枯草芽孢杆菌分泌表达极耐热木聚糖酶及其酶学性质

摘要

To heterogeneously express the gene xynB encoding extremely thermostable xylanase from Thermotoga ma⁃ritima in Bacillus subtilis916( Bs916) and characterize the properties of recombinant enzymes, the xynB gene with the pro⁃moter of a novel gene cluster for locillomycin were fused and resulted in a recombinant expression plasmid pXYNB2 which was then transformed into Bs916 and achieved the genetically engineered strain B. subtilis(Bs916/pXYNB2). SDS⁃PAGE revealed a protein with 43 000 molecular mass yielded by Bs916/pXYNB2, and the protein showed thermostable xylanase activity. The recombinant xylanase exerted activity at pH 5�0-5�8 above 100℃, and kept 84% of its activity after incuba⁃ted at 90 ℃ for 2 h. The strain B. subtilis 916 is able to heterologouly express xynB, which lays a foundation for co⁃express⁃ing cellulose and semi⁃cellulose.%为将海栖热袍菌极耐热木聚糖酶基因xynB（ GenBank 登录号AY340789）在枯草芽孢杆菌Bs916中进行异源分泌表达，并阐明工程菌分泌重组木聚糖酶的酶学性质。克隆极耐热木聚糖酶基因xynB，将其与罗克霉素启动子融合，构建融合质粒载体pXYNB2，将其转化到枯草芽孢杆菌Bs916中，获得重组枯草芽孢杆菌（ Bs916／pXYNB2），并对重组芽孢杆菌分泌表达的极耐热木聚糖酶的酶学性质进行了研究。 SDS⁃PAGE法测定重组枯草芽孢杆菌分泌的蛋白在相对分子质量43000处有明显的蛋白表达条带。重组木聚糖酶的最适反应pH值为5�0～5�8，重组木聚糖酶的最适反应温度大于或等于100℃，重组极耐热木聚糖酶在pH值4�6～7�8比较稳定，重组木聚糖酶的温度稳定性在90℃下保温2 h后残存酶活83％。可见，生防枯草芽孢杆菌Bs916能有效分泌表达极耐热木聚糖酶，为以后构建多种纤维素和半纤维素在其中的协同表达，拓宽其碳源利用范围奠定基础。