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Article Abstract
In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (k /K ) for COBE was 51.36 s ·mM . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.
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