Protein engineering of a Pseudomonas fluorescens esterase [Elektronische Ressource] : alteration of substrate specificity and stereoselectivity / vorgelegt von Anna Schließmann

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 T Melting temperature M
H Hour Tris Tris-(hydroxymethyl)-
HPLC High pressure liquid aminomethane
chromatography UV Ultraviolet
kb Kilobase V Volt
k Turnover number wt Wild type cat
kDa KiloDalton °C Degree Celsius
K Michaelis constant µg Microgram M
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 i