Research Collection 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 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. 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..............................................................................................