Nishizawa M, Shimizu M, Ohkawa H, Kanaoka M
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Appl Environ Microbiol. 1995 Sep;61(9):3208-15. doi: 10.1128/aem.61.9.3208-3215.1995.
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.
从球形节杆菌SC-6-98-28中克隆出编码一种新型酯酶的基因，该酯酶能立体选择性地水解菊酸乙酯的(+)-反式(1R,3R)立体异构体，并在大肠杆菌中实现了过量表达。通过高密度细胞培养，重组大肠杆菌JM105细胞中活性酶的细胞含量达到总可溶性蛋白的33%，每升培养物中活性酶含量达到5.6克。重组大肠杆菌细胞对菊酸乙酯的水解活性达到每克干细胞每分钟605微摩尔菊酸，这比球形节杆菌细胞的水解活性高约2500倍。通过超滤去除生成的菊酸，重组大肠杆菌细胞在底物浓度高达40%时的立体选择性水解效率很高。生成的(+)-反式菊酸的光学纯度为100%。发现该酯酶的氨基酸序列与几种C类β-内酰胺酶、D,D-羧肽酶、D-氨肽酶、6-氨基己酸二聚体水解酶和假单胞菌酯酶的氨基酸序列相似。序列比较还表明，该酯酶中的Ser-X-X-Lys基序位于酶的活性位点。
