多粘类芽孢杆菌β-葡萄糖苷酶bglA、bglB和bgl基因在大肠杆菌中的表达

发布时间：2018-01-28 00:40

  本文关键词： β-葡萄糖苷酶 多粘类芽孢杆菌 克隆和表达 共表达 酶活　出处：《草业学报》2017年05期 　论文类型：期刊论文

【摘要】：为了有效地提高β-葡萄糖苷酶的活性,本实验将多粘类芽孢杆菌(Bacillus polymyxa)β-葡萄糖苷酶bglA、bglB和bgl(bglA和bglB基因)基因分别连接在pET-28a上并在大肠杆菌C41中表达。将这3株重组菌分别命名为EA、EB及共表达菌株EAB,并将构建好的工程菌EA和EB按1∶1,1∶2,2∶1进行混合培养,分别比较单一酶组分、共表达及混合表达菌株的酶活。SDS-PAGE电泳图显示BglA和BglB的大小都为50ku,EA和EB混合培养的蛋白大小也为50ku,Bgl大小为100ku,表明BglA和BglB在体外不能形成蛋白复合物,只有在生物体内才能形成Bgl复合蛋白。酶活测定结果表明共表达Bgl与多粘类芽孢杆菌的酶活差异不显著,但显著高于其他组分酶活(P0.05)。刚果红染色结果也表明Bgl酶活比单一酶组分的酶活明显提高。本实验为纤维素酶多组分人工组装及集成生物工艺菌种的构建奠定实验基础。
[Abstract]:In order to improve the activity of 尾 -glucosidase, Bacillus polymyxa- 尾 -glucosidase bglA was used in this experiment. BglB, bgl(bglA and bglB genes were linked to pET-28a and expressed in Escherichia coli C41. The three recombinant strains were named EA, respectively. EB and EAB were co-expressed, and the constructed engineering bacteria EA and EB were mixed by 1: 1 and 1: 1: 2: 1 to compare the single enzyme components. The enzyme activity. SDS-PAGE electrophoresis of co-expressed and mixed expression strains showed that the size of BglA and BglB were both 50ku and 50ku in mixed culture of EA and EB. The size of Bgl was 100ku. indicating that BglA and BglB could not form protein complexes in vitro. Only in vivo can Bgl complex protein be formed. The results of enzyme activity test showed that there was no significant difference between the enzyme activity of co-expressed Bgl and that of Bacillus polymyxides. But significantly higher than that of other components (P0.05). The results of Congo red staining also showed that the enzyme activity of Bgl was significantly higher than that of single enzyme component, which laid the experimental foundation for the artificial assembly of cellulase multicomponent and the construction of integrated biotechnological bacteria.
【作者单位】： 甘肃农业大学动物医学学院;
【基金】：甘肃省科技支撑计划项目(1204NKCA103) 国家自然科学基金青年项目(31500067)资助
【分类号】：Q55;Q78
【正文快照】： 纤维素是植物材料的主要成分,植物通过光合作用的形式使光能以生物能的形式固定下来,其生成量每年高达2000亿t,其中50%以上为纤维素和半纤维素,这些能量相当于全球人类每年能源消耗量的20倍,食物中所含能量的200倍,是永远不会枯竭的可再生资源[1]。但由于纤维素具有水不溶性的，

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