A Novel Lipase from Pseudomonas fluorescens HU380: Gene Cloning, Overproduction, Renaturation-Activation, Two-Step Purification, and Characterization(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
- 論文の詳細を見る
The extracellular lipase gene (lipA) from Pseudomonas fluorescens HU380 was cloned from a genomic library constructed in pBluescript SK+. Nucleotide sequence analysis revealed an open reading frame of 1854 bp encoding the lipase. Its deduced amino acid sequence included internal amino acid sequences of the lipase from this strain. The lipase showed significant sequence similarity to lipases of Serratia marcescens strains and P. fluorescens strains. In Escherichia coli, lipA was expressed in the form of inclusion bodies, which were subsequently solubilized by urea followed by dialysis. The refolded protein was soluble and biologically active. The lipase purified from the E. coli transformant by this denaturation-renaturation procedure followed by only two steps of column chromatographs exhibited the same electrophoretic mobility as did the enzyme purified from P. fluorescens HU380, and both enzymes were quite similar in physicochemical properties such as specific activity, suggesting that the recombinant lipase protein has an intrinsic folding capability in vitro. The function of its C-terminal region is also discussed.
- 社団法人日本生物工学会の論文
- 2003-09-25
著者
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Kojima Yuzo
Medical Enzyme Division, Amano Enzyme Co., Ltd.
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Shimizu Sakayu
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
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Kojima Yuzo
Medical Enzyme Division Amano Enzyme Co. Ltd.
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Shimizu Sakayu
Division Of Applied Life Science Graduate School Of Agriculture Kyoto University
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KOBAYASHI Michihiko
Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The Univer
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Kobayashi Michihiko
Institute Of Applied Biochemistry The University Of Tsukuba
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Kobayashi Michihiko
Institute Of Applied Biochemistry And Graduate School Of Life And Environmental Sciences The Univers
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