Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01.
A novel lipase gene, lipase A, of Acinetobacter species SY-01 (A. species SY-01) was cloned, sequenced, and expressed in Bacillus subtilis 168. The deduced amino acid (aa) sequences for the lipase A and its chaperone, lipase-specific chaperone, were found to encode mature proteins of 339 aa (37.2 kDa) and 347 aa (38.1 kDa), respectively. The aa sequence of lipase A and lipase-specific chaperone shared high homology 82 and 67% identity with the lipase A and the lipase B of A. species RAG-1. This new lipase was defined as a group I Proteobacterial lipase family. The expressed lipase A was purified through sequential treatment with Q-Sepharose, Resource Q, and Superdex-S75 columns. The maximal activity was observed at 50 degrees C for hydrolysis of p-nitrophenyl monoesters and found to be stable at pH 9-11, with optimal activity at pH 10. Lipase A hydrolyzed wide range of fatty acid esters of p-nitrophenyl, but preferentially hydrolyzed short length acyl chains (C2 and C4). Moreover, lipase A from A. species SY-01 catalyzed hydrolysis of the two acetate isomers of cis-(+/-)-2-(bromomethyl)-2-(2,4-dichloro phenyl)-1,3-dioxolane-4-methyl acetate, an intermediate required for the synthesis of Itraconazole which was an anti-fungal drug, at different rate and yielded cis-(-)-isomer in 81.5% conversion with 91.9% enantiomeric excess.
Han
Soo-Jin
SJ
Structural Biology Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul, South Korea. yshan2@kist.re.kr
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Jung Ho
JH
Yoon
Moon Young
MY
Shin
Pyong Kyun
PK
Cheong
Chan Seong
CS
Sung
Moon-Hee
MH
Hong
Seung-Pyo
SP
Chung
Il Yup
IY
Han
Ye Sun
YS
D000150:Acinetobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006868:Hydrolysis; D008049:Lipase / Q000502:physiology*; D018832:Molecular Chaperones / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D008049:Lipase