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Cloning, Expression, Characterization and Immobilization of Carbonyl Reductase from Candida Parapsilosis

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Cloning, Expression, Characterization and Immobilization of Carbonyl Reductase from Candida Parapsilosis Cloning, expression, characterization and immobilization of carbonyl reductase from Candida parapsilosis Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch- Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science Monideepa Bhattacharjee aus Dibrugarh, Indien Berichter: Universitätsprofessor Dr.-Ing. Winfried Hartmeier Universitätsprofessor Dr. rer. nat. Klaus Wolf Tag der mündlichen Prüfung: 08.06.2006 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Table of Contents I Table of Contents Table of Contents ......................................................................................................................I Glossary of Abbreviations and Symbols ..............................................................................IV 1 Introduction ...................................................................................................................... 1 1.1 Chirality and biocatalysis........................................................................................... 1 1.2 Alcohol dehydrogenases ............................................................................................ 2 1.3 Carbonyl reductase from Candida parapsilosis......................................................... 2 1.4 Versatility of CPCR to other alcohol dehydrogenases............................................... 4 1.5 Secondary alcohol dehydrogenase from Candida parapsilosis ................................. 5 1.6 Cofactor regeneration................................................................................................. 6 1.7 Alcohol dehydrogenases in organic solvents ............................................................. 8 1.8 Immobilization ......................................................................................................... 10 1.9 Aim and motivation of the research work................................................................ 15 2 Materials and Methods .................................................................................................. 17 2.1 Materials................................................................................................................... 17 2.1.1 Chemicals......................................................................................................... 17 2.1.2 Devices............................................................................................................. 18 2.1.3 Cultivation media ............................................................................................. 19 2.1.4 Microorganisms................................................................................................ 20 2.1.5 Vectors ............................................................................................................. 21 2.1.6 Materials for Molecular biology work ............................................................. 21 2.2 Microbiological methods.......................................................................................... 22 2.2.1 Conservation and storage of microbiological strains ....................................... 22 2.2.2 Cell cultivation ................................................................................................. 23 2.2.3 Cell disruption and preparation of cleared E.coli lysates under native conditions ......................................................................................................... 24 2.2.4 Preparation of competent cells of E.coli .......................................................... 25 2.3 Molecular biology methods...................................................................................... 27 2.3.1 Preparation of C.parapsilosis genomic DNA (gDNA) .................................... 27 2.3.2 Construction of genomic DNA libraries .......................................................... 27 2.3.3 Synthetic DNA-oligonucleotides ..................................................................... 28 2.3.4 Polymerase chain reaction (PCR) .................................................................... 29 2.3.5 Agarose gel electrophoresis ............................................................................. 32 Table of Contents II 2.3.6 Plasmid DNA isolation from E.coli ................................................................. 33 2.3.7 DNA restriction digestion ................................................................................ 34 2.3.8 5’ Dephosphorylation of DNA fragments........................................................ 34 2.3.9 Ligation of DNA fragments ............................................................................. 34 2.3.10 Transformation and selection........................................................................... 35 2.3.11 Preparation of DNAse free RNAse .................................................................. 36 2.3.12 Sequencing ....................................................................................................... 36 2.3.13 Description of cloning set-up of recombinant CPCR and CpSADH ............... 37 2.4 Biochemical and analytical methods........................................................................ 37 2.4.1 Determination of protein .................................................................................. 37 2.4.2 Determination of enzyme activity.................................................................... 38 2.4.3 Protein purification........................................................................................... 40 2.4.4 Gel electrophoresis........................................................................................... 43 2.4.5 Transfer of protein (Western Blot)................................................................... 44 2.4.6 Chromatographic analysis of substrate and product ........................................ 45 2.4.7 Inhibition study ................................................................................................ 45 2.4.8 Temperature optimum and activation energy................................................... 46 2.4.9 Temperature stability and half-life................................................................... 46 2.4.10 pH optimum and pH stability........................................................................... 47 2.4.11 Stability in hexane............................................................................................ 47 2.4.12 Synthesis in aqueous system ............................................................................ 48 2.4.13 Synthesis in liquid two-phase system............................................................... 48 2.4.14 Substrate spectrum of recombinant CpSADH ................................................. 49 2.5 Immobilization of recombinant CpSADH ............................................................... 49 2.5.1 Immobilization in polyvinyl alcohol (PVA) .................................................... 49 2.5.2 Immobilization in Sepabeads ........................................................................... 50 2.5.3 Stability of the immobilized CpSADH in Sepabeads ...................................... 52 3 Results and Discussion................................................................................................... 53 3.1 Cloning of CPCR and CpSADH .............................................................................. 53 3.1.1 Isolation of genomic DNA from C.parapsilosis .............................................. 53 3.1.2 Construction of libraries................................................................................... 53 3.1.3 Analysis of the partial amino acids sequences of wtCPCR ............................. 54 3.1.4 PCR amplification of CPCR from the libraries and subcloning in pDrive vector .......................................................................................................................... 57 Table of Contents III 3.1.5 Cloning of the 1212 bp CPCR gene into expression vectors ........................... 66 3.1.6 Cloning of the 1011 bp CpSADH gene into expression vectors...................... 69 3.1.7 Conclusion of the cloning of CPCR and CpSADH.......................................... 75 3.2 Expression of recombinant CPCR and CpSADH .................................................... 77 3.2.1 Expression of recombinant CPCR.................................................................... 78 3.2.2 Expression of recombinant CpSADH .............................................................. 80 3.2.3 Conclusion of the expression of recombinant CPCR and CpSADH ............... 90 3.3 Characterization of recombinant CpSADH.............................................................. 91 3.3.1 Temperature optimum and temperature stability ............................................. 91 3.3.2 pH optimum and pH stability........................................................................... 94 3.3.3 Stability in hexane............................................................................................ 95 3.3.4 Inhibition study ................................................................................................ 97 3.3.5 Substrate spectrum of recombinant CpSADH ................................................. 98 3.3.6 Conclusion of the characterization of recombinant CpSADH......................
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