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 hydrolyzes the (+)-trans (1R,3R) stereoisomer of ethyl chrysanthemate was cloned from Arthrobacter globiformis SC-6-98-28 and overexpressed in Escherichia coli. The cellular content of the active enzyme reached 33% of the 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 reached 605 mumol of chrysanthemic acid per min per g of dry cells, which 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 produced chrysanthemic acid by ultrafiltration. The (+)-trans-chrysanthemic acid produced had 100% optical purity. The amino acid sequence of the esterase was found to be similar to that of several class C beta-lactamases, D,D-carboxypeptidase, D-aminopeptidase, 6-aminohexanoate-dimer hydrolase, and Pseudomonas esterase. The sequence comparison also suggested that the Ser-X-X-Lys motif in the esterase was at the active site of the enzyme.
Nishizawa
M
M
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Shimizu
M
M
Ohkawa
H
H
Kanaoka
M
M
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011722:Pyrethrins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D015342:DNA Probes; D007306:Insecticides; D011722:Pyrethrins; C007051:chrysanthemic acid; D004950:Esterases