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[1]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[J].应用与环境生物学报,2013,19(06):990-996.[doi:10.3724/SP.J.1145.2013.00990]
 WANG Yuan,GAO Qiuqiang,XIN Xiujuan,et al.Expression of Endoglucanase Gene and β-Glucosidase Genes in Bacillus subtilis[J].Chinese Journal of Applied & Environmental Biology,2013,19(06):990-996.[doi:10.3724/SP.J.1145.2013.00990]
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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()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年06期
页码:
990-996
栏目:
研究论文
出版日期:
2013-12-25

文章信息/Info

Title:
Expression of Endoglucanase Gene and β-Glucosidase Genes in Bacillus subtilis
作者:
王远高秋强辛秀娟鲍杰
(华东理工大学生物反应器工程国家重点实验室 上海 200237)
Author(s):
WANG Yuan GAO Qiuqiang XIN Xiujuan BAO Jie
(State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China)
关键词:
枯草芽孢杆菌β-葡萄糖苷酶内切葡聚糖酶胞外分泌共表达
Keywords:
Bacillus subtilis β-glucosidase endoglucanase extracellular secretion coexpression
分类号:
Q786 : Q814
DOI:
10.3724/SP.J.1145.2013.00990
文献标志码:
A
摘要:
为了实现多种纤维素酶基因的分泌型共表达,利用含有枯草芽孢杆菌中性蛋白酶NprB信号肽的穿梭质粒pP43JM2作为表达载体,将来源于热纤梭菌的内切葡聚糖酶基因celA与来源于多粘芽孢杆菌的β-葡萄糖苷酶基因bglA和bglB在枯草芽孢杆菌WB800中进行了分泌型的单独表达及共表达. 结果表明,内切葡聚糖酶基因celA与β-葡萄糖苷酶基因bglA和bglB均能够在枯草芽孢杆菌中实现分泌表达,而且检测到了celA和bglB基因在枯草芽孢杆菌中的分泌型共表达,其中,共表达菌株的胞外酶液分别与PASC和纤维二糖底物反应时,释放出674 mg/L和24 mg/L的葡萄糖. 而bglA基因只检测到在胞内表达,其胞内酶液以纤维二糖为底物时生成938.7 mg/L的葡萄糖. 本研究为实现在枯草芽孢杆菌中纤维素酶多组分人工组装并为集成生物工艺菌种的构建奠定了一定的实验基础.
Abstract:
In order to archive the secretory coexpression of cellulase genes, this study used the pP43JM2 shuttle vector carrying NprB signal peptide of the Bacillus subtilis neutral protease as expression vector to express in B. subtilis WB800 the celA gene from Clostridium thermocellum DSM 1237 encoding endoglucanase and bglA and bglB genes from B. polymyxa encoding β-glucosidases. The results showed that the endoglucanase encoded by celA and the β-glucosidases encoded by bglB were secreted into the culture broth successfully; the two cellulase genes could be coexpressed and secreted; the extracellular enzyme solution reacted with PASC and cellobiose, producing 674 and 24 mg/L glucose, respectively; on the contrary, the β-glucosidases encoded by bglA did not react with PASC, only with cellobiose, its intracellular enzyme solution producing 938.7 mg/L glucose. The current work suggested a possible method for multiple cellulase secretion in B. subtilis and may provide a way of economical construction of bioprocessing strains for bulk chemical production.

参考文献/References:

1 Sun Y, Cheng JY. Hydrolysis of lignocellulosic materials for ethanol production: a review [J]. Bioresour Technol, 2002, 83 (1): 1-11 2 Bhatia Y, Mishra M, Bisaria VS. Microbial β-glucosidases: cloning, properties, and applications [J]. Crit Rev Biotechnol, 2002, 22 (4): 375-407 3 洪剑辉, 张梁, 石贵阳, 王正祥, 章克昌. 利用纤维二糖的酵母工程菌构建[J]. 应用与环境生物学报,?2006, 12 (3): 391-394 [Hong JH, Zhang L, Shi GY, Wang ZX, Zhang KC. Construction of recombinant yeast strain using cellobiose as sole carbon source [J]. Chin?J?Appl?Environ?Biol,?2006, 12 (3): 391-394] 4 Candelas LG, Aristoy MC, Polaina J, Flors A. Cloning and characterization of two genes from Bacillus polymyxa expressing β-glucosidase activity in Escherichia coli [J]. Appl Environ Microbiol, 1989, 55 (12): 3173-3177 5 Vazana Y, Mora?s S, Barak Y, Lamed R, Bayer EA. Interplay between Clostridium thermocellum family 48 and family 9 cellulases in the cellulosomal versus noncellulosomal states [J]. Appl Environ Microbiol, 2010, 76 (10): 3236-3243 6 Yao Q, Sun TT, Chen GJ, Liu WF. Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum [J]. Biotechnol Lett, 2007, 29: 1243-1247 7 Zhang XZ, Zhang YH. One-step production of biocommodities from lignocellulolytic biomass by recombinant cellulolytic Bacillus subtilis: opportunities and challenges [J]. Eng Life Sci, 2010, 10 (5): 398-406 8 Petit MA, Joliff G, Mesas JM, Klier A, Rapoport G, Ehrlich SD. Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification [J]. Nat Biotechnol, 1990, 8: 559-563 9 Aminov RI, Golovchenko NP, Ohmiya K. Expression of a celE gene from Clostridium thermocellum in Bacillus [J]. J Ferment Bioeng, 1995, 79 (6): 530-537 10 Cho HY, Yukawa H, Inui M, Doi RH, Wong SL. Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800 [J]. Appl Environ Microbiol, 2004, 70 (9): 5704-5707 11 Romero S, Merino E, Bolívar F, Gosset G, Martinez A. Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism [J]. Appl Environ Microbiol, 2007, 73: 5190-5198 12 Zhang XZ, Sathitsuksanoh N, Zhu ZG, Zhang YH. One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis [J]. Metab Eng, 2011, 13 (4): 364-372 13 Wu SC, Yeung JC, Duan YJ, Ye RQ, Szarka SJ, Habibi HR, Wong SL. Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production [J]. Appl Environ Microbiol, 2002, 68 (7): 3261-3269 14 Dartois V, Coppee JY, Colson C,.Baulard A. Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis [J]. Appl Environ Microbiol, 1994, 60: 1670-1673 15 Liu JM, Xin XJ, Li CX, Bao J. Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis [J]. Appl Biochem Biotechnol, 2012, 166: 652-662 16 Luria SE, Delbruck M. Mutations of bacteria from virus sensitivity to virus resistance [J]. Genetics, 1943, 28: 491-511 17 John S. Transformation of biochemically de?cient strains of Bacillus subtilis by deoxyribonucleotide [J]. Proc Natl Acad Sci USA, 1958, 44: 1072-1078 18 Zhang YH, Cui J, Lynd LR, Kuang LR. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidences from enzymatic hydrolysis and supramolecular structure [J]. Biomacromolecules, 2006, 7 (2): 644-648

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备注/Memo

备注/Memo:
国家“973计划”项目(2011CB707406)、“863计划”项目(2012AA022301)、中国博士后基金(2011M500742/2012T50380/2012M520850)、中央高校基本科研业务费专项资金(WF0913005/1114054/1214025)和上海市重点学科建设项目(B505)资助
更新日期/Last Update: 2014-01-03