STEREOSELECTIVE PRODUCTION OF (-TRANS-CHRYSANTHEMIC ACID BY A MICROBIAL ESTERASE - CLONING, NUCLEOTIDE-SEQUENCE, AND OVEREXPRESSION OF THE ESTERASE GENE OF ARTHROBACTER-GLOBIFORMIS IN ESCHERICHIA-COLI())

The gene coding for a novel esterase which stereoselectively hydrolyze s the (+)-trans (1R,3R) stereoisomer of ethyl chrysanthemate was clone d from Arthrobacter globiformis SC-6-98-28 and overexpressed in Escher ichia coli, The cellular content of the active-enzyme reached 33% of t he total soluble protein in the recombinant E. coli JM105 cells and 5. 6 g/liter of culture by high-density cell cultivation. The hydrolytic activity of the recombinant E. coli cells for ethyl chrysanthemate rea ched 605 mu mol of chrysanthemic acid per min per g of dry cells, whic h is approximately 2,500-fold higher than that of A, globiformis cells . The stereoselective hydrolysis by the recombinant E, coli cells was efficient at substrate concentrations of up to 40% by removing the pro duced chrysanthemic acid by ultrafiltration. The (+)-trans-chrysanthem ic acid produced had 100% optical purity, The amino acid sequence of t he esterase was found to be similar to that of several class C beta-la ctamases, D,D-carboxypeptidase, D-aminopeptidase, 6-aminohexanoate-dim er hydrolase, and Pseudomonas esterase, The sequence comparison also s uggested that the Ser-X-X-Lys motif in the esterase was at the active site of the enzyme.