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Biotransformations Using Nitrile Hydrolysing Enzymes for Stereoselective Organic Synthesis

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BIOTRANSFORMATIONS USING NITRILE HYDROLYSING ENZYMES FOR STEREOSELECTIVE ORGANIC SYNTHESIS Being a thesis submitted for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY To Waterford Institute of Technology By Tracey M. Coady B.Sc. Based on research carried out under the supervision of Dr. Claire Lennon Ph.D, And Dr. Catherine O’Reilly Ph.D. Department of Chemical and Life Sciences, Waterford Institute of Technology. Waterford, Ireland. May 2014 DECLARATION I hereby certify that this material, is entirely my own work and has not been taken from the work of others, save and to the extent that such work has been cited and acknowledged within the text. It is based on research carried out within the department of Chemical and Life Science at Waterford Institute of Technology. Signed: _______________________ Date: __________________________ ACKNOWLEDGEMENTS I would like to express my special appreciation and thanks to my supervisor Dr. Claire Lennon, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and allowing me to grow as a research scientist. A special thanks also for motivating me when things got hard, for that I owe you big time. To Dr. Catherine O’Reilly thanks for the guidance and for being so patient with me through the Bio work, I really appreciate it. Thanks also to Dr. Lee Coffey for his guidance and support. Thank you to Prof. Gary Black and his group in Northumbria Uni. for helping out with the protein work. Thanks to the technicians; Aidan, Steve, Hubert and last but not least mammy Pat. I appreciate you guys turning a blind eye on the consumables and glass ware I’ve managed to acquire. To the bio techs; Kaz, Walter and now Erica for the endless supply of plates and tips... Cheers. To the past and present members of the PMBRC, you’ve all been amazing. Thanks to Jenny for being an amazing level-headed friend, I think your outlook on life has kept me sane through-out the past four years. From the crazy nights out to the soppy nights in, I know I’ve made a friend for life. Thanks to Dave, Steve, Marc and Jen for adopting me into B21, the organic lab gets very lonely. To John Power and the “spo” crew, for the fresh air and banter breaks, a nicer bunch of pals I couldn’t have asked for. Thanks to Erica for being the sentimental girly girl, and for the molecular bio work. A special thanks to Marc for the love, friendship, support, encouragement, humour and endlessly sorting out my computer issues. Thank you for listening to me and somehow managing to reassure me that things will be okay <3. Thanks to my undergrad and non-science friends Claire, Ash, Rachel and Jen for providing a means of escapism and refusing to talk shop. Not to forget the best adventure holiday ever.... memories of a life time. Finally a special thank you to my Family whose support I could not have done without, John, Maria, Mags, Nan and Grandad. Thanks to my dad for being so understanding and caring, “you’re the bestest”. And lastly to mam, I couldn’t have asked for more support, financially or emotionally. It is because of your encouragement (and nagging) that I got this far <3. So I would like to dedicate this thesis to my family, those present and past, I am blessed to be part of such a loving clan. ABSTRACT Nitrile hydrolysing enzymes have found wide use in the pharmaceutical industry for the production of fine chemicals. This work presents a strategy that facilitates the rapid identification of bacterial isolates demonstrating nitrile hydrolysing activity. The strategy incorporates toxicity, starvation and induction studies along with subsequent colorimetric screening for activity, further focusing the assessment towards the substrates of interest. This high-throughput strategy uses a 96 well plate system, and has enabled the rapid biocatalytic screening of 256 novel bacterial isolates towards β- hydroxynitriles. Results demonstrate the strategy’s potential to rapidly assess a variety of β-hydroxynitriles including aliphatic, aromatic and dinitriles. A whole cell catalyst Rhodococcus erythropolis SET1 was identified and found to catalyse the hydrolysis of 3-hydroxybutyronitrile with remarkably high enantioselectivity under mild conditions, to afford (S)-3-hydroxybutyric acid in 42% yield and >99.9% ee. The biocatalytic capability of this strain including the variation of parameters such as temperature and time were further investigated and all results indicate the presence of a highly enantioselective if not enantiospecific nitrilase enzyme within the microbial whole cell. We present substrate evaluation with 34 chiral nitriles of Rhodococcus erythropolis SET1. These substrates consist primarily of β-hydroxy nitriles with varying alkyl and aryl groups at the β-position containing in some cases, various substituents at the α- position. In the case of β-hydroxy nitriles unsubstituted at the α-position, acids were the major products as a result of suspected nitrilase activity of the isolate. Unexpectedly, amides were found to be the major products when β-hydroxynitriles were substituted at the α-position with a vinyl group. Therefore this novel isolate has demonstrated additional NHase behaviour which is dependent on the functionality at the α-position. In order to probe this mechanism further related substrates were evaluated and amide was observed where other electron withdrawing groups were present at the α-position. Additionally various parameters which may influence the biocatalytic hydrolysis by SET1 were studied and are presented herein. ABBREVIATIONS Ar Aryl t-Bu tert-Butyl Bz Benzyl COSY Correlated Spectroscopy DABCO 1,4-diazobicyclo[2.2.2.]octane DEPT Distortionless Enhancement by Polarization Transfer DMAP 4-Dimethylaminopyridine DMF N,N-Dimethylformamide DMF-DEA N,N-Dimethylformamide diethylacetal DMF-DMA N,N-Dimethylformamide dimethylacetal EDCI 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide ee Enantiomeric excess ESI electrospray ionization Et Ethyl HPLC High performance liquid chromatography LC-MS Liquid chromatography- mass spectrometry HMQC Heteronuclear Multiple-Quantum Coherence Me Methyl MTBE Methyl tert-butyl ether NMR Nuclear magnetic resonance NOESY Nuclear Overhauser Enhancement Spectroscopy Ph Phenyl rt Room temeprature TBDMS tert-butyldimethylsilyl TFA Trifluoroacteic acid THF Tetrahydrofuran TLC Thin layer chromatography TMS Trimethylsilyl CONTENTS ACKNOWLEDGEMENTS ABSTRACT ABBREVIATIONS CHAPTER 1: NITRILE HYDROLYSING ENZYMES FOR STEREOSELECTIVE ORGANIC SYNTHESIS 1.1 Introduction 2 1.2 Asymmetric Synthesis for the Production of Enantiopure Compounds 3 1.2.1 Resolution of Racemic Mixtures to Generate Single Enantiomers 3 1.2.2 Asymmetric Synthesis using the Chiral Pool 4 1.2.3 Chiral Auxiliaries to Influence Reaction Selectivity 5 1.2.4 Chiral Reagents in Asymmetric Synthesis 6 1.2.5 Chiral Catalysts in asymmetric synthesis 7 1.2.6 Chiral Organocatalysts in Asymmetric Synthesis 8 1.3 Enzymatic Reactions in Organic Synthesis 10 1.3.1 Enzymatic resolution of racemic mixtures 10 1.4 Biotransformations of nitrile substrates 14 1.4.1 Types of Nitrile degrading enzymatic pathways 14 1.4.2 Nitrile Hydratase enzymes 15 1.4.3 Amidase enzymes 17 1.4.4 Biotechnological potential of NHase and amidase enzymes 18 1.4.5 NHase/amidase catalysed hydrolysis of chiral substrates 19 1.5 Nitrilase as a member of the nitrilase superfamily 23 1.5.1 Nitrilase Enzymes in Biocatalysis 25 1.5.2 Nitrilase substrate specificity and activity 27 1.6 Nitrilase catalysed hydrolysis of substrates containing a chiral centre. 32 1.6.1 Nitrilase catalysed hydrolysis of substrates bearing an α-stereocentre 32 1.6.2 Nitrilase hydrolysis of chiral substrates bearing a β-stereocentre 38 1.6.3 Nitrilases displaying NHase activity 44 1.7 Screening strategies for nitrile hydrolyzing enzymes 48 1.7.1 Selection and Enrichment techniques 48 1.7.2 Nitrile Hydrolysing activity assays 50 1.7.3 High throughput screening strategies 54 CHAPTER 2: DEVELOPMENT OF A HIGH-THROUGHPUT SCREENING STRATEGY TO IDENTIFY NOVEL NITRILE HYDROLYSING ENZYMES 2.1 Introduction 69 2.2 Project Aim 69 2.3 Isolate Source 70 2.4 Substrates of interest 71 2.5 Identification of Nitrile hydrolysing isolates 72 2.5.1 Toxicity screening 72 2.5.2 Isolate Induction and Activity Screening 73 2.5.3 Temperature growth studies as an induction guideline 77 2.5.4 Investigation into the effect of temperature of growth on activity 79 2.5.5 Alternative induction methods 80 2.6 Screening for enantioselective isolates active towards β-hydroxynitriles. 85 2.6.1 Synthesis of standards for chromatographic method development 85 2.6.2 Enantioselectivity screening of isolates towards 3-hydroxybutyronitrile 89 CHAPTER 3: ISOLATE EVALUATION AND ATTEMPTED PROTEIN PURIFICATION 3.1 Introduction 101 3.2 Influence of temperature during the biotransformation of 3-hydroxybutyronitrile 101 3.3 Influence of pH on the nitrilase activity of SET1 103 3.4 Influence of metal ions and other reagents on the activity of SET1 105 3.5 Effect of organic solvents on nitrilase activity of SET1 109 3.6 Substrate concentration and its effect on activity and enantioselectivity 113 3.7 Protein purification of the nitrilase in R. erythropolis SET1 115 3.7.1 Preparation of a cell free extract 116 3.7.2 SDS-PAGE analysis of cell free extracts 118 3.7.3 Protein Precipitation 119 3.7.4 Hydrophobic interaction chromatography, Phenyl sepharose CL-4B 120 3.7.5 Gel filtration Sephacryl S-200 121 3.7.6 Protein identification using trypsin 123 CHAPTER 4: SUBSTRATE EVALUATION OF R. ERYTHROPOLIS SET1 4.1 Introduction
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