Abstract
We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg2+, MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the Km and kcat values for (S)-CPPA were 0.92±0.17 mM and 0.28±0.026 s−1 respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C8). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.
