Protein engineering of a Pseudomonas fluorescens esterase Alteration of substrate specificity and stereoselectivity I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Anna Schließmann geboren am 01.03.1981 in Husum Greifswald, im Mai 2010 II Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Karl-Erich Jaeger Tag der Promotion: 27.07.2010 III „Today is your day! Your mountain is waiting. So… get on your way.” - Theodore Seuss Geisel IV Table of contents Table of contents 1. Introduction.................................................................................................................... 1 1.1. Enantioselectivity ................................................................................................... 1 1.2. Biocatalysis............................................................................................................ 2 1.3. Sources of suitable biocatalysts ............................................................................. 4 1.3.1. Isolation of new enzymes ............................................................................... 4 1.3.2. Protein engineering ........................................................................................ 4 1.3.3. Rational protein design................................................................................... 5 1.3.4. Directed evolution........................................................................................... 6 1.3.5. Focused directed evolution............................................................................. 7 1.4. Screening and selection systems........................................................................... 9 1.4.1. Selection .......................................................................................................10 1.4.2. Screening......................................................................................................10 1.5. Catalytic promiscuity.............................................................................................13 1.6. Hydrolases............................................................................................................14 1.6.1. Esterases......................................................................................................14 1.6.2. Pseudomonas fluorescens Esterase I ...........................................................16 1.6.3. (-)-γ-Lactamase from Microbacterium spec ....................................................17 1.7. Biogenic amides....................................................................................................19 2. Aims .............................................................................................................................21 3. Results .........................................................................................................................22 3.1. Chain-length selectivity .........................................................................................22 3.2. Enantioselectivity ..................................................................................................26 3.3. Amidase activity....................................................................................................31 3.4. Biogenic amides....................................................................................................42 3.4.1. Avenanthramides ..........................................................................................44 3.4.2. Chlorogenate esterase from Acinetobacter bailii ADP1 .................................47 4. Discussion....................................................................................................................52 5. Summary......................................................................................................................58 6. Materials and Methods .................................................................................................60 6.1. Materials ...............................................................................................................60 6.1.1. Bacterial Strains............................................................................................60 6.1.2. Plasmids .......................................................................................................60 6.1.3. Chemicals and Disposables ..........................................................................61 6.1.4. Enzymes .......................................................................................................61 6.1.5. Primers..........................................................................................................61 Table of contents V 6.1.6. Laboratory Equipment ...................................................................................63 6.1.7. Computer Programs and Databases .............................................................63 6.1.8. Cultivation Media, Buffers and Solutions .......................................................64 6.2. Methods................................................................................................................69 6.2.1. Microbiological Methods................................................................................69 6.2.2. Molecular Biology Methods ...........................................................................71 6.2.3. Biochemical and Chemical Methods..............................................................75 6.2.4. Analytical Methods ........................................................................................78 7. References...................................................................................................................80 8. Appendix ......................................................................................................................89 1. VI Abbreviations Abbreviations A. dest. Distilled water oligo oligonucleotide Amp Ampicillin PAGE Polyacrylamide gel APS Ammonium persulfate electrophoresis bp Base pairs PCR Polymerase chain reaction BSA Bovine serum albumin PFE I Pseudomonas fluorescens c Conversion esterase I CAS Cassette pG pGaston (plasmid) cm Centimetre pNPA p-Nitrophenyl acetate Da Dalton pNPB p-Nitrophenyl butyrate DMSO Dimethylsulfoxide pNPC p-Nitrophenyl caprylate DNA Deoxyribonucleic acid pNPL p-Nitrophenyl laurate dNTP Deoxyribonucleoside Rha Rhamnose triphosphate RM Roti Mark Standard ® E Enantioselectivity s Second E. coli Escherichia coli SDS Sodium dodecyl sulphate ee Enantiomeric excess TEMED N, N, N‘,N‘- epPCR Error prone PCR Tetramethylethylendiamine g Gram TLC Thin-layer chromatography GC Gas Chromatography TM Melting temperature H Hour Tris Tris-(hydroxymethyl)- HPLC High pressure liquid aminomethane chromatography UV Ultraviolet kb Kilobase V Volt kcat Turnover number wt Wild type kDa KiloDalton °C Degree Celsius KM Michaelis constant µg Microgram l Liter µl Microliter LB Lysogeny Broth %(w/v) Masspercent M Mole per liter %(v/v) Volumepercent mA Milliampere MES 2-(N-Morpholino)ethane Additionally the conventional abbreviations sulfonic acid for amino acids and nucleotides are used . mg Milligram min Minute ml Milliliter mM Millimole per liter mmol Millimole MS Mass spectrometry MTP Microtiter plate n.d. Not determined nm Nanometer OD Optical density Introduction 1 1. Introduction 1.1. Enantioselectivity Molecules which lack an internal plane of symmetry are called chiral molecules; they cannot be superimposed with their mirror images. Asymmetric centers (e.g. a carbon atom with four different substituents) are the most common causes for chirality in a molecule (see Figure 1-1), but there is also axial chirality (e.g. allenes), planar chirality (e.g. (E) -cyclooctene), and inherent chirality (e.g. calixarenes, fullerenes). The two isomers of a chiral molecule rotate the plane of polarized light by the same amount (they are said to be optically active), but in opposite direction; they are called enantiomers. In achiral environments, their chemical behaviour is identical; however, they react differently in the presence of other chiral compounds, such as enzymes. The Cahn Ingold Prelog rule is widely used for the designation of enantiomers. It assigns the four substituents of the chiral center a priority based on the atomic number. When the lowest priority substituent is rotated behind the chiral center, and the priority of the remaining substituents decreases in clockwise direction, the enantiomer is labelled R, if it decreases in counterclockwise direction, the enantiomer is labelled S [1, 2]. A A C C D B B D Figure 1-1: Example for a chiral center (A-D are different substituents) As many pharmaceutically active compounds are chiral and will thus act differently in the chiral environment of a living cell, it is very important to supply optically pure compounds. A prominent example for the different biological activities of enantiomers is Thalidomide. Between 1957 and 1961 it was sold under the name Contergan ® as anti-depressant and to alleviate the morning sickness of pregnant women. The intake of Thalidomide during a pregnancy leads to children born with deformities, therefore the drug was forbidden (it is now in use as drug against leprosy, but no longer administered to pregnant women). It is thought that the ( R)-enantiomer is responsible