Expression of Lipase cDNA from Fusarium heterosporum by Saccharomyces cerevisiae : High-Level Production and Purification
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概要
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Fusarium heterosporum produces a solvent-tolerant lipase, and we previously succeeded in cloning the lipase cDNA. The present study was undertaken to express the cDNA using Saccharomyces cerevisiae. The expression plasmid was constructed by ligating the cDNA (coding signal peptide, propeptide, and mature lipase) between the GAP (the gene coding glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae) promoter and its terminator. Five S. cerevisiae strains transformed with the resulting plasmid secreted the lipase into the culture supernatant, although their productivities differed. The transformant producing lipase at the highest level, 10 U/ml, was selected, and the medium compositioin was examined with the aim of further elevating production. The lipase was produced most effectively in a medium containing 3% yeast extract, 1% peptone, and 4% sucrose; the production attained 78 U/ml, corresponding to 39 μg/ml lipase. The lipase produced by S. cerevisiae was purified by DEAE-Sephadex A-50 ion-exchange chromatography and Sephardex G-75 gel filtration, and by SP-Sephadex C-50 ion-exchange chromatography. At the final purification step, the activity was separated into two fractions. One fraction contained a homogeneous 32-kDa protein and the other was composed of several proteins (34-38 kDa). Since the properties of the 32-kDa enzyme agreed with those of the lipase from F. heterosporum, including solvent-tolerance, it could be confirmed that the cloned cDNA coded the lipase. It was further suggested that S. cerevisiae possesses processing tools, which contain a singal peptidase and a trypsin-like proteinase cleaving a propeptide, similar to those existing in F. heterosporum. The finding that the 34-38-kDa enzymes did not contain carbohydrate, suggests that they were generated by post-translational modificatioin and not by glycosylation.
- 社団法人日本生物工学会の論文
- 1996-06-25
著者
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Tominaga Y
Dep. Of Applied Biochemistry And Food Sci. Saga Univ.
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Yanagita Teruyoshi
Lab. Of Nutrition Biochemistry Dep. Of Applied Biochemistry And Food Sci. Saga Univ.
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Shimada Y
Department Of Biochemistry Osaka Municipal Technical Research Institute
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Shimada Y
Department Of Physics Chuo University:tecmo
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Shimada Yuji
Osaka Municipal Technical Research Institute
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NAGAO Toshihiro
Osaka Municipal Technical Research Institute
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SUGIHARA Akio
Faculty of Engineering, Tokushima Bunri University
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SUGIHARA Akio
Osaka Municipal Technical Research Institute
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TOMINAGA Yoshio
Osaka Municipal Technical Research Institute
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Yanagita Teruyoshi
Dep. Of Applied Biochemistry And Food Sci. Saga Univ.
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Tominaga Yoshio
The Organ Transplant Center Nagoya Second Red Cross Hospital
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Sugihara A
Faculty Of Engineering Tokushima Bunri University
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Nagao Toshihiro
Biomaterials And Commodity Chemicals Res. Div. Osaka Municipal Technical Res. Inst.
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Nagao Toshihiro
Department Of Biochemistry Osaka Municipal Technical Research Institute
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