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

doi:10.11686/cyxb2016220

草业学报 ›› 2017, Vol. 26 ›› Issue (5): 189-196.DOI: 10.11686/cyxb2016220

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

王艳, 马亚茹, 万学瑞, 王川^*, 吴润^*, 刘桂林, 刘原子, 吴自祥

甘肃农业大学动物医学学院,甘肃兰州 730070

收稿日期:2016-05-27 修回日期:2016-07-06 出版日期:2017-05-20 发布日期:2017-05-20
作者简介:王艳(1991-),女,甘肃嘉峪关人,在读硕士。E-mail:409193838@qq.com
基金资助:
甘肃省科技支撑计划项目(1204NKCA103)和国家自然科学基金青年项目(31500067)资助

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

WANG Yan, MA Ya-Ru, WAN Xue-Rui, WANG Chuan^*, WU Run^*, LIU Gui-Lin, LIU Yuan-Zi, WU Zi-Xiang

College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2016-05-27 Revised:2016-07-06 Online:2017-05-20 Published:2017-05-20

摘要/Abstract

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50 ku,EA和EB混合培养的蛋白大小也为50 ku,Bgl大小为100 ku,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。

Abstract: To improve the enzyme activity of β-glucosidase, two β-glucosidase genes from Bacillus polymyxa were introduced separately (bglA and bglB) and together (bgl) into the pET-28a vector 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∶1 and their total β-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 bgl in 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.

中图分类号:

王艳, 马亚茹, 万学瑞, 王川, 吴润, 刘桂林, 刘原子, 吴自祥. 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[J]. 草业学报, 2017, 26(5): 189-196.

WANG Yan, MA Ya-Ru, WAN Xue-Rui, WANG Chuan, WU Run, LIU Gui-Lin, LIU Yuan-Zi, WU Zi-Xiang. Expression of β-glucosidase genes bglA, bglB, and bgl from Bacillus polymyxa in Escherichia coli[J]. Acta Prataculturae Sinica, 2017, 26(5): 189-196.

参考文献

[1] Xiong L G, Chai S M, Li W. Research progress of cellulase and its application. Jiangxi Feed, 2008, (4): 23-27.
熊立根, 柴士名, 李旺. 纤维素酶及其应用研究进展. 江西饲料, 2008, (4): 23-27.
[2] Wang W Y, Zhu J H, Wu S Y. Research progress of cellulose science and cellulose. Journal of Jiangsu University of Science and Technology, 1998, (3): 20-28.
汪维云, 朱金华, 吴守一. 纤维素科学及纤维素酶的研究进展. 江苏理工大学学报, 1998, (3): 20-28.
[3] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual[M]. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.
[4] Hu S, Wang W, Zhan F Q, et al . Screening and identification of a strain producing cellulase.Biotechnology, 2008, (5): 36-38.
胡爽, 王炜, 詹发强, 等. 一株产纤维素酶细菌的筛选鉴定. 生物技术, 2008, (5): 36-38.
[5] Shewale J G. β-Glucosidase : Its role in cellulase synthesis and hydrolysis of cellulose. International Journal of Biochemistry, 1982, 14(6): 435-443.
[6] Wilson K. Preparation of genomic DNA from bacteria[M]//Ausubul F M, Bent R. Current Protocols in Molecular Biology. New York(NY): John Wiley & Sons, 1987.
[7] Sambrook J, Russell D W. Molecular Cloning: a Laboratory Manual[M]. New York: Cold Spring Harbor Laboratory Press, 2001.
[8] Guo H, Feng Y, Mo X C, et al . Cloning, expression and characterization of EG gene umcel 3 G from the rumen of Buffalo.Chinese Journal of Biotechnology, 2008, (2): 232-238.
郭鸿, 封毅, 莫新春, 等. 水牛瘤胃宏基因组的一个新的β-葡萄糖苷酶基因 umcel 3 G 的克隆、表达及其表达产物的酶学特性. 生物工程学报, 2008, (2): 232-238.
[9] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 . Nature, 1970, 227: 680-685.
[10] Cui Y, Chen H M, Shang H L, et al . Screening of neutral cellulase producing strain and optimization of its culture medium and characterization of the enzyme. Journal of Zhejiang Agricultural Sciences, 2006, (2): 214-217.
崔琰, 陈红漫, 尚宏丽, 等. 中性纤维素酶产生菌的筛选及其培养基的优化和酶学性质研究. 浙江农业科学, 2006, (2): 214-217.
[11] Ding K, Luo W G, Ding P P, et al . Fused secretory expression of double cellulase gene in Lactic acid bacteria. Food Science, 2014, (15): 127-131.
丁轲, 罗伟光, 丁盼盼, 等. 双纤维素酶基因在乳酸杆菌中的融合分泌表达. 食品科学, 2014, (15): 127-131.
[12] Yang P X, Zheng R D, Luo J F, et al . Screening and identification of 1 strains of cellulase producing fungi and optimization of enzyme production conditions. Jiangsu Journal of Agricultural Sciences, 2015, (1): 186-191.
杨培新, 郑锐东, 罗集丰, 等. 1株产纤维素酶真菌的筛选鉴定及其产酶条件优化. 江苏农业学报, 2015, (1): 186-191.
[13] Gonzalez Candelas L, Aristoy M C, Polania J. Cloning and characterization of two genes from Bacillus polymyxa expressing β-glucosidase activity in Escherichia coli . Applied and Environmental Microbiology, 1989, 55(12): 3173-3177.
[14] Hao S O, Cho H Y, Yukawa H. Regulation of expression of cellulosomes an noncellulosomal (hemi) cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources. Journal of Bacteriology, 2004, 186: 4218-4227.
[15] Zhao Y, Liu W F, Mao A J, et al . Expression, purification and characterization of Bacillus polymyxa BG gene in Escherichia coli . Chinese Journal of Biotechnology, 2004, (5): 741-744.
赵云, 刘伟丰, 毛爱军, 等. 多粘芽孢杆菌 ( Bacillus polymyxa ) β-葡萄糖苷酶基因在大肠杆菌中的表达、纯化及酶学性质分析. 生物工程学报, 2004, (5): 741-744.
[16] Hu K L, Han J, Liu W F, et al . Cloning and recombinant expression and characterization of recombinant protein from Bacillus sp. Microbiology, 2012, (7): 891-900.
胡开蕾, 韩剑, 刘伟丰, 等. 嗜热脱氮土壤芽孢杆菌β-葡萄糖苷酶的克隆与重组表达及其酶学性质研究. 微生物学通报, 2012, (7): 891-900.
[17] Li X H, Zhu M J, Guo Q J. Establishment of a method for measuring the activity of β-glucosidase in crude cellulose. Modern Food Science and Technology, 2010, 26: 323-325.
李旭辉, 朱明军, 郭启军. 一种测定粗纤维素酶中β-葡萄糖苷酶酶活方法的建立. 现代食品科技, 2010, 26: 323-325.
[18] Chang J J, Ho C Y, Ho F J, et al . A synthetic biology tool for engineering a cellulolytic yeast. Biotechnology for Biofuels, 2012, 5: 53.
[19] Wang Y, Gao Q Q, Xin X J, et al . The expression of beta-glucosidase gene and endo-glucanase gene in Bacillus polymyxa . Chinese Journal of Applied and Environmental Biology, 2013, (6): 990-996.
王远, 高秋强, 辛秀娟, 等. β-葡萄糖苷酶基因和内切葡聚糖酶基因在枯草芽孢杆菌中的表达. 应用与环境生物学报, 2013, (6): 990-996.
[20] Tang Z Z, Liu S, Han X Y, et al . Co-expression of recombinant beta-glucosidase gene and endo-glucanase gene in Escherichia coli . Science and Technology of Food Industry, 2013, 34(24): 189-194.
唐自钟, 刘姗, 韩学易, 等. 重组β-葡萄糖苷酶基因和内切葡聚糖苷酶基因在大肠杆菌中的共表达. 食品工业科技, 2013, 34(24): 189-194.

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

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