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Directed evolution of a thermostable l-aminoacylase biocatalyst

Paper ID Volume ID Publish Year Pages File Format Full-Text
23963 43485 2011 10 PDF Available
Title
Directed evolution of a thermostable l-aminoacylase biocatalyst
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.

► A structural homology model of l-aminoacylase from Thermococcus litoralis was created. ► Two different directed evolution strategies identified improved variants. ► Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance. ► Mutations favouring N-benzoyl-valine occurred in the dimerization and zinc-binding domains.

Keywords
Biocatalysis; l-Aminoacylase; Directed evolution; Saturation mutagenesis; Amino acids
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Directed evolution of a thermostable l-aminoacylase biocatalyst
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 155, Issue 4, 10 October 2011, Pages 396–405
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us