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Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM)

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posted on 2003-08-26, 00:00 authored by Grace DeSantis, Kelvin Wong, Bob Farwell, Kelly Chatman, Zoulin Zhu, Geoff Tomlinson, Hongjun Huang, Xuqiu Tan, Lisa Bibbs, Pei Chen, Keith Kretz, Mark J. Burk
Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.

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