Abstract
DpkA possesses N-methyl-L-amino acid dehydrogenase and Δ 1-piperideine-2-carboxylate/Δ1 -pyrroline-2-carboxylate reductase activities. DpkA is useful for the synthesis of N-alkyl-L-amino acids and L-pipecolic acid, which serve as a chiral building block for medicines. To construct an industrial synthetic process of these chemicals with DpkA, thermostabilization of DpkA using error-prone PCR was performed. Mutations of V117M and Q302R improved the thermostability of the enzyme. A gene encoding a mutant DpkA (V117M/Q302R) with simultaneous mutations of V117M and Q302R was constructed by site-directed mutagenesis. Native DpkA was stable up to 30°C and lost its activity at 50°C, whereas the V117M/Q302R was stable up to 45°C and lost its activity at 55°C. Because the mutations of V117M and Q302R had only a modest effect on the kinetic parameters of DpkA, the V117M/Q302R was able to be utilized for the enzymatic synthesis of N-methyl-L-phenylalanine. Since DpkA requires NADPH as a coenzyme, glucose dehydrogenase from Bacillus subtilis was thermostabilized by simultaneous mutations of E170K/Q252L and used as an NADPH regenerator to synthesize N-methyl-L-phenylalanine. After a 24-h reaction, 16.4 mM N-methyl-L-phenylalanine was produced by the native enzymes, whereas 130 mM N-methyl-L-phenylalanine was produced by V117M/Q302R and the thermostabilized glucose dehydrogenase.
