摘要
通过生物信息学分析,在本实验室分离得到的1株羽毛高效降解菌微白黄链霉菌Fea-10基因组中发现基因gm2886(GenBank Accession Number:KY368946)可能编码一新的角蛋白酶,通过在该基因5'端和3'端分别连接红霉素抗性基因启动子(PermE)和组氨酸标签编码序列并构建在大肠杆菌-链霉菌穿梭质粒pSET152上,接合转入密旋链霉菌Streptomyces pactum ACT12,从而实现了异源表达,蛋白纯化后对其酶学性质进行了研究。实验结果表明,带有组氨酸标签编码序列的gm2886在密旋链霉菌ACT12中可以表达分泌得到1个大小约为36 kDa的蛋白。多种底物检测表明异源表达得到的重组蛋白GM2886-His6具有蛋白酶活性,可以降解水不溶性的天青角蛋白和羽毛粉;其最适温度和pH分别为50℃和pH 10.0。PMSF可抑制GM2886-His6的酶活,而EDTA不能,说明该酶为丝氨酸蛋白酶。本研究为从分子水平上解析羽毛高效降解菌Fea-10的活性机理,从而进一步开发其应用潜力提供了基础,同时可为该类蛋白酶的研究提供借鉴。
By bioinformatics analysis, a putative keratinase gene gm2886（Accession number： KY368946） was discovered in the genome of a feather-degrading strain, Streptomyces albidoflavus Fea-10. gm2886 was ligated into integrative Escherichia coli-Streptomyces shuttle vector pSET152 under the promoter Perm E and added with C-terminal His-tag. The expression vector was transformed into Streptomyces pactum ACT12 by conjugal transfer and the recombinant protein GM2886-His6 was detected in fermentation broth. GM2886-His6 was purified and characterized. Its size was nearly 36 kDa. GM2886-His6 showed proteolytic activity towards a variety of substrates and could even degrade insoluble substrates, such as azure keratin and chicken feathers. The optimal p H and temperature of GM2886-His6 for proteolysis of casein was pH 10.0 and 50 ℃, respectively. The enzyme activity was inhibited by PMSF, but not EDTA, indicating that GM2886-His6 was a serine proteinase. Our results laid the foundation for the research of the molecular biological mechanism on feather-degrading and for the further utilization of Fea-10.
作者
马怡茗
柯欣
李晓霞
舒伟学
杨文翰
刘亚勇
颜霞
贾良辉
颜华
Yiming Ma;Xin Ke;Xiaoxia Li;Weixue Shu;Wenhan Yang;Yayong Liu;Xia Yan;Lianghui Jia;Hua Yan(College of Life Sciences, Northwest A＆F University, Yangling 712100, Shaanxi, China;Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2017年第12期1968-1978,共11页
Chinese Journal of Biotechnology
基金
陕西省农业科技创新与攻关(No.2015NY066)
上海交通大学微生物代谢国家重点实验室开放基金(No.MMLKF14-09)
中央高校基本科研业务费(Nos.Z109021426
Z109021432)
国家自然科学基金(No.31601700)资助~~
关键词
角蛋白酶
微白黄链霉菌Fea-10
密旋链霉菌
异源表达
重组酶
keratinase, Streptomyces albidoflavus Fea-10, Streptomyces pactum, heterologous expression, recombinant enzyme
