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Front Matter UvA-DARE (Digital Academic Repository) Expanding the catalytic activity of amine dehydrogenases Rational enzyme engineering via computational analysis and application in organic synthesis Tseliou, V. Publication date 2020 Document Version Other version License Other Link to publication Citation for published version (APA): Tseliou, V. (2020). Expanding the catalytic activity of amine dehydrogenases: Rational enzyme engineering via computational analysis and application in organic synthesis. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:25 Sep 2021 Rational Enzyme Engineering via Computational Analysis and Application in Organic Synthesis Analysis and Rational Enzyme Engineering via Computational ACTIVITY OF AMINEEXPANDING DEHYDROGENASES: THE CATALYTIC EXPANDING THE CATALYTIC ACTIVITY OF AMINE DEHYDROGENASES: RATIONAL ENZYME ENGINEERING VIA COMPUTATIONAL ANALYSIS AND APPLICATION IN ORGANIC SYNTHESIS V. Tseliou V. VASILEIOS TSELIOU Expanding the catalytic activity of amine dehydrogenases: rational enzyme engineering via computational analysis and application in organic synthesis Tseliou Vasileios 2020 Expanding the catalytic activity of amine dehydrogenases: rational enzyme engineering via computational analysis and application in organic synthesis ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen op dinsdag 12 mei 2020, te 12:00 uur door Vasileios Tseliou geboren te Rodos Promotiecommissie: Promotores: dr. F. G. Mutti Universiteit van Amsterdam Prof. dr. J. H. van Maarseveen Universiteit van Amsterdam Overige leden: Prof. dr. R. Wever Universiteit van Amsterdam Prof. dr. T.N. Grossmann VU Amsterdam Prof. dr. S. Brul Universiteit van Amsterdam dr. I.V. Pavlidis University of Crete dr. M. A. Fernández Ibáñez Universiteit van Amsterdam Faculteit der Natuurwetenschappen, Wiskunde en Informatica (FNWI) This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme (ERC-StG, grant agreement No 638271, BioSusAmin). Dutch funding from the NWO Sector Plan for Physics and Chemistry is also acknowledged. ‘Η Ιθάκη σ’ έδωσε τ’ωραίο ταξίδι. Χωρίς αυτήν δεν θα ‘βγαινες στον δρόμο. Αλλά δέν έχει να σε δώσει πια. Κι αν πτωχική την βρείς, η Ιθάκη δεν σε γέλασε. Έτσι σοφός που έγινες, με τόση πείρα, ήδη θα κατάλαβες οι Ιθάκες τι σημαίνουν.’ Κωνσταντίνος Π. Καβάφης (1863-1933) ‘Ithaka gave you the beautiful journey. without her you wouldn't have set out. But there's nothing else to give you anymore. And if you find her poor, Ithaka won’t have fooled you. Wise as you will have become, so full of experience, you’ll have understood by then what these Ithakas mean.’ Constantine P. Cavafy (1863-1933) Table of Contents 1 Chapter 1 13 1.1 Towards greener production of α-chiral amines 14 1.2 Engineered AmDHs from L-AADHs for the asymmetric synthesis of amines 18 1.2.1 Evolution of Bacillus stearothermophilus Leu-DH to Bs-AmDH 18 1.2.2 Evolution of Bacillus badius Phe-DH to Bb-AmDH 20 1.2.3 Generation of a chimeric amine dehydrogenase from Bs-AmDH and Bb-AmDH 21 1.2.4 Generation of Rs-AmDH from Rhodococcus sp. M4 Phe-DH 23 1.2.5 Generation of Es-AmDH from Exiguobactertium sibiricum Leu-DH 24 1.2.6 Generation of Cal-AmDH from Caldalkalibacillus thermarum Phe-DH 25 1.2.7 Generation of Lf-AmDH and Bsp-AmDH from Leu-DHs 26 1.2.8 Generation of LE-AmDHs from ε-deaminating L-Lysine dehydrogenase 28 1.2.9 Discovery of Pm-AmDH-4 and engineering for the acceptance of 2-pentanone 30 1.3 Native AmDHs for the asymmetric synthesis of amines 31 1.4 Applicability of AmDHs 42 1.4.1 Kinetic resolution and deracemization of racemic amines 45 1.4.2 Biocatalytic cascades for the asymmetric amination of alcohols 49 1.4.3 Synthesis of secondary and tertiary amines by AmDHs 57 1.5 Outline of the thesis 61 1.6 References 64 2 Chapter 2 69 2.1 Abstract 70 2.2 Introduction 71 2.3 Results and discussion 74 2.3.1 Screening of carbonyl compounds and amine donors 74 2.3.2 Influence of the enzyme and amine donor concentration 83 2.3.3 Initial biochemical and computational studies towards the understanding of the reaction mechanism 85 2.3.4 Elucidation of the stereoselective properties of AmDHs with the aid of computational studies 98 2.3.5 Proposed catalytic mechanism 107 2.4 Conclusions 110 2.5 Methods 111 2.6 References 117 3 Chapter 3 123 3.1 Abstract 124 3.2 Introduction 125 3.3 Results and Discussion 128 3.3.1 Molecular modelling and preliminary activity studies 128 3.3.2 Asymmetric synthesis of amines catalyzed by LysEDH variants 132 3.3.3 Thermal stability of LE-AmDH-v1 140 3.3.4 Biocatalytic optimization studies on LE-AmDH-v1 141 3.3.5 Substrate scope of LE-AmDH-v1 142 3.3.6 Reaction intensification 144 3.3.7 Reductive amination reactions in preparative scale 145 3.3.8 Steady state kinetic data and inhibition studies 146 3.3.9 Computational studies 150 3.3.10 Enzymatic synthesis of a-chiral amines as reference compounds using ωTAs 152 3.4 Conclusions 157 3.5 Methods 158 3.6 References 168 4 Chapter 4 173 4.1 Abstract 174 4.2 Introduction 175 4.3 Results and discussion 177 4.3.1 Response factors 177 4.3.2 Influence of the pH in the oxidative deamination of α-methyl-benzylamine 178 4.3.3 Influence of the type of buffer 180 4.3.4 Influence of the temperature into the stereoselective outcome 181 4.3.5 Kinetic resolution of pharmaceutical relevant racemic amines employing LE-AmDH-v1 182 4.3.6 Influence of the substrate concentration in the kinetic resolution of α­methyl­benzylamine 183 4.3.7 Kinetic resolution in semi-preparative scale 184 4.3.8 Dynamic kinetic resolution of α­methyl­benzylamine using amine boranes 185 4.3.9 Biocatalytic deracemization cascades employing LE-AmDH-v1 and (S)­selective ω-TAs 186 4.4 Conclusions 187 4.5 Methods 188 4.6 References 192 5 Chapter 5 195 5.1 Abstract 196 5.2 Introduction 197 5.3 Results and discussion 199 5.3.1 Substrate selection 199 5.3.2 Reductive amination of aldehydes 202 5.3.3 Reductive amination of ketones 204 5.3.4 Investigation of the optimal reaction conditions for the reduction of aldehydes to alcohols 206 5.3.5 Reduction of aldehydes to alcohols 208 5.3.6 Reduction of benzaldehyde at higher concentrations 210 5.3.7 Investigation of the oxidation of benzyl alcohol to benzaldehyde 211 5.3.8 Investigation of direct conversion of benzylalcohol to benzylamine 213 5.4 Conclusions 217 5.5 Methods 218 5.6 References 228 6 Chapter 6 231 6.1 Abstract 232 6.2 Introduction 233 6.3 Results and discussion 226 6.3.1 Electrocompetent cells and electroporation 236 6.3.2 Development of an expression and purification assay in 96-deep well blocks 236 6.3.3 Small scale expression and purification of D-AADH 238 6.3.4 Assay for the detection of (S)-configured amines using chromotropic acid 240 6.3.5 Assay for the detection of (S)-configured amines using 2,4,6-tribromo-3-hydroxybenzoic acid 243 6.4 Conclusions 247 6.5 Methods 248 6.6 References 254 Summary 255 Samenvatting 257 Acknowledgements 259 List of publications 262 List of abbreviations AADH Amino acid dehydrogenase AAP 4-aminoantipyrine ADH Alcohol dehydrogenase AlaDH Alanine dehydrogenase AmDH Amine dehydrogenase API Active pharmaceutical ingredient Cb-FDH Formate dehydrogenase from Candida boidinii CTA Chromotropic acid DCM Dichloromethane DKR Dynamic kinetic resolution DMSO Dimethyl sulfoxide E. coli Escherichia coli ee Enantiomeric excess EtOAc Ethyl acetate FAD Flavin adenine dinucleotide FDA Food and drug administration FDH Formate dehydrogenase GC Gas cromatography GDH Glucose Dehydrogenase GST Glutathione S­transferase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High pressure liquid chromatography HRP Horseradish peroxidase IPTG Isopropyl β-D-thiogalactoside IS Internal standard KPi Phosphate buffer KR Kinetic resolution LB Lysogeny broth medium LDH Lactate Dehydrogenase LE-AmDH Variant of L-lysine ε-dehydrogenase from Geobacillus stearothermophilus LysEDH L-lysine ε-dehydrogenase from Geobacillus stearothermophilus MAO Monoamine oxidase MD Molecular dynamics MOPS 3-(N-morpholino)propanesulfonic acid MTBE Methyl tert-butyl ether MW Molecular weight N.a. Not applicable N.d Not detected N.m Not measured NAD+ Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate Ni­NTA Nickel-nitrilotriacetic acid NOx NADH-oxidase from Streptococcus mutans NPC Non protein control OD600 Optical density at 600 nm ONC Overnight culture PLP Pyridoxal phosphate rac Racemic RF Response factor rpm Revolutions per minute SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TFA Trifluoroacetic acid THF Tetrahydrofuran Tris Tris(hydroxymethyl)aminomethane UV-Vis Ultraviolet-visible spectroscopy WT Wild type ωTA ω-transaminase .
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