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Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase
M Nojiri1, S Yoshida1, H Kanamaru1
1Biotechnology Development Laboratories, Kaneka Corporation, Takasago, Hyogo, Japan.
Aims:
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.
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