P-aminosalicylic acid production by enzymatic kolbeschmitt reaction using salicylic acid decarboxylases improved through site-directed mutagenesis

A reversible salicylic acid decarboxylase (Sdc) catalyzes the carboxylation of m-aminophenol (m-AP) to paminosalicylic acid (PAS) as an antituberculous agent, through an enzymatic KolbeSchmitt reaction. To develop a highyield PAS production system through such an enzymatic reaction, we generated Sdc mutants by site-directed mutagenesis and succeeded in generating several mutants showing increased carboxylation specific activities. Among them, a Y64T-F195Y-Sdc mutant showed a 12-fold higher carboxylation specific activity toward m-AP than wild-type Sdc. By the whole-cell reaction of recombinant Escherichia coli BL21(DE3) expressing the gene encoding Y64T-F195Y-Sdc, 70mM PAS was produced from 100 mM m-AP within 2 h. This reaction time was shortened to one-twelfth that of the PAS production using E. coli BL21(DE3) expressing the gene encoding wild-type Sdc (24 h). Moreover, 140 mM PAS was produced from 200 mM m-AP within 9 h by the whole-cell reaction of recombinant E. coli BL21(DE3) expressing the gene encoding Y64T-F195Y-Sdc.