文章基本信息

标题：Expression of Bacillus Protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and Enhancement of Its Specific Activity by Site-Directed Mutagenesis—Improvement in Productivity of Detergent Enzyme—
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作者：Seiichi Tobe ; Hisao Shimogaki ; Motoyasu Ohdera 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2006
卷号：29
期号：1
页码：26-33
DOI：10.1248/bpb.29.26
出版社：The Pharmaceutical Society of Japan
摘要：An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis , DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.
关键词：protease BYA;subtilisin;expression;Bacillus;bacteriolysis;detergent protease