[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.%[Objective] |3-mannanase and xylanase are hemicellulase, which are already used in many areas of industrial and agricultural. The aim of this study was to coexpress two hemicellulase genes in E. Coli (Escherichia coli). [Methods] The P-mannanase and xylanase genes were cloned from Bacillus subtilis BE-91, then linked together by a common restriction enzyme site and inserted into the pET28a(+), establishing a coexpression strain named B.pET28a-man-xyl for the extracellular production of p-mannanase and xylanase in E.coli. [Results] After being induced 21 h, the specific activities of P-mannanase and xylanase were 713.34 U/mL and 1455.83 U/mL, respectively. [Conclusion] The result of SDS-PAGE analysis, hydrolytic ring detection and enzyme activity determination showed that each enzyme was expressed extracellularly as individual functional proteins. In addition, comparing with p-mannanase and xylanase degraded hemicellulose alone, the effection of compound enzymes are much better. The successful construction of a strain which produces xylanase and P-mannanase will be helpful for the study and production of hemicellulases preparation.
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