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Novel Steroid Reductase from Escherichia Coli: from Identification and Characterization Towards the Design of a Whole-Cell Biocatalyst

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Novel Steroid Reductase from Escherichia Coli: from Identification and Characterization Towards the Design of a Whole-Cell Biocatalyst Novel steroid reductase from Escherichia coli: from identification and characterization towards the design of a whole-cell biocatalyst Inaugural dissertation for the attainment of the title of doctor in the Faculty of Mathematics and Natural Sciences at the Heinrich Heine University Düsseldorf presented by Agne Tubeleviciute from Vilnius Düsseldorf, June 2014 from the institute for Pharmaceutical and Medicinal Chemistry at the Heinrich Heine University Düsseldorf Published by permission of the Faculty of Mathematics and Natural Sciences at Heinrich Heine University Düsseldorf Supervisor: Prof. Dr. Joachim Jose Co-supervisor: Prof. Dr. Jörg Pietruszka Date of the oral examination: 2 TABLE OF CONTENTS Summary .................................................................................................................................... 7 Zusammenfassung...................................................................................................................... 8 1 Introduction ............................................................................................................................. 9 1.1 Steroids: structure and biological functions ..................................................................... 9 1.2 Production of steroid drugs by microbial transformations ............................................. 10 1.3 Microbial reduction of C20 carbonyl of 20-ketosteroids ............................................... 12 1.4 Enzymes catalyzing C20 carbonyl reduction of 20-ketosteroids ................................... 17 1.4.1 Hydroxysteroid dehydrogenases (HSDs) ................................................................ 19 1.4.1.1 HSDs with 20-ketosteroid reductase activity in vertebrates ............................. 19 1.4.1.2 20-HSDs from gut microbiota .......................................................................... 21 1.4.1.3 Other microbial 20-HSDs ................................................................................. 22 1.4.2 Miscellaneous enzymes possessing 20-ketosteroid reductase activity .................... 23 1.5 Short-chain dehydrogenases/reductases and aldo-keto-reductases ................................ 24 1.5.1 Short-chain dehydrogenases/reductases (SDRs) ..................................................... 24 1.5.2 Aldo-keto-reductases (AKRs) ................................................................................. 28 1.6 Examples of applications of 20-ketosteroid reductases in biocatalysis ......................... 29 1.7 Unidentified 20-ketosteroid reductase in Escherichia coli and a whole-cell biocatalysis .............................................................................................................................................. 29 1.8 Autodisplay .................................................................................................................... 31 1.9 Aims of the work ............................................................................................................ 35 2 Materials and methods .......................................................................................................... 36 2.1 Materials ......................................................................................................................... 36 2.1.1 Instruments .............................................................................................................. 36 2.1.2 Software ................................................................................................................... 37 2.1.3 Chemicals and Reagents .......................................................................................... 37 2.1.4 Kits........................................................................................................................... 41 3 2.1.5 Antibodies ................................................................................................................ 41 2.1.6 Enzymes and proteins .............................................................................................. 41 2.1.7 Oligonucleotides ...................................................................................................... 42 2.1.8 Plasmids ................................................................................................................... 43 2.1.9 Bacterial strains ....................................................................................................... 44 2.1.10 Media ..................................................................................................................... 44 2.1.11 Buffers and other solutions .................................................................................... 45 2.1.12 Protein chromatography media .............................................................................. 48 2.1.13 Other materials ...................................................................................................... 48 2.2 Methods .......................................................................................................................... 50 2.2.1 Partial purification of E. coli steroid reductase ....................................................... 50 2.2.2 Identification of proteins by mass spectrometry ...................................................... 51 2.2.3 Enzyme and protein assays ...................................................................................... 52 2.2.4 Analytical HPLC ..................................................................................................... 52 2.2.5 Cloning and expression of ydbC gene ..................................................................... 53 2.2.6 Cloning and expression of kduD, hdhA and uxuB genes ......................................... 54 2.2.7 Whole cell biotransformation assay ........................................................................ 54 2.2.8 Evaluation of an effect of 11-DOC on the expression level of KduD in E. coli ..... 55 2.2.9 Cloning, expression and affinity purification of KduD ........................................... 55 2.2.10 Determination of pH and temperature optimum.................................................... 55 2.2.11 Determination of substrate specificity ................................................................... 56 2.2.12 Evaluation of effect of salts, reducing/oxidizing and denaturing agents ............... 57 2.2.13 Kinetic assays ........................................................................................................ 57 2.2.14 Construction of pAT002, pAT004, pMATE-AT001 and pMATE-AT002 plasmids .......................................................................................................................................... 58 2.2.15 Whole-cell activity assays with E. coli cells expressing FP, FPΔβ1, FP1 and FP2 .......................................................................................................................................... 59 2.2.16 Preparation of membrane protein fraction ............................................................. 60 4 2.2.17 Protease assays ...................................................................................................... 61 2.2.17.1 Proteinase K assay .......................................................................................... 61 2.2.17.2 IgA1 protease assay ........................................................................................ 61 2.2.18 Immunoblot analysis.............................................................................................. 62 2.2.19 LC-MS analysis of steroid conversions ................................................................. 63 2.2.20 DNA sequence analysis ......................................................................................... 63 2.2.21 Other basic methods .............................................................................................. 63 3 Results ................................................................................................................................... 68 3.1 Identification of 11-deoxycorticosterone reductase in Escherichia coli ........................ 68 3.1.1 A bioinformatics approach ...................................................................................... 68 3.1.1.1 Investigation of steroid reductase activity of enzyme encoded by ydbC gene . 72 3.1.2 Partial purification of enzyme from E. coli DH5α .................................................. 73 3.1.2.1 Ammonium sulfate precipitation step ............................................................... 73 3.1.2.2 Column chromatography steps ......................................................................... 74 3.1.3 Identification of NADH-dependent oxidoreductases in partially purified enzyme sample ............................................................................................................................... 81 3.1.3.1 Investigation of 11-DOC reductase activity of NADH-dependent oxidoreductases ............................................................................................................. 82 3.2 Characterization of KduD oxidoreductase ..................................................................... 84 3.2.1 Purification of KduD and optimization of reaction conditions ............................... 84 3.2.2 Determination of the substrate spectrum ................................................................. 86 3.2.2.1 Reduction of doxorubicin
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