A Cysteine-Dependent Serine Protease Associated with the Dormant Spores of Bacillus cereus : Purification of the Protein and Cloning of the Corresponding Gene
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概要
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Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2_M KCl and purified to homogeneity by carbobenzoxy-_D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl_2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5_M urea at 4℃ for at least 2 months, and the specific activity was 50 times that of subtilisin BPN' when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-_L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions ; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.
- 社団法人日本農芸化学会の論文
- 1998-02-23
著者
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SUGIMOTO Kazuhiro
Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical
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MORIYAMA Ryuichi
Department of Food and Nutritional Sciences, College of Bioscience and Biotechnology, Chubu Universi
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MAKINO Shio
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya Uni
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INOUE Toshihiko
Department of Multidisciplinary Radiotherapy, Osaka University Graduate School of medicine
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Makino S
Nagoya Univ. Nagoya Jpn
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Makino Shio
Department Of Applied Biological Science Faculty Of Agriculture Nagoya University
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Makino S
Nagoya Univ. Aichi Jpn
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Moriyama Ryuichi
Department Of Applied Biological Science School Of Agriculture Nagoya University
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Moriyama Ryuichi
Department Of Applied Biological Sciences School Of Agricultural Sciences Nagoya University
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Inoue Toshihiko
Department Of Matallurgy And Ceramics Science Graduate School Of Science And Engineering Tokyo Insti
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Sugimoto Kazuhiro
Faculty of Horticulture, Minamikyushu University
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ZHANG Haishuo
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University
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Sugimoto K
Minamikyushu Univ. Miyazaki Jpn
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Sugimoto Kazuhiro
Department Of Applied Chemistry Shibaura Institute Of Technology
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Inoue Toshihiko
Department Of Applied Biological Sciences School Of Agricultural Sciences Nagoya University
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Zhang Haishuo
Department Of Applied Biological Sciences School Of Agricultural Sciences Nagoya University
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