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

Expression of β-glucosidase genes bglA,bglB,and bgl from Bacillus polymyxa in Escherichia coli

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(P<0.05)。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。

To improve the enzyme activity ofβ-glucosidase,twoβ-glucosidase genes from Bacillus polymyxa were introduced separately（bglAand bglB）and together（bgl）into the pET-28 avector and expressed in Escherichia coli C41.The three recombinant strains were designated as EA,EB,and co-expression EAB.Strains EA and EB were mixed at ratios of 1∶1,1∶2,and 2∶1and their totalβ-glucosidase activity was compared with those of each single enzyme group,the co-expression strain,and mixed expression strains.The results of SDS-PAGE analyses showed that both BglA and BglB were 50 ku,and the size of these proteins in the EA and EB mixed cultures was also 50 ku.The size of Bgl in the co-expression strain EAB was 100 ku.These results indicated that a Bgl complex was able to form in cells,but not in vitro.In an enzyme activity assay,the activity of Bgl from the co-expression strain was not significantly different from that of bglin B.polymyxa,but it was significantly higher than those of BglA in EA and BglB in EB（P〈0.05）.The results of Congo red staining also showed that the enzyme activity of Bgl was significantly higher than those of BglA and BglB.This study lays the foundation for the construction of artificial assemblies,and for the integration of biological technologies in cellulose processing.