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KMID : 0613820190290040492
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Journal of Life Science
2019 Volume.29 No. 4 p.492 ~ p.498
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Expression and Purification of Three Lipases (LipAD1, LipAD2, and LipAD3) and a Lipase Chaperone (LipBD) from Acinetobacter schindleri DYL129
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Kim Sun-Hee
Lee Yong-Suk
Jeong Hae-Rin
Pyeon Hyo-Min
You Ju-Soon
Choi Yong-Lark
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Abstract
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Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ρ-nitrophenyl acetate and ρ-nitrophenyl butyrate, LipAD2 showed high activity toward ρ-nitrophenyl acetate and ρ-nitrophenyl myristate, and LipAD3 showed high activity toward ρ-nitrophenyl acetate, ρ-nitrophenyl butyrate, and ρ-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at 50℃ using ρ-nitrophenyl butyrate, as substrate.
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KEYWORD
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Acinetobacter schindleri, lipase, lipase chaperone, protein expression, purification
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