Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli.
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 (C8) 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.
Lee
Y P
YP
Department of Biotechnology, Korea Advanced Institute of Science and Technology, Taejon.
Chung
G H
GH
Rhee
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008066:Lipolysis; D008670:Metals / Q000737:chemistry; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000737:chemistry
D008670:Metals; D014280:Triglycerides; D008049:Lipase