Biocatalytic conversion of avermectin to 4''-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution.

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.

Alpha-galactosyl fluoride in transfer reactions mediated by the green coffee beans alpha-galactosidase in ice.

We show that the yields in saccharide synthesis by tranglycosylation with alpha-galactosidase from green coffee beans can be greatly enhanced when working in ice. Thus, methyl alpha-D-galactopyranosyl-(1-->3)-alpha-D-galactopyranoside (3a) produced by reaction of alpha-D-galactopyranosyl fluoride 1 with methyl alpha-D-galactopyranoside (2) is obtained with 51% yield in ice while only 29% is synthesized at 37 degrees C. This result, already previously found by others with proteases and by us with a beta-galactosidase appears to be a general property of hydrolases.