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首页> 外文期刊>Journal of bacteriology >Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes
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Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes

机译：枯草芽孢杆菌枯草芽孢杆菌的异源生产梭状芽胞杆菌，并制备活性重组纤维素体。

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摘要

In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a “designer” cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli. The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.
机译：在 Clostridium spp。产生的纤维素小体中，dockerin域和cohesin域之间的高亲和力相互作用是酶亚基组装成复合物的原因。因此，含有“ dockerin”结构域和支架单元的全长酶亚基的异源表达对于“设计”纤维素酶体或具有特定功能的重组纤维素体的体外组装是必不可少的。我们报告制备的梭菌纤维素重组纤维素酶体，其包含酶亚基EngB和支架单元mini-CbpA，其中包含纤维素结合域，推定的细胞壁结合域和两个黏附素单元。枯草芽孢杆菌WB800可以表达含有dockerin结构域的全长EngB，它缺乏8种细胞外蛋白酶，可以防止在催化结构域和dockerin结构域之间发生酶促亚基的蛋白水解切割。以前尝试用大肠杆菌表达EngB。重组EngB与mini-CbpA的组装通过免疫染色，纤维素结合实验和天然聚丙烯酰胺凝胶电泳分析得以确认。

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