Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes
文献类型： 外文期刊
作者： Wang, Lin 1 ; Zhou, Ying 1 ; Huang, Ying 1 ; Wei, Qishun 1 ; Huang, Hongying 2 ; Guo, Chengbao 1 ;
作者机构： 1.Nanjing Inst Agr Sci Jiangsu Hilly Area, Nanjing 210046, Jiangsu, Peoples R China
2.Jiangsu Acad Agr Sci, Circular Agr Res Ctr, Nanjing 210014, Jiangsu, Peoples R China
关键词： Keratinase; Heterologous expression; Thermoactinomyces; Escherichia coli
期刊名称：WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY （ 影响因子：3.312； 五年影响因子：3.58 ）
ISSN： 0959-3993
年卷期： 2019 年 35 卷 9 期
页码：
收录情况： SCI
摘要： The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 degrees C and 8.5, respectively, and the protein remained stable from 50 to 60 degrees C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and beta-mercaptoethanol (beta-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.
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