Broad specificity amino acid racemase (E.C. 188.8.131.52) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive L-amino acids to DL-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of D-amino acids, especially D-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. L-Trp (20 mM) was successfully racemized, and the proportion of D-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial L-Trp.
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