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|Title:||Scaling up Asymmetric Biocatalysis with Cofactor Regeneration by Heterologous Expression of a Supra-active Carbonyl Reductase from Candida glabrata|
|Keywords:||Enzyme Kinetics;Cofactor Regeneration;Biotransformation;Enantiomeric Excess;Binding Site|
|Abstract:||A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been cloned, over-expressed, characterised and subsequently employed in biotransformation of a prochiral keto ester (COBE) to a chiral alcohol (ethyl-4-chloro-3-hydroxybutanoate or CHBE). Using NADPH as cofactor and as substrate, the isolated enzyme (CR) exhibited a towering specific activity of 173.49 ± 6.08 Umin-1mg-1 with Km and Kcat as 0.45 ± 0.02 mM and 112.77 ± 3.95 s-1 respectively. Unlike other proteins of this class which usually show substrate inhibition at high substrate concentration (≥ 230 mM), the CR enzyme exhibited marked velocity at substrate concentration as high as 363 mM with highest turnover number (112.77 ± 3.95 s-1). This advocated utility of the enzyme in a batch reactor where maximum COBE conversion has been achieved (161.04 g.L-1 CHBE per g of dry cell weight) compared to the reported so far (1.51~ 149 g.L-1 CHBE per g of dry cell weight). The reaction yielded sparingly available yet greatly important (R) isomer in over 99% enantiomeric excess (e.e) with 88.30% molar bioconversion. Although numerous proteins have been investigated to accomplish the prochiral COBE to chiral CHBE bioconversion, we present our finding as a highly efficient choice for conversion of COBE into CHBE through an efficient batch reaction system.|
|ISSN:||0975-1084 (Online); 0022-4456 (Print)|
|Appears in Collections:||JSIR Vol.77(09) [September 2018]|
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