To improve the efficiency of asymmetric hydrolysis of 3-(4-chlorophenyl) glutaric acid diamide (CGD) using a recombinant Comamonas sp. KNK3–7 amidase (CoAM) produced in Escherichia coli.Methods and Results:
The CoAM gene was cloned, sequenced and found to comprise 1512 bp, encoding a polypeptide of 54 054 Da. CoAM-transformed E. coli were able to perform R-selective hydrolysis of CGD; however, complete conversion of 166·2 mmol l−1 CGD in 28 h could not be obtained. We attempted to optimize the reactivity of CoAM by mutating single amino acids in the substrate-binding domain. Notably, the methionine-substituted L146M mutant enzyme showed increased reactivity, completing the conversion of 166·2 mmol l−1 CGD in just 4 h. The Km value for L146M was lower than that of CoAM.Conclusions:
We succeeded in creating the L146M mutant of CoAM with increased substrate affinity and found that this was the best mutant for the hydrolysis of CGD.Significance and Impact of the Study:
Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.