蜡样芽孢杆菌中甲酸脱氢酶基因产氢研究
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摘要
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 CO_2 from formate, is a critical enzyme in hydrogen production by bacteria. In this study the formate dehydrogenase(fdh F) gene from Bacillus cereus strain XN12 was cloned. The sequencing analysis revealed that the cloned fdh F gene contained 2937 base pairs, 39.3% GC content and shared 100% identity with the fdh F gene of Bacillus cereus strain Q1(genebank No. CP000227.1). To characterize the fdh F gene product of Bacillus cereus strain XN12, the fdh F gene was then subcloned into p ET32 a and the resulting p ET32-FDHF-His plasmid was transformed into Escherichia coli BL21 cells. Through the IPTG induction, the cloned fdh F gene was efficiently overexpressed. The recombinant Fdh F protein was highly functional as demonstrated by BV reduction experiment. It was found that the hydrogen production rate of recombinant Fdh F protein was greatly influenced by the presence of various metal ions, among which MoO_4~(2-) and SeO_3~(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.
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 CO_2 from formate, is a critical enzyme in hydrogen production by bacteria. In this study the formate dehydrogenase(fdh F) gene from Bacillus cereus strain XN12 was cloned. The sequencing analysis revealed that the cloned fdh F gene contained 2937 base pairs, 39.3% GC content and shared 100% identity with the fdh F gene of Bacillus cereus strain Q1(genebank No. CP000227.1). To characterize the fdh F gene product of Bacillus cereus strain XN12, the fdh F gene was then subcloned into p ET32 a and the resulting p ET32-FDHF-His plasmid was transformed into Escherichia coli BL21 cells. Through the IPTG induction, the cloned fdh F gene was efficiently overexpressed. The recombinant Fdh F protein was highly functional as demonstrated by BV reduction experiment. It was found that the hydrogen production rate of recombinant Fdh F protein was greatly influenced by the presence of various metal ions, among which MoO_4~(2-) and SeO_3~(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.
引文
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[13]Pinske C,Sawers R G.The importance of iron in the biosynthesis and assembly of[Ni Fe]-hydrogenases[J].Biomolecular Concepts,2014,5(1):55-70.
[14]Shimizu K.Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism[J].ISRN Biochemistry,2013,2013(6):645983.
[15]Lara A R,Taymaz-Nikerel H,Mashego M R,et al.Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses[J].Biotechnology and Bioengineering,2009,104(6):1153-1161.
[16]Asano M,Basieva I,Khrennikov A,et al.Quantum-like model for the adaptive dynamics of the genetic regulation of E.coli's metabolism of glucose/lactose[J].Systems and Synthetic Biology,2012,6(1/2):1-7.
[17]Marx G,Miskolci F.The CO2 greenhouse effect and the thermal history of the atmosphere[J].Advances in Space Research,1981,1(14):5-18.
[18]Knox S H,Sturtevant C,Matthes J H,et al.Agricultural peatland restoration:effects of land-use change on greenhouse gas(CO2and CH4)fluxes in the Sacramento-San Joaquin Delta[J].Global Change Biology,2015,21(2):750-765.
[19]Mandal B,Nath K,Das D.Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae[J].Biotechnology Letters,2006,28(11):831-835.
[20]Li X,Wang Y,Zhang S,et al.Effects of light/dark cycle,mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5[J].Bioresource Technology,2011,102(2):1142-1148.
[21]Kraemer J T,Bagley D M.Improving the yield from fermentative hydrogen production[J].Biotechnology Letters,2007,29(5):685-695.
[22]周灿灿,唐波,罗玮,等.电子载体对丁醇发酵的影响[J].生物加工过程,2012,10(5):1-6.
[2]Zhang S C,Lai Q H,Lu Y,et al.Enhanced biohydrogen production from corn stover by the combination of Clostridium cellulolyticum and hydrogen fermentation bacteria[J].Journal of Bioscience and Bioengineering,2016,122(4):482-487.
[3]Dhar B R,Elbeshbishy E,Hafez H,et al.Hydrogen production from sugar beet juice using an integrated biohydrogen process of dark fermentation and microbial electrolysis cell[J].Bioresource Technology,2015,198:223-230.
[4]刘洪艳,朱大玲,王文磊.一株耐酸产氢突变株Pantoea agglomerans的筛选与产氢特性[J].中国环境科学,2012,32(1):125-129.
[5]Wagner R,Andreesen J R.Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation[J].Archives of Microbiology,1977,114(3):219-224.
[6]Maeda T,Sanchez-Torres V,Wood T K.Metabolic engineering to enhance bacterial hydrogen production[J].Microbial Biotechnology,2008,1(1):30-39.
[7]王海燕,程水源,孟静.蜡状芽孢杆菌的筛选及其产氢性能[J].北京工业大学学报,2014,40(10):1540-1546.
[8]胡春辉,于浩,赵阳国,等.高效耐盐柴油降解菌的筛选、鉴定及降解基因[J].中国环境科学,2017,37(11):4251-4258.
[9]张鹏幸,许静,卢剑功,等.含荧光素酶四膜虫B2086-LUC全细胞生物传感器的构建及其对重金属离子的响应[J].中国环境科学,2013,33(6):1075-1080.
[10]Lester R L,Demoss J A.Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli[J].Journal of Bacteriology,1971,105(3):1006-1014.
[11]Enoch H G,Lester R L.Effects of molybdate,tungstate,and selenium compounds on formate dehydrogenase and other enzyme systems in Escherichia coli[J].Journal of Bacteriology,1972,110(3):1032-1040.
[12]Leonhardt U,Andreesen J R.Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum[J].Archives of Microbiology,1977,115(3):277-284.
[13]Pinske C,Sawers R G.The importance of iron in the biosynthesis and assembly of[Ni Fe]-hydrogenases[J].Biomolecular Concepts,2014,5(1):55-70.
[14]Shimizu K.Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism[J].ISRN Biochemistry,2013,2013(6):645983.
[15]Lara A R,Taymaz-Nikerel H,Mashego M R,et al.Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses[J].Biotechnology and Bioengineering,2009,104(6):1153-1161.
[16]Asano M,Basieva I,Khrennikov A,et al.Quantum-like model for the adaptive dynamics of the genetic regulation of E.coli's metabolism of glucose/lactose[J].Systems and Synthetic Biology,2012,6(1/2):1-7.
[17]Marx G,Miskolci F.The CO2 greenhouse effect and the thermal history of the atmosphere[J].Advances in Space Research,1981,1(14):5-18.
[18]Knox S H,Sturtevant C,Matthes J H,et al.Agricultural peatland restoration:effects of land-use change on greenhouse gas(CO2and CH4)fluxes in the Sacramento-San Joaquin Delta[J].Global Change Biology,2015,21(2):750-765.
[19]Mandal B,Nath K,Das D.Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae[J].Biotechnology Letters,2006,28(11):831-835.
[20]Li X,Wang Y,Zhang S,et al.Effects of light/dark cycle,mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5[J].Bioresource Technology,2011,102(2):1142-1148.
[21]Kraemer J T,Bagley D M.Improving the yield from fermentative hydrogen production[J].Biotechnology Letters,2007,29(5):685-695.
[22]周灿灿,唐波,罗玮,等.电子载体对丁醇发酵的影响[J].生物加工过程,2012,10(5):1-6.