Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03 |
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Authors: | Hiroyasu Ogino Yoshikazu Katou Rieko Akagi Takashi Mimitsuka Shinichi Hiroshima Yuichi Gemba Noriyuki Doukyu Masahiro Yasuda Kosaku Ishimi Haruo Ishikawa |
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Institution: | (1) Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan;(2) Department of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan |
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Abstract: | Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the
lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino
acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase
gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase
was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific
foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which
the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively
activated by lipase-specific foldase. |
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Keywords: | Lipase Lipase-specific foldase Molecular chaperone Organic solvent-stable Pseudomonas aeruginosa |
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