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 PermE 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 pH 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.
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。.
Keywords: Streptomyces albidoflavus Fea-10; Streptomyces pactum; heterologous expression; keratinase; recombinant enzyme.