Purification and Characterization of the Lipase from Pseudomonas fluorescens HU380(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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A lipase, which markedly splits polyunsaturated fatty acid ester (PUFA) bonds, from newly isolated Pseudomonas fluorescens HU380 was purified. The purification procedure included Phenyl-Toyopearl fractionation, DEAE-Sepharose chromatography, and Superdex-200HR chromatography. The enzyme was purified 24.3-fold with a yield of 14% and a specific activity of 9854 U/mg. Its molecular weight was estimated on SDS-PAGE to be 64,000. The optimum pH and temperature were 8.5 and 45℃, respectively. The lipase was stable over the pH range of 6.0-7.0 at 30℃ for 24 h, and up to 40℃ at pH7.0 for 60min, when 0.1% Triton X-100 was present. The lipase preferably acted on short to middle-chain fatty acid simple methyl-esters and triglycerides, and cleaved mainly 1,3-ester bonds and to a lesser extent the 2-position ester bond of triolein. The lipase was inhibited by Co^<2+>, Ni^<2+>, Fe^<3+>, Fe^<2+>, and EDTA, and activated by Ca^<2+>. Its N-terminal amino acid sequence was determined to be GVYDYKNFGTADSKALFSDAMAITLY, which exhibited considerable similarity with those of the lipases from other P. fluorescens strains, but no significant homology with other lipases. This lipase was able to decompose fats and oils that contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) without significantly affecting the contents of these fatty acids. The results suggest that the lipase may be useful when applied to the processing of industrial fats and oils containing EPA and DHA, such as fish oil splitting.
- 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
Department Of Bioscience Faculty Of Bioresources Center For Molecular Biology And Genetics Mie Unive
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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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Kojima Y
Medical Enzyme Division Amano Enzyme Co. Ltd.
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