Directed evolution of a thermostable l-aminoacylase biocatalyst |
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Authors: | Parker Brenda M Taylor Ian N Woodley John M Ward John M Dalby Paul A |
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Affiliation: | a The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK b Chirotech Technology Ltd., Dr. Reddy's Laboratories (EU) Ltd., 410 Cambridge Science Park, Milton Road, Cambridge CB4 0PE, UK c Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark d Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK |
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Abstract: | Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65 °C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of kcat values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases. |
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Keywords: | Biocatalysis smallcaps" >l-Aminoacylase Directed evolution Saturation mutagenesis Amino acids |
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