Tools in Biocatalysis

Home , Alanine dehydrogenase, Alanine racemase, Biocatalysis

Enzyme Immobilisation on Silica and Synthesis of Enantiopure Amines

Karim Engelmark Cassimjee

Licentiate thesis

Royal Institute of Technology

School of Biotechnology

Stockholm 2010

Royal Institute of Technology School of Biotechnology AlbaNova University Centre SE-106 91 Stockholm Sweden

ISBN 978-91-7415-653-9 TRITA-BIO Report 2010:11 ISSN 1654-2312

Printed in Stockholm, April 2010 Universitetsservice US-AB Drottning Kristinas väg 53 B SE-100 44 Stockholm Sweden

Abstract

This thesis presents two techniques in the field of biocatalysis:

An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol.

An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines.

Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development.

Sammanfattning

I denna avhandling presenteras två tekniker inom ämnet biokatalys:

En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod.

Ett jämviksförskjutningssystem som möjliggör fullständig omsätt- ning i reaktioner med ω-transaminaser där isopropylamin är amino- donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer.

Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling.

List of publications

This thesis is written based on the work in the following publications, referred to by the index roman numerals:

I Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent Karim Engelmark Cassimjee, Martin Trummer, Cecilia Branneby and Per Berglund 2008, Biotechnology and Bioengineering, 99, 3, 712-716

II HisSi-immobilization, a one step extraction and immobilization method for enzymes Karim Engelmark Cassimjee, Robert Kourist, Diana Lindberg, Marianne Wittrup Larsen, Nguyen Hong Thanh, Mikael Widersten, Uwe T Bornscheuer and Per Berglund Submitted manuscript

III Transaminations with isopropyl amine: Equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration Karim Engelmark Cassimjee, Cecilia Branneby, Vahak Abedi, Andrew Wells and Per Berglund 2010, ChemComm, accepted manuscript

Contents

1 INTRODUCTION 1

1.1 Enzymes, natural catalysts 1

1.2 Enzymes used in this thesis 2 1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR) 2 1.2.2 Arthrobacter citreus ω‐transaminase 3 1.2.3 Candida antarctica lipase B (CalB) 4 1.2.4 Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum tubersum epoxide hydrolase (StEH1) 4 1.2.5 Bacillus subtilis L‐alanine dehydrogenase (AlaDH), yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) and Candida boidinii formate dehydrogenase (FDH) 5

1.3 Biocatalysis 6 1.3.1 Enzyme Immobilisation 7 1.3.2 Enzymatic Synthesis of Enantiopure Amines 7

2 PRESENT INVESTIGATION 11

2.1 Objectives 11

2.2 Enzyme Immobilisation on modified TLC plates (Paper I) 12

2.3 Enzyme immobilisation and extraction with modified silica oxide beads and employment in organic solvent, HisSi‐immobilisation (Paper II) 15

2.4 Efficient Enzymatic Synthesis of Chiral Amines (Paper III) 18

3 CONCLUSIONS 21

3.1 Short summary of the publications 21

3.2 Discussion and future outlook 21

4 ACKNOWLEDGEMENTS 23

5 REFERENCES 25

1 Introduction

1.1 Enzymes, natural catalysts An amino acid is a molecule which contains an amino group and a carboxylic acid group. In nature such molecules are present in many different versions, twenty of which are commonly used for the formation of chains with subsequent folding which are crucial for any living cell, proteins. Each amino acid residue contributes with a distinct side chain with properties elegantly chosen by evolution. The protein will spontaneously fold in an ordered manner to form a biologically functional unit. The study of proteins has resulted in many astounding findings, including observations of the vital contributions proteins serve for the prevalence of life on earth. A protein which is capable of catalysing a chemical reaction is called an enzyme. Enzymes are the foundation of chemical life; they uphold essentially all chemical reactions at a viable rate within a living organism.[1]

Observed at macro scale proteins behave in unexpected ways. E.g. with rising temperature a protein solution (such as egg white) will turn into a semi solid (an omelette[2]) whereas other compounds typically will melt and/or lower in viscosity. The reason is the unfolding and subsequent aggregation of the protein chains. Looking at proteins in micro scale also yields fascinating findings. An enzyme is able to bind one or more molecules or atoms (substrates) and create an environment which is favourable for a desired chemical reaction. The manner of how this is done is specific for each distinct enzyme and to some extent remains a matter of debate; theories of near attack conformers (NAC[3]) and favourable electrostatic interactions[4] present plausible explanations of function. Consensus lies in the transition state theory, applicable for all types of catalysis, which is based on the lowering of the activation energy of a given reaction.

A molecule is defined as chiral if it cannot be superimposed upon its mirror image, like your left or right hand. A molecule is not chiral (or achiral) if it has symmetry elements of the second kind, i.e. a mirror

1 plane, a centre of inversion, or a rotation-reflection axis.[5] The chiral mirror versions of the otherwise identical compounds are named enantiomers. In nature such molecules are predominantly existent as one of the possible versions. Nineteen of the twenty amino acids which are the common building blocks of proteins are chiral and are with few exceptions present as the L-enantiomer, as opposed to the D-enantiomer.[1] In organic chemistry the common way of denoting the enantiomers are with an S (left handed) or R (right handed).[5]

When the substrate is chiral, the enzyme interacts differently with the different enantiomers. The enzyme, composed of only L-amino acid residues, is often specific for the enantiomeric version present in the environment in which it was evolved. This is possible since the enzyme is itself chiral and enantiopure. A lower or sometimes virtually inexistent catalytic power for the other enantiomer is often observed. The understanding of this is of paramount importance in the production of pharmaceuticals since the enantiomeric version of a pharmaceutically active compound is often of critical importance for the function in the human body.[1, 6] Enzymes also exhibit other forms of specificity; substrate specificity, chemospecificity and regiospecificity. The use of enzymes in chemical synthesis can provide the chemist with a means of creating enantiopure compounds, performing specific reactions in the presence of other reactive compounds and/or steering of reactions to specific groups, in an environmentally compatible setting.[7] Enzyme catalysis has proven to be a success story for life, of which the qualities remain to be fully utilised in chemical engineering. Many enzymes are presently being utilised in industry and academic research, but the current exploitation does not by any means reach the full potential.

1.2 Enzymes used in this thesis

1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR) Alanine racemase (AlaR) is present in all bacteria; in this work the gene from the thermophilic bacterium G. stearothermophilus was used. Alanine racemase interconverts the enantiomers of the amino acid alanine (see Figure 1). In nature it generally exists in the L-form as all amino acids, and is by the enzyme converted into the D-form.

The D-alanine is thereafter combined with other compounds to form peptidoglycan, the fabric of the stable bacterial cell wall.

O O AlaR H N H N 3 O 3 O

L-alanine D-alanine Figure 1: Racemisation of alanine catalysed by alanine racemase (AlaR). The interconversion of enantiomers is referred to as racemisation, hence the name alanine racemase. Although it remains to be shown, it is conceivable that this enzyme or a mutant thereof, may well be able to racemise other compounds of pharmaceutical interest. If this would be achieved a dynamic kinetic resolution[8] could be constructed, a process in which one of the enantiomers is reacted while continuously produced by the enzyme from the corresponding enantiomer. Theoretically, this produces an enantiomerically pure product with a yield of one hundred percent.

1.2.2 Arthrobacter citreus ω-transaminase Transamination is the process in which an amine and a ketone react to interchange their functional groups, producing the corresponding ketone and amine (see Figure 2). This is common biochemistry and is the manner in which an amino acid is produced from a ketoacid.

O NH NH O 2 ω-TA 2

Figure 2: A transamination reaction where S‐methylbenzylamine and acetone is produced from acetophenone and isopropylamine, catalysed by an ω‐transaminase (ω‐TA).

A cofactor is needed for the reaction, the transaminases utilises the versatile PLP (pyridoxal-5’-phosphate) for this purpose. One can portray the reaction as the amination of PLP which gives PMP (pyridoxamine-5’-phosphate) which then donates the amino group to another ketone, thereby restoring the PLP.[9] The transaminases labelled with the Greek letter omega (ω) are able to accept aliphatic ketones as their substrates, i.e. non-acids.[10] For a synthetic chemist this property is rather interesting, together with the enantiospecific nature of these enzymes. Typically only one amine enantiomer is accepted as substrate or product, whereas the standard chemical synthesis will produce the racemic mixture. In

3 this work an optimised mutant gene of A. citreus ω-transaminase (Cambrex Karlskoga AB) was used.

Although ω-transaminases inherently are able to accept non-natural substrates, many obstacles remain for the synthetic chemist to conquer. E.g. the transamination reaction is an equilibrium (runs in both ways) which is often shifted to the substrate side for the reactions in question (represented by the unbalanced double arrow in Figure 2), with low product yield as a consequence. Also, many substrates and/or products tend to inhibit the enzyme which practically stops the reaction at high concentrations. These are very limiting factors in large scale synthesis.

1.2.3 Candida antarctica lipase B (CalB) Candida antarctica lipase B (CalB), also known as Pseudozyma antarctica lipase B (PalB)[11], is probably the most commonly used enzyme in industry. It was isolated in Antarctica, hence the name. It is thermostable and displays high enantioselectivity.[12] Lipases are enzymes that catalyse the hydrolysis of the ester bonds in triacylglycerols (fats), as a part of fat catabolism.[1] In vitro lipases can be used for transacylation[13] if employment in non-aqueous media is possible, for applications such as polymer synthesis[14, 15] and resolution (enantiomeric purification) of chiral secondary alcohols[16]. CalB has proven to be a stable and versatile catalyst in many different solvents. Additionally, rational redesign (see section 1.3) of the enzyme has resulted in broadening of its reaction range.[17-20]

1.2.4 Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum tubersum epoxide hydrolase (StEH1) Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum tubersum epoxide hydrolase (StEH1) are all readily cloned and overexpressed in Escherichia coli. Esterases and lipases essentially carry out the same reaction, hydrolysis of ester bonds. Lipases however, function in the interface between water and lipid (fat) which is necessary in fat catabolism.[1] Use of these enzymes for transacylation requires employment in non-aqueous media.

An interesting feature with Bacillus subtilis esterase (BS2) is its promiscuous activity towards amides.[21] Also, it is active towards tertiary alcohols.[22-24] These traits make the enzyme attractive for use in synthesis applications, and interesting for exploring the possibility of use in organic solvent. Some activity in 1-propanol has been shown with this enzyme, but none in non-polar solvents.

Bacillus subtilis lipase A (BslA) is a rather typical small lipase[25], which has never been employed in non-aqueous environments.

Pseudomonas fluorescence esterase (PFE) was in other works selected for engineering, and subsequently used for synthesis of attractive compounds.[26-28] Still, it remains to be successfully immobilised and employed in organic solvent.

Solanum tuberosum epoxide hydrolase 1 (StEH1) converts epoxides to diols, and is of synthetic interest because of the enantioselectivity.[29, 30] Large scale employment is hindered by the low solubility of the substrates, aromatic epoxides, in water. Activity in an organic solvent may well enable industrial use.

1.2.5 Bacillus subtilis L-alanine dehydrogenase (AlaDH), yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) and Candida boidinii formate dehydrogenase (FDH) Several enzymes were in this thesis utilised for analysis and reaction engineering purposes.

Bacillus subtilis L-alanine dehydrogenase (AlaDH) specifically oxidises L-alanine to pyruvate with concomitant production of the spectrophotometrically detectable reduced form of nicotine adenine dinucleotide (NADH from NAD+). This can be used in an activity assay for alanine racemase.[31]

Yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) is selective for oxidation/reduction of small alcohols and ketones with NAD+/NADH as cofactor.[32] Ethanol and acetaldehyde are the best substrates, methanol and formaldehyde and also isopropanol and acetone can be converted. Extremely low or virtually no activity has been observed for other alcohols or ketones.

Candida boidinii formate dehydrogenase (FDH) is an enzyme which is used for cofactor regeneration. In the exothermic oxidation reaction of converting formate to carbon dioxide the enzyme reduces NAD+ to NADH. Cofactors are relatively expensive compounds and need to be regenerated so as to keep the total amount at a low level for an industrially feasible process.[33]

1.3 Biocatalysis Enzymatic synthesis refers to any chemical process in which an enzyme is involved in the production of a given compound, synonymous with biosynthesis (in vivo). The term also refers to the in vitro use of enzyme catalysis in synthetic chemistry or biocatalysis; the production of chemicals such as pharmaceuticals, cosmetics, polymers, household reagents etc. This is a fairly new application of biotechnology. Biocatalysis together with other uses of natures tools for industrial production of materials, chemicals or energy form white biotechnology, an important and growing field.[34, 35]

Classic synthetic chemistry is generally associated with organic solvents (i.e. non-aqueous environments), high temperatures and metal catalysts. Numerous efficient processes have been developed in this field, and it is undoubtedly the foundation of today’s chemical industry. Still, nature has been performing advanced chemical transformations in aqueous environments at moderate temperature by employment of enzymes since the beginning of life. The harnessing, mimicking and modification of these natural processes remain an engineering challenge, but with great promise of reward by an environmentally sustainable chemical industry.[36-38]

The modification of enzymes for adaptation to the unnatural environment present in a chemical synthesis is often considered necessary, for which many strategies have been developed. Often the compounds in the process are poor substrates, or a more heat stable or solvent tolerable enzyme is desired. By using an experimentally determined structure of the enzyme (determined by X-ray chrystallography or NMR) the enzyme engineer can locate specific amino acid residues suitable for mutation, this is called rational redesign. When such attempts are unsuccessful, or a structure is

6 unavailable, the amino acid chain can be altered in a randomised fashion and a library of mutants can be screened for the desired new properties. An improved clone can be further randomised and a new library can be screened. The iterative process is repeated for the potential discovery of the required enzyme. This strategy is called directed evolution. In the work presented here the conditions were engineered to enable the enzyme to function and/or several enzymes were used to form an enzymatic cascade reaction to obtain an efficient synthesis.

1.3.1 Enzyme Immobilisation The employment of an enzyme in a synthetic chemical process may be facilitated if the enzyme in question can be immobilised. The advantages of immobilising the enzyme include: the initiation and discontinuation of the reaction is easily accomplished by simple addition and removal of the catalyst, reusability can be ensured if the enzyme is sufficiently stable, the absence of protein in the product can be assured and reaction condition promiscuity[39] (e.g. the activity of an enzyme in non-aqueous conditions) can be induced. The latter is mainly a result of structural stabilisation, hindrance of aggregation and unfolding of the enzyme. There is no general immobilisation method valid for all enzymes and applicable for synthesis applications, though many methods have been developed. E.g. enzyme molecules can be chemically cross-linked to form what is called a cross-linked enzyme crystal or aggregate (CLEC or CLEA), which create a material to the most part consisting of enzyme, hence giving a high activity to volume ratio. More straightforward methods typically include the single or multipoint binding of enzyme to a support material. This can be done in both reversible and irreversible ways.[40-42] None of the methods are general in the sense that they are directly useable for any enzyme of choice. The positioning of the active site (the binding spot of the substrates), surface properties (i.e. hydrophobicity or reactive amino acid residues) and flexibility are factors that are crucial for the immobilised enzyme to function.

1.3.2 Enzymatic Synthesis of Enantiopure Amines Amines are common motifs in pharmaceuticals. Such molecules are also prevalent as active entities in cells. In mammals e.g. dopamine, epinephrine (adrenaline) and serotonin are amines present as signal

7 substances. Synthesis of a wide range of enantiopure amines are of great interest for the pharmaceutical industry. The use of enzymes, ω-transaminases (section 1.2.2), for this purpose has been shown to be effective[43-47] although with many obstructing parts that need to be addressed.

Largely, one can group the synthetic transamination reactions by denoting them as being performed in synthesis mode or resolution mode (see Figure 3). The latter is referring to a process in which the racemate of an amine is used; one enantiomer is removed by the enantiospecific transaminase leaving the other, which can be extracted in enantiopure form. In this case the maximum yield is fifty percent. In synthesis mode the enantiopure amine is synthesised from the corresponding ketone, a reaction which in theory can reach a yield of one hundred percent if it is run to completion. However, as mentioned in section 1.2.2, in synthesis mode the equilibrium most often favours the substrates.[10]

A Resolution mode

O NH3 O ω-TA NH2 NH2

B Synthesis mode

O NH2 ω-TA NH2 O

Figure 3: Examples of two different types of transamination reactions employed in industry. In A, the reaction denoted as run in resolution mode. This equilibrium reaction greatly favours the products. In B, the reaction in synthesis mode, as described in Figure 2. Here the equilibrium is strongly shifted towards the substrates.

The impediment of the transamination reaction by substrate and/or product inhibition has been tackled by means of directed evolution with some success.[48-50] The problem with the unfavourable equilibrium has been partly solved by removal of the products or large excess of amino donor, thus shifting the equilibrium.[10, 51-58] When using the industrial chemical isopropylamine as donor acetone is produced beside the enantiopure amine. Acetone is rather

8 volatile and can be removed by distillation. But, in larger scale distillation is not always feasible since it requires efficient mixing and heating. Raising the temperature also requires a heat stable enzyme.

2 Present Investigation

2.1 Objectives As in all fields of research, and even more so in the rather uncharted field of biocatalysis there is a need for method development. In this thesis two methods are presented. The enzyme immobilisation method and the equilibrium displacement system that is described here were solutions to problems at hand in the current work.

Immobilised metal affinity chromatography (IMAC) is a technique commonly used for purification of proteins with a His6-tag, six histidine residues inserted at either terminal of the protein chain. The structure formed by the histidine residues can be compared with heme, the iron-ion binding molecule in red blood cells. The

His6-tag is an effective binder of metal ions. This enables separation of the tagged protein from a solution by introduction of a matrix containing e.g. nickel or cobalt ions. It is conceivable that these features enable the use of the His6-tag immobilisation for synthesis applications in a variety of solvents. In this work a procedure for

His6-tag immobilisation onto a modified silica support and subsequent reaction engagement was evaluated. The use of this protocol for enzyme immobilisation from crude cell extract was also tested.

ω-transaminases are generally enantioselective and have a broad substrate range (section 1.2.2 and 1.3.2). The synthetic chemist is thereby provided with a catalyst for producing pharmacologically interesting amines of high enantiopurity. However, the transamination reaction comes with many bottlenecks as mentioned in section 1.3.2. An alternative solution to the problem of the unfavourable equilibrium in synthesis mode was constructed, where isopropylamine is the amino donor. An enzymatic cascade reaction where enzyme selectivity was used to form an equilibrium displacement system is presented in this work.

2.2 Enzyme Immobilisation on modified TLC plates (Paper I)

OH O OH O OH + O Si O Si NH2 NH2 OH O OH O

O Δ OH

O O OH O Si N Si O NH2 O OH O

1. Co(II) 2. His6-Enzyme

O O Si N His O O- His 2+ His Co His -O His His

Figure 4: Modification of silica oxide for subsequent binding of cobalt(II) and His6‐tagged enzyme.

Thin layer chromatography (TLC) plates are frequently used as a quick analysis method for separation and visualisation of different compounds. They are usually assembled by grafting silica oxide on a metal surface. Here, such plates were instead used as a carrier for enzyme. After modification the plates were able to efficiently bind cobalt ions, and thereafter His6-tagged enzyme. The plates were treated with 3-aminopropyltriethoxysilane, then resacetophenone (2’,4’-dihydroxyacetophenone). At this point the colour was yellow (due to Schiff base formation, see Figure 6). Resacetophenone is an efficient chelator where cobalt(II) could be bound, which turned the

O O H N AlaR H N 3 O 3 O

D-Ala L-Ala

NAD+

AlaDH

NADH,H+

O O

NH3 + O pyruvate Figure 5: Spectrophotometric assay for the racemisation of D‐alanine. The produced NADH is detected at 340 nm. colour into deep purple. The material is porous; the area on which the cobalt binds greatly exceeds the apparent area of the flat surface. The reaction scheme used for the modification of the silica is described in Figure 4. Geobacillus stearothermophilus alanine racemase was used as a test enzyme for the immobilisation method, which also opens for further testing of this enzyme for synthetic purposes. The enzyme was tethered to the modified plates and tested for activity in different conditions. In aqueous environment (100 mM CHES buffer, pH 9.1) the enzyme was active with retained kinetic properties compared to the solubilised free enzyme. The assay (see Figure 5) was based on the racemisation of D-alanine and the selective oxidation of L-alanine by L-alanine dehydrogenase (AlaDH), producing NADH which is spectrophotometrically detectable at 340 nm. Since an assay for non-aqueous media could not be found (i.e. the available substrates were insoluble in organic solvent) the solvent tolerability of the immobilised enzyme was evaluated by immersion in different media, drying, and thereafter testing in aqueous buffer. It was found that the enzyme was more tolerant towards the more hydrophobic solvent hexane than ethyl acetate, possibly due to stripping of structural water molecules from the enzyme causing unfolding and irreversible aggregation.[59, 60] Table 1 shows the activity of the immobilised enzyme (tested in aqueous buffer) after immersion in n-hexane, ethyl acetate, or dry storage in 4 °C and -20 °C.

Table 1: Remaining enzyme activity after immersion in solvent or cold storage. Remaining enzyme activity (%) Time n‐hexane ethyl acetate 4 °C ‐20 °C 15 min 100 10 100 100 1 h 100 0 100 100 4 days 53 0 100 100 4 months 0 0 100 100

This practical technique, based on the well established His6-tag, was aimed as a first step towards a general immobilisation method for a wide range of enzymes for synthetic applications.

Figure 6: Photograph of the modified silica oxide plates prepared for binding metal ions and thereafter His6‐ tagged enzyme.

2.3 Enzyme immobilisation and extraction with modified silica oxide beads and employment in organic solvent, HisSi-immobilisation (Paper II) Section 2.2 describes the modification of TLC plates. The leap to applying the same scheme for silica oxide micro- beads is not very long. The same method as depicted in Figure 4 was used Figure 7: Photographs after synthesis and washing of and a chelating pow- the modified silica oxide beads. On the left, the yellow der was obtained powder obtained after binding of resacetophenone, which effectively the colour is a result of the Schiff base formation. On the right, the powder now blue after adsorption of bound cobalt(II) and cobalt ions. His6-tagged enzyme. This method was named HisSi-immobilisation. Figure 7 shows photographs of the silica oxide powder after modification (yellow) and binding of cobalt(II) (blue).

O O R3 R2 R2 HO R3 HO R1 O R1 O

CalB: R1: n‐heptyl R2: ethyl R3: n‐hexyl BS2, PFE: R1: methyl R2: vinyl R3: 1‐phenylethyl BslA: R1: ethyl R2: methyl R3: propyl

Figure 8: A general transacylation reaction. The details of the reactions are specified as definitions of the R‐groups for the different cases.

The enzymes C. antarctica lipase B (CalB), B. subtilis esterase 2 (BS2), B. subtilis lipase A (BslA), P. fluorescence esterase (PFE) and

S. tubersum epoxide hydrolase (StEH1) were cloned with a His6-tag, overexpressed in E. coli and bound to the constructed carrier matrix. The initial tests with immobilised CalB in aqueous buffer showed activity, as with the alanine racemase in paper I. HisSi-immobilised CalB effectively hydrolysed 4’-nitrophenylacetate in MOPS-buffer at pH 7.0. The possibility of using enzyme immobilised in this way for

15 chemical synthesis in organic solvent was then evaluated. The lipases and esterases were directly employed in organic solvent for

O OH StEH1 OH Figure 9: Hydrolysis by StEH1 of S,S‐trans‐stilbene oxide to form meso‐hydrobenzoin. transacylation reactions (see Figure 8). CalB was used for the acylation of 1-hexanol. With the BS2 and PFE an enantioselective acylation of the secondary alcohol 1-phenylethanol was tested. The epoxide hydrolase, StEH1, was used for catalysis of the reaction in Figure 9. The powder with bound StEH1 was mixed with water to create a paste which was used to cover the inside of a reaction vessel; then the substrate, trans-stilbene oxide, was dissolved in organic solvent (cyclohexane) and added. This created a two phase system with high substrate concentration in the liquid phase. The results of the different reactions are summarised in table 2.

Table 2: A Summary of the results from employment of HisSi‐immobilised enzyme in organic solvent. The figures show the maximum conversion achieved after 24 h. CalB BS2 BslA PFE StEH1 Conversion 100% 20% 6% 5% 3% Solvent cyclohexane n‐hexane cyclohexane toluene water/cyclohexane

CalB, an enzyme known for its stability in organic solvents, was active and gave complete conversion in the chosen reaction. The transacylation reaction was the synthesis of hexyloctanoate from ethyloctanoate and hexanol in cyclohexane.

The 20% conversion with BS2 is rather surprising since this enzyme never has been successfully employed in any organic solvent. The other lipase and the esterases were inactivated with time in the organic solvents tested. However, low conversions with enantioselectivity could be achieved (the measurement of the enantiomeric excess, the ee (%), was not exact at these low conversions due to the signal-to-noise ratio in the gas chromatograph). It is conceivable that the polar surface of the silica oxide results in unfolding of the enzyme when submersed in non-polar solvent. Still, the existence of activity and enantioselectivity in this rather unoptimised system

16 with these bacterial enzymes is promising. For the StEH1 the conversion was very low, but detectable. Assuming that the formed diol can be adsorbed to the silica the actual conversion could be higher; the detectable amount in the solvent corresponded to a conversion of 3%.

To further simplify the handling, using the modified silica oxide beads for binding of enzyme from a crude extract was tested. CalB was expressed and obtained as a periplasmic preparation from E. coli to which the powder was added and gently stirred. After separation the powder could be directly used for synthesis. This enables the chemist to obtain a usable catalyst without extensive purification and/or lyophilisation. The yield of immobilised enzyme was measured by an active site titration. The yield in this case describes the fraction of active enzyme on the beads compared to the loss of activity in the solution from which it was immobilised. In other words, the yield in this case refers to the eventual loss of activity upon immobilisation. The immobilised CalB had a yield of 58%.

2.4 Efficient Enzymatic Synthesis of Chiral Amines (Paper III) A transamination equilibrium reaction in synthesis mode with isopropylamine as donor produces acetone as a by-product; removal of the acetone and thus shifting of the equilibrium was performed with YADH This alcohol dehydrogenase is selective for small alcohols and ketones. The amine acceptor is most often a larger ketone, hence unaffected by the presence of YADH..

O NH2 NH2 O ω-TA O CO NADH,H+ YADH FDH NAD+ OH -O O Figure 10: A transamination reaction with isopropylamine as donor and acetophenone as acceptor, catalysed by an ω‐transaminase (ω‐TA). The unfavourable equilibrium is displaced by selectively removing the formed acetone with yeast alcohol dehydrogenase (YADH, S. cerevisiae) and regenerating the cofactor NADH with C. boidinii formate dehydrogenase (FDH).

This is an example of utilisation of enzyme selectivity for practical use in an enzymatic cascade reaction. The cofactor NADH was regenerated with FDH, producing carbon dioxide which effectively drives the reaction to completion. Figure 10 shows such a displaced transamination reaction where the acceptor is acetophenone, the desired product in this case is (S)-methylbenzylamine.

The gene for an A. citreus ω-transaminase variant (Cambrex Karlskoga AB) was cloned and successfully overexpressed in E. coli BL21(DE3), and purified by IMAC. The use of purified enzyme facilitated the measurement of kinetic parameters and the influence of excess concentration of the cofactor PLP. It was found that exceeding the concentration beyond that of the enzyme was detrimental to the reaction. Figure 11 shows the activity versus the excess concentration of PLP, it is clear that the activity is greatly decreased with increasing amount of cofactor. Since the enzyme concentration is usually very low, in the micromolar range, this effect is of importance. Already at millimolar concentrations the PLP will practically completely inhibit the proceeding of the reaction.

1 0.8 0.6 (U/mg) 0.4 0.2 Rate 0 0 200 400 [PLP]/[E]

Figure 11: The graph shows the lowering in activity as a result of excess [PLP], i.e. adding more PLP than enzyme.

The reason for this effect remains unknown; it is plausible that PLP (which is an aldehyde) can function as a good amine acceptor and thereby inhibit the reaction with the desired acceptor. It should be noted that the figure shows initial rates, the complete progress of the reaction with different concentrations of PLP was not evaluated.

A balanced ratio of PLP to enzyme was obtained by dissolving an excess of PLP with the purified enzyme solution and then changing the buffer. Assuming that no enzyme dissociates from the PLP during this process this provides an enzyme ready for use with the correct amount of cofactor. No cofactor was thereafter added in the synthesis reactions.

2 Equilibrium reaction mM Displaced reaction

[acetophenone] 0 0 6 12 18 Time (h)

Figure 12: Comparison of a displaced versus a non‐displaced transamination reaction, followed spectrophotometrically by the consumption of acetophenone.

The equilibrium displacement system was verified with acetophenone as the amine acceptor. Though it is a poor substrate, it was chosen because the reaction can easily be followed spectrophotometrically.[61] Figure 12 shows a comparison of the displaced versus the non-displaced reaction as the consumption of acetophenone over time. The displaced reaction continues towards completion while the non-displaced stops at equilibrium conversion. Here the initial concentration of acetophenone was low, only 1.8 mM, hardly a useable large scale process. This concentration was chosen to keep the starting absorbance at a reasonable level and to have a practical equilibrium conversion point for comparison. Even with this large excess of amine donor, 564 mM of isopropylamine was used, the conversion (without displacement) only reached 82%. The amount of formed product, (S)-methylbenzylamine, and the enantiomeric excess was measured with high pressure liquid chromatography (HPLC). Indeed, the displaced reaction had virtually reached completion and the product amine was enantiomerically pure.

The displacement system was explored for other amino acceptors. Table 3 shows the results; with 20 mM substrate concentration the displaced reactions also reached completion.

Table 3: Results from synthesis reactions of four different amines, with and without the displacement system. Conversion (%) No Conversion (%) with Product amine ee (%) p displacement displacement

NH2 >99.9 82 a >99 a

NH2 >99.9 63 b >99 b

O2N NH2 >99.9 89 c >99 c

NH2 >99.9 68 c >99 c HO OH a 1.8 mM acetophenone. Conversions were calculated based on formed product. b 20 mM 4‐nitroacetophenone (two phase system as saturation is reached at ~2 mM). Conversions were calculated based on formed product. c20 mM ketone substrate. Conversions were calculated based on consumed substrate.

3 Conclusions

3.1 Short summary of the publications Largely, this thesis describes two new tools in the field of biocatalysis:

Paper I and II: An employment expansion of the His6-tag for use in synthesis applications is presented. By using modified silica oxide for binding cobalt(II) and thereafter

His6-enzyme a versatile carrier matrix was created, which is also applicable as a one step extraction and immobilisation matrix. This provides a rapid and facile means of obtaining an enzyme catalyst in a suitable form for use in synthesis applications.

Paper III: An equilibrium displacement system for shifting the otherwise unfavourable equilibrium reaction with ω-transaminases in synthesis mode for production of pharmaceutically important enantiopure amines was devised. Isopropylamine as donor is preferably used by the industrialist. The formed acetone in such a reaction is selectively removed by yeast alcohol dehydrogenase, in the presence of larger ketones. With cofactor regeneration where carbon dioxide is irreversibly produced the equilibrium can be fully shifted to the desired products.

3.2 Discussion and future outlook The immobilisation method which has been devised here is based on the use of silica oxide, not a cost efficient chemical. Still the process is suitable for evaluation and small scale synthesis. The same idea can be employed with another carrier, e.g. porous glass or an alternative material that can be tailored into a chelator. Further development of the technique is needed, what is shown here is the facile usage of the His6-tag for enzyme extraction and immobilisation for synthesis applications. The method includes the use of cobalt(II), which is toxic. Use of other metal ions, e.g. iron(II), will offer a more environmentally sustainable method should they provide enough affinity. Cobalt(II) was the only tested ion in the experiments here.

Many times the practical threshold of obtaining, purifying and immobilising the protein catalyst deters the organic chemist from starting the experiments. Another catalyst might be the final choice, from which the entire process is later optimised. This method could be the basis for facilitating the use of enzymes rather than other less environmentally sustainable alternatives.

The synthesis of enantiopure amines is a topic into which much research effort is currently invested. Already biocatalytic synthesis of enantiopure amines is employed in industry, still with room for a lot of improvement and further development. The work done here is an example of how an efficient synthesis can be made; another tool in the box. A plethora of pharmaceuticals and fine chemicals contain chiral amines, and more could be made with increased availability of methods of their production.

Biocatalysis as a research area is in a sense in a battle of proving its worth and value as opposed to established methods in organic chemistry. New successful methods are needed to show that enzymes can perform as catalytic tools that are in deed industrially feasible, to open for more investment. Biocatalysis is predicted to be a vital part in meeting the increasing environmental responsibility put on the chemical industry.

4 Acknowledgements

I would like to express my sincere gratitude to my supervisor, Prof. Per Berglund, for kindness, patience, scientific teaching and pedagogic approach, and for providing an open and very stimulating work environment. Thanks for letting me test my crazy ideas! Maybe I have some more in store…

Prof. Karl Hult, my teacher in biochemistry (among other things) since I started at KTH, has always kept the main focus on teaching and learning more, a great scientist and humanist. I never stop learning from you. Thanks!

My office mates! Anders, thanks for rocking! And for welcomed musical breaks after office hours. Magnus (now Doc!), thanks for listening to my grunts and providing yours, and all the funny signs in the lab. Cecilia Hedfors, thanks for always smiling and explaining techs and methods.

Ol’ members of the biocat group, thanks for lunches and coffee breaks! Maria, thanks for the great shift-work and for being a great travelling companion! Marianne, I’m still angry that you made me run Midnattsloppet and missing it yourself, but I like you anyway. Thanks for teaching me about Danes and beer. Linda, you’re the most awesome physicist to ever become chemist! Thanks for enlightening me with more doubts about the world. Per-Olof, since HPR, no more sterilisations of fridges and unwilling alcohol intoxications. Mats, thanks for good advice and discussions! Mohamad, thanks for being bearded like me!

My great Erasmus students! You make coming to work a true joy! Carla, there’s no salsa like chimichurri for microwaved meat balls. Valentina, I hope you find a bicycle wherever you go! Silvia, enzymes are great, especially ADH!

Co-authors (here, there, AstraZeneca, Cambrex, and elsewhere, not named now) and all the people on the floors 1, 2 and 3, and “the other” buildings, thanks for helping out! Oliver, amasing that you always have a minute to spare, thanks a lot! Lotta, you are the queen of f-n everything, the one and only! Ela, without you I would

23 still be looking for my socks in the tumble dryer, thanks, you are great!! Erik, or should I say Markoolio, or DaddyCool? Since Bio-00 until Dr, let’s do it! The same goes for Jens, who knows about loosing keys and finding couches, let’s do it! Henrik, sing it baby! Harry, you always have time for a good explanation, thanks. Christian, the duke is always ready!! Gea, you make a great Japanese curry! (Yes, I do appreciate a good curry.) Johanna, the lab could not survive without your cakes! Fredrika, you will conquer the world with science, or run around it ten times. Cortwa, you’re almost as nutty as me, smiling Cortwa! Nomchit, great Christmas curry! (I love curry!) Johan, thanks for understanding the enjoyment of living in the HPLC-room. Felicia, Panthera leo Leijon of the Felidae? Qi, thanks to you I have a UV-detector, great! Gustav, the MASSive man, thanks for all your efforts! Marcus, thanks for talks and help in dark moments! Martin, AlaR to ω-TA, PLP for ever man! Anton, there’s no other way to party, the dude! Fahmi, we shall cooperate! Either in chemistry or at re-orient. Ernesto, I can exchange some enzyme for Spanish lessons, ok?

All my friends! I love you guys and gals! No names here, but you already know who you are! (Elsewhere, and at AlbaNova) You helped out a lot with my work by talking about completely different things than science. Thanks!!

I thank my mom for always being a great support and understanding everything except science. I thank dad for cooking the best curry on the planet, and feeding me after late work days. (There’s nothing in the world as good as curry!)

Julia! My wonderful crazy cat!! Thanks for this year, and all the years to come. I love you!

Financial support from The Swedish Governmental Agency for Innovation Systems (VINNOVA) is gratefully acknowledged. EU- COST Action CM0701 is acknowledged for travel funds and for the opportunity to join the COST Training School in Cascade Chemoenzymatic Processes – New Synergies between Chemistry and Biochemistry - in Italy 2009. KTH is acknowledged for travelling scholarships in 2006, 2007 and 2008 that enabled conference participation.

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