Purification and properties of lipases/esterases from a Bacillus strain for enantioselective resolution of (S)-Ketoprofen.
A Bacillus strain having a certain extent of asymmetric resolution for (S)-ketoprofen was studied for its culture and conversion condition. The distribution and properties of lipases/esterases from its bacterial cell and the change of asymmetric resolution ability were also investigated. After purification by hydrophobic interaction chromatography and gel filtration, the lipase/esterase terase activity of active fraction was 61.7 times higher than the crude enzyme and the purity of 23 kDa protein increased more than 400 times. Hydrolytic activity of lipase/esterase using ketoprofen chloroethyl ester and pNPA as substrate remained essentially constant in the reaction system during the purification procedure. The above result clearly indicated that the application of hydrophobic interaction chromatography-gel filtration was fast, useful and effective when compared to the difficulties in purification of low content crude enzyme prepared directly from bacterial cells. Experimental results also indicated that there are several lipases/esterases in the bacillus cell selective to the optically active ketoprofen and the conversion result was derived from the comprehensive function of all these enzymes. This is a new way to understand the bacterial asymmetric resolution with high conversion rate and low optical purity. The ESI-QUAD-TOF mass spectrum was used to analyze 23 kDa protein. Protein identities were revealed by searching sequence databases (MSDB) with peptide sequence tags. The result showed that 23 kDa protein is a novel protein.
Zhang
Jinhong
J
Institute for Molecular Biology, Nankai University, Tianjin, PR China. jinhzhang@nankai.edu.cn
Guan
Rong
R
Tan
Zhilei
Z
Yu
Yaoting
Y
Hou
Zhibo
Z
Qi
Zhong
Z
Wang
Shanwei
S
D001407:Bacillus / Q000201:enzymology*; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004950:Esterases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007660:Ketoprofen / Q000031:analogs & derivatives / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D013237:Stereoisomerism
D007660:Ketoprofen; D004950:Esterases; D008049:Lipase