PURIFICATION AND CHARACTERIZATION OF PSEUDOMONAS-FLUORESCENS SIK-W1 LIPASE EXPRESSED IN ESCHERICHIA-COLI

被引:0
作者
LEE, YP
CHUNG, GH
RHEE, JS
机构
[1] KOREA ADV INST SCI & TECHNOL, DEPT BIOTECHNOL, 373-1 KUSONG DONG, TAEJON 305701, SOUTH KOREA
[2] DOOSAN TECH CTR, KYONGGI DO, SOUTH KOREA
关键词
LIPASE; REFOLDING; ENZYME PURIFICATION; CHARACTERIZATION; (P-FLUORESCENS);
D O I
暂无
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Pseudomonas fluorescens SIK W1 lipase was expressed as a form of inclusion bodies in Escherichia coli, which was equivalent to 46% of total cell protein. The inclusion bodies isolated from other cell components were solubilized in the buffer containing 8 M urea and then refolded by diluting urea. The lipase with active conformation was purified by hydrophobic interaction chromatography, gel filtration, anion-exchange chromatography and hydroxyapatite chromatography from the refolded sample. By these purification steps, a single band for active lipase was detected on non-reducing SDS-PAGE and 10-fold purification was attained on the basis of specific activity. Specific activity of the purified lipase toward olive oil emulsion was found to be 7395 units per mg protein. The optimum pH and temperature of the lipase were pH 8.5 and 45-55-degrees-C, respectively. The lipase showed higher lipolytic activity toward tricaproin (C6) and tricaprylin (C-8) among the triacylglycerols examined and preferentially hydrolyzed ester bond of 1- and 3-position of triolein. Lipase activity was greatly increased by approx. 6-fold and stability for pH was shifted to alkaline pH by Ca2+ ion. The lipase was inhibited by Hg2+, Ag2+, p-chloromercuribenzoate, diethylpyrocarbonate and sodium dodecyl sulfate.
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页码:156 / 164
页数:9
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