p-Aminosalicylic Acid Production by Enzymatic Kolbe-Schmitt 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 p-aminosalicylic acid (PAS) as an antituberculous agent, through an enzymatic Kolbe-Schmitt reaction. To develop a high-yield 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 100mM 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 (24h). Moreover, 140mM PAS was produced from 200mM m-AP within 9h by the whole-cell reaction of recombinant E. coli BL21(DE3) expressing the gene encoding Y64T-F195Y-Sdc.