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Tools in Biocatalysis Tools in Biocatalysis Enzyme Immobilisation on Silica and Synthesis of Enantiopure Amines Karim Engelmark Cassimjee Licentiate thesis Royal Institute of Technology School of Biotechnology Stockholm 2010 © Karim Engelmark Cassimjee 2010 Royal Institute of Technology School of Biotechnology AlbaNova University Centre SE-106 91 Stockholm Sweden ISBN 978-91-7415-653-9 TRITA-BIO Report 2010:11 ISSN 1654-2312 Printed in Stockholm, April 2010 Universitetsservice US-AB Drottning Kristinas väg 53 B SE-100 44 Stockholm Sweden Abstract This thesis presents two techniques in the field of biocatalysis: An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol. An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines. Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development. Sammanfattning I denna avhandling presenteras två tekniker inom ämnet biokatalys: En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod. Ett jämviksförskjutningssystem som möjliggör fullständig omsätt- ning i reaktioner med ω-transaminaser där isopropylamin är amino- donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer. Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling. List of publications This thesis is written based on the work in the following publications, referred to by the index roman numerals: I Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent Karim Engelmark Cassimjee, Martin Trummer, Cecilia Branneby and Per Berglund 2008, Biotechnology and Bioengineering, 99, 3, 712-716 II HisSi-immobilization, a one step extraction and immobilization method for enzymes Karim Engelmark Cassimjee, Robert Kourist, Diana Lindberg, Marianne Wittrup Larsen, Nguyen Hong Thanh, Mikael Widersten, Uwe T Bornscheuer and Per Berglund Submitted manuscript III Transaminations with isopropyl amine: Equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration Karim Engelmark Cassimjee, Cecilia Branneby, Vahak Abedi, Andrew Wells and Per Berglund 2010, ChemComm, accepted manuscript Contents 1 INTRODUCTION 1 1.1 Enzymes, natural catalysts 1 1.2 Enzymes used in this thesis 2 1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR) 2 1.2.2 Arthrobacter citreus ω‐transaminase 3 1.2.3 Candida antarctica lipase B (CalB) 4 1.2.4 Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum tubersum epoxide hydrolase (StEH1) 4 1.2.5 Bacillus subtilis L‐alanine dehydrogenase (AlaDH), yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) and Candida boidinii formate dehydrogenase (FDH) 5 1.3 Biocatalysis 6 1.3.1 Enzyme Immobilisation 7 1.3.2 Enzymatic Synthesis of Enantiopure Amines 7 2 PRESENT INVESTIGATION 11 2.1 Objectives 11 2.2 Enzyme Immobilisation on modified TLC plates (Paper I) 12 2.3 Enzyme immobilisation and extraction with modified silica oxide beads and employment in organic solvent, HisSi‐immobilisation (Paper II) 15 2.4 Efficient Enzymatic Synthesis of Chiral Amines (Paper III) 18 3 CONCLUSIONS 21 3.1 Short summary of the publications 21 3.2 Discussion and future outlook 21 4 ACKNOWLEDGEMENTS 23 5 REFERENCES 25 1 Introduction 1.1 Enzymes, natural catalysts An amino acid is a molecule which contains an amino group and a carboxylic acid group. In nature such molecules are present in many different versions, twenty of which are commonly used for the formation of chains with subsequent folding which are crucial for any living cell, proteins. Each amino acid residue contributes with a distinct side chain with properties elegantly chosen by evolution. The protein will spontaneously fold in an ordered manner to form a biologically functional unit. The study of proteins has resulted in many astounding findings, including observations of the vital contributions proteins serve for the prevalence of life on earth. A protein which is capable of catalysing a chemical reaction is called an enzyme. Enzymes are the foundation of chemical life; they uphold essentially all chemical reactions at a viable rate within a living organism.[1] Observed at macro scale proteins behave in unexpected ways. E.g. with rising temperature a protein solution (such as egg white) will turn into a semi solid (an omelette[2]) whereas other compounds typically will melt and/or lower in viscosity. The reason is the unfolding and subsequent aggregation of the protein chains. Looking at proteins in micro scale also yields fascinating findings. An enzyme is able to bind one or more molecules or atoms (substrates) and create an environment which is favourable for a desired chemical reaction. The manner of how this is done is specific for each distinct enzyme and to some extent remains a matter of debate; theories of near attack conformers (NAC[3]) and favourable electrostatic interactions[4] present plausible explanations of function. Consensus lies in the transition state theory, applicable for all types of catalysis, which is based on the lowering of the activation energy of a given reaction. A molecule is defined as chiral if it cannot be superimposed upon its mirror image, like your left or right hand. A molecule is not chiral (or achiral) if it has symmetry elements of the second kind, i.e. a mirror 1 plane, a centre of inversion, or a rotation-reflection axis.[5] The chiral mirror versions of the otherwise identical compounds are named enantiomers. In nature such molecules are predominantly existent as one of the possible versions. Nineteen of the twenty amino acids which are the common building blocks of proteins are chiral and are with few exceptions present as the L-enantiomer, as opposed to the D-enantiomer.[1] In organic chemistry the common way of denoting the enantiomers are with an S (left handed) or R (right handed).[5] When the substrate is chiral, the enzyme interacts differently with the different enantiomers. The enzyme, composed of only L-amino acid residues, is often specific for the enantiomeric version present in the environment in which it was evolved. This is possible since the enzyme is itself chiral and enantiopure. A lower or sometimes virtually inexistent catalytic power for the other enantiomer is often observed. The understanding of this is of paramount importance in the production of pharmaceuticals since the enantiomeric version of a pharmaceutically active compound is often of critical importance for the function in the human body.[1, 6] Enzymes also exhibit other forms of specificity; substrate specificity, chemospecificity and regiospecificity. The use of enzymes in chemical synthesis can provide the chemist with a means of creating enantiopure compounds, performing specific reactions in the presence of other reactive compounds and/or steering of reactions to specific groups, in an environmentally compatible setting.[7] Enzyme catalysis has proven to be a success story for life, of which the qualities remain to be fully utilised in chemical engineering. Many enzymes are presently being utilised in industry and academic research, but the current exploitation does not by any means reach the full potential. 1.2 Enzymes used in this thesis 1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR) Alanine racemase (AlaR) is present in all bacteria; in this work the gene from the thermophilic bacterium G. stearothermophilus was used. Alanine racemase interconverts the enantiomers of the amino acid alanine (see Figure 1). In nature it generally exists in the L-form as all amino acids, and is by the enzyme converted into the D-form. 2 The D-alanine is thereafter combined with other compounds to form peptidoglycan, the fabric of the stable bacterial cell wall. O O AlaR H N H N 3 O 3 O L-alanine D-alanine Figure 1: Racemisation of alanine catalysed by alanine racemase (AlaR). The interconversion of enantiomers is referred to as racemisation, hence the name alanine racemase. Although it remains to be shown, it is conceivable that this enzyme or a mutant thereof, may well be able to racemise other compounds of pharmaceutical interest. If this would be achieved a dynamic kinetic resolution[8] could be constructed, a process in which one of the enantiomers is reacted while continuously produced by the enzyme from the corresponding enantiomer. Theoretically, this produces an enantiomerically pure product with a yield of one hundred percent. 1.2.2 Arthrobacter citreus ω-transaminase Transamination is the process in which an amine and a ketone react to interchange their functional groups, producing the corresponding ketone and amine (see Figure 2). This is common biochemistry and is the manner in which an amino acid is produced from a ketoacid. O NH NH O 2 ω-TA 2 Figure
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