Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01

• Published in: Biochimie 2003-05, Vol.85 (5), p.501-510
• Main Authors: Han, Soo-Jin, Back, Jung Ho, Yoon, Moon Young, Shin, Pyong Kyun, Cheong, Chan Seong, Sung, Moon-Hee, Hong, Seung-Pyo, Chung, Il Yup, Han, Ye Sun
• Format: Article
• Language: English
• Subjects: Acinetobacter; Acinetobacter - enzymology; Acinetobacter species SY-01; Amino Acid Sequence; Animals; Bacillus subtilis; Bacterial Proteins - genetics; Base Sequence; cis-(±)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-methyl acetate; Cloning, Molecular; Enantioselective; Evolution, Molecular; Hydrolysis; Lipase; Lipase - physiology; Lipase-specific chaperone; Molecular Chaperones - genetics; Molecular Sequence Data; Recombinant Proteins - genetics; Sequence Homology, Amino Acid; Species Specificity; Stereoisomerism; Substrate Specificity
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(03)00057-9

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 °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.