of phosphate-selective CHEM- FETs. Sensors and Actuators B-Chemical 78(1-3), 315-319.

Wurth, C., Kessler, U., Vogt, J., Schulz, G.E., Folkers, G., and Scapozza, L. (2001) The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase. Protein Science, 10, 63–73.

Wurth, C., Thomas, R.M., Folkers, G., and Scapozza, L. (2001) Folding and self-assembly of herpes simplex virus type 1 thymidine kinase. Journal of Molecular Biology, 313(3), 657- 670.

Xiao, D., Yuan, H. Y., Li, J., and Yu, R. Q. (1995). Surface-Modified Cobalt-Based Sensor as a Phosphate-Sensitive Electrode. Analytical Chemistry 67(2), 288-291.

Yao, N., Ledvina, P. S., Choudhary, A., and Quiocho, F. A. (1996). Modulation of salt link does not affect binding of phosphate to its specific active transport receptor. Biochemistry 35, 2079.

Zaks, A., and Klibanov, A. (1988). The effect of water on enzyme action in organic media. J. Biol. Chem. 263(17), 8017-8021.

172 Zapata-Bacri, A. M., and Burstein, C. (1987). Enzyme electrode composed of the pyruvate oxidase from pediococcus species coupled to an oxygen electrode for measurements of pyruvate in biological media. Biosensors 3(4), 227-237.

Zhang, W., Chang, H. D., and Rechnitz, G. A. (1997). Dual-enzyme fiber optic biosensor for pyruvate. Analytica Chimica Acta 350(1-2), 59-65.

Zhao, M., Bada, J. L., and Ahern, T. J. (1989). Racemization rates of asparagine-aspartic acid residues in lysozyme at 100[deg]C as a function of pH. Bioorganic Chemistry 17(1), 36- 40.

Zschokke, S. (2003). Spider-web silk from the Early Cretaceous. Nature 424, 636-637.

173 174 Danksagung

hne die Mithilfe und Unterstützung von vielen Personen wäre diese Arbeit nicht möglich Ogewesen. Ihnen allen, die mir diese Arbeit ermöglicht haben und mich während meiner Dissertationszeit unterstütz haben gilt mein ganz grosser Dank. Als erstes möchte ich mich bei meinen beiden Doktorvätern Prof. Dr. Gerd Folkers und Prof. Dr. Leonardo Scapozza bedanken. Sie haben mir in Ihrer Arbeitsgruppe das freie wissen- schaftliche Arbeiten ermöglicht und mir so viele Einblicke in die Proteinchemie aber auch in die Wissenschaft ganz allgemein ermöglicht. Ich konnte in dieser Zeit vieles lernen, eigene Ideen einbringen und Projekte umsetzen. Ausserdem haben sie mich in den vielen Diskussionen mit ihrer optimistischen Einstellung dem Projekt gegenüber und ihrer motivierenden Art sehr unterstützt. Bei Prof. Dr. Ursula Spichiger bedanke ich mich für die Unterstützung und Diskussionen im Projekt in allen Belangen der Bio-Sensoren und für die Übernahme des Koreferates. Her- zlich bedanken möchte ich mich auch bei Prof. Dr. August Schubiger für die freundliche Über- nahme des Koreferates. Ein herzliches Dankeschön gilt PD Dr. Vivianne Otto, die mich gelehrt hat, den Blick auf das Wesentliche zu konzentrieren, und mich auch in schwierigeren Projektphasen immer wieder motiviert hatte. Bei Sämi möchte ich mich für die geleistete Vorarbeit, die vielen Tips im Labor und die Einführung in die Bio-Sensor-Technik bedanken. Bei Beatrice und Leah, die je eine Se- mesterarbeit zu diesem Thema verfasst haben, bedanke ich mich für ihren Einsatz und ihre Mi- tarbeit. Regula, Anja, Thomas und Markus, meinen Bürokolleginnen und –kollegen, danke ich für das kollegiale und gute Arbeitsklima. Ein Dankeschön geht auch an Remo, Eleonora und Loris für die vielen Tipps im Labor. Thomas K., Vanvan, Remo, Andi, Ingrid, Patrik, Pavel, Mine, Marc, Maria-Vittoria, Katia, Gabi und allen weiteren Mitgliedern im grossen Arbeitskreis auf dem ehemaligen M-Stock am Irchel Campus vielen Dank für die gute Stimmung im Labor, im Büro und bei den Meetings. Meiner Familie Yvonne, Fiona und meinen Eltern danke ich herzlich für die Geduld und die Zeit, in der sie mich entbehren mussten. Und ausserdem ein Dankeschön für die grosse Un- terstützung und Motivation während der ganzen Zeit. Bei Prof. Dr. Roger Schibli und Dr. Thomas Minth bedanke ich für die Aufnahme in ihrer Arbeitsgruppe am Hönggerberg. Dem CCS-Team am Technopark, allen voran Gleb, danke ich für die Mithilfe bei den Sensormessungen und die kollegiale Stimmung während und nach den Meetings. Zugleich ein Dankeschön an Herrn Bemsel und dem Team vom Pharma-Schalter für die ausserordentlich wertvolle Unterstützung und die stets funktionierende Dienstleistung der Ma- terial- und Chemikalienbestellung.

175 176 Curriculum Vitae

Personal:

Name: Matthias Kramer Date of birth: May 25, 1975 Citizenship: Full-Reuenthal, AG

Education:

1982 – 1990 Primar- und Sekundarschule Wigoltingen 1990 – 1995 Kantonsschule Frauenfeld, Typus C 1995 – 2001 Undergratuate studies in Pharmaceutical Sciences, ETH Zurich 1997 – 1998 12 months practical training, Deutweg Apotheke, Winterthur March 2001- July 2001 Diploma thesis with Prof. Dr. U. Spichiger title: Konzepte für die Entwicklung von Biosensoren auf der Basis planarer Wellenreiter November 2001 Final Examinations Degree: Eidg. Dipl. Apotheker 2001 – 2008 Graduate studies at the Institute of Pharmaceutical Sciences, ETH Zurich (Proff. G. Folkers, L. Scapozza, U. Spichiger) title: Protein Engineering of Pyruvate Oxidase from Lactobacillus plantarum for Application in Biosensors April 2006 – January 2008 Head of Research and Development, Streuli Pharma AG, Uznach SG Since March 2008 Disease Area Specialist / Medical Science Manager in Immunology, Bristol-Myers Squibb, Baar

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