蜡样芽孢杆菌中甲酸脱氢酶基因产氢研究

Studies on hydrogen production via formate dehydrogenase from Bacillus cereus strains

通过基因筛选,成功分离并克隆到蜡样芽胞杆菌XN12(Bacillus cereus XN12)的甲酸脱氢酶基因fdh F(formate dehydrogenase),该基因全长2937bp,GC含量39.3%,编码978个氨基酸,与已报道的蜡样芽孢杆菌Q1的fdh F基因(Gen Bank No.CP000227.1)同源性达到100%.将其连接在表达载体p ET32a上并融合His标签,构建了重组质粒p ET32a-FDHF-His,转入大肠杆菌BL21(Escherichia coli BL21)后获得了高效表达.重组菌株经IPTG诱导后经Western Blot分析表明,重组蛋白分子量约为108k Da.通过对重组菌株产氢性能试验表明,重组菌对提高产氢率具有一定促进作用,产氢量为每消耗1mol的葡萄糖和甲酸盐分别能产生0.73mol和0.20mol的氢气.

Hydrogen is a promising clean energy resource.However,the biohydrogen production efficiency needs to be significantly improved to make it competitive to fossil fuels.Formate dehydrogenase,which is a catalyst in the production of 2H+,2e-,and CO2 from formate,is a critical enzyme in hydrogen production by bacteria.In this study the formate dehydrogenase(fdhF)gene from Bacillus cereus strain XN12 was cloned.The sequencing analysis revealed that the cloned fdhF gene contained 2937base pairs,39.3%GC content and shared 100%identity with the fdhF gene of Bacillus cereus strain Q1(genebank No.CP000227.1).To characterize the fdhF gene product of Bacillus cereus strain XN12,the fdhF gene was then subcloned into pET32a and the resulting pET32-FDHF-His plasmid was transformed into Escherichia coli BL21 cells.Through the IPTG induction,the cloned fdhF gene was efficiently overexpressed.The recombinant FdhF protein was highly functional as demonstrated by BV reduction experiment.It was found that the hydrogen production rate of recombinant FdhF protein was greatly influenced by the presence of various metal ions,among which MoO4 2-and SeO3 2-increased the hydrogen production mainly by increase recombinant protein expression.The hydrogen production was also higher when glucose used as the substrate than formate used as the substrate.The results suggested that recombinant Bacillus cereus formate dehydrogenase protein was a promising solution for improving biohydrogen production.