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[1]刘柏宏,张娟,方真,等.来源于地衣芽胞杆菌的角蛋白酶在大肠杆菌中的表达、理化性质及其发酵优化[J].应用与环境生物学报,2013,19(06):997-1002.[doi:10.3724/SP.J.1145.2013.00997]
 LIU Baihong,ZHANG Juan,FANG Zhen,et al.Expression, Characterization and Fermentation Optimization of Keratinase from Bacillus licheniformis in Escherichia coli[J].Chinese Journal of Applied & Environmental Biology,2013,19(06):997-1002.[doi:10.3724/SP.J.1145.2013.00997]
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来源于地衣芽胞杆菌的角蛋白酶在大肠杆菌中的表达、理化性质及其发酵优化()
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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:
997-1002
栏目:
研究论文
出版日期:
2013-12-25

文章信息/Info

Title:
Expression, Characterization and Fermentation Optimization of Keratinase from Bacillus licheniformis in Escherichia coli
作者:
刘柏宏张娟方真刘文涛堵国成陈坚廖祥儒
(1江南大学工业生物技术教育部重点实验室 无锡 214122) (2江南大学粮食发酵工艺与技术国家工程实验室 无锡 214122) (3江南大学糖化学与生物技术教育部重点实验室 无锡 214122) (4江南大学生物工程学院 无锡 214122)
Author(s):
LIU BaihongZHANG JuanFANG ZhenLIU WentaoDU GuochengCHEN JianLIAO Xiangru
(1Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China) (2National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China) (3Key Laboratory of Carbohydrate and Biotechnology, Jiangnan University, Wuxi 214122, China) (4School of Biotechnology, Jiangnan University, Wuxi 214122, China)
关键词:
角蛋白酶地衣芽胞杆菌大肠杆菌重组表达酶学性质发酵优化
Keywords:
keratinase Bacillus licheniformis Escherichia coli recombinant expression enzymatic property fermentation optimization
分类号:
Q939.9 : Q796
DOI:
10.3724/SP.J.1145.2013.00997
文献标志码:
A
摘要:
为了提高角蛋白酶在大肠杆菌中的胞外表达量并了解其酶学性质,从一株具有羽毛降解能力的地衣芽胞杆菌(Bacillus licheniformis BBE11-1)中克隆获得全长为1 140 bp的角蛋白酶基因ker,将去掉自身信号肽全长为1 053 bp的ker基因与质粒pET-22b (+)进行连接并转化到大肠杆菌BL21宿主中获得重组菌株. 此后,采用疏水苯基层析柱对所表达的角蛋白酶进行纯化,获得分子量(Mr)大小约32×103的重组角蛋白酶. 酶学性质研究表明,该重组角蛋白酶最适温度为50 ℃,最适pH值为10,属于丝氨酸蛋白酶,金属离子Mg2+和K+对酶活力有促进作用,而Mn2+对其有抑制作用. 经优化后,该酶胞外酶活在诱导温度20 ℃、IPTG 0.05 mmol/L、甘氨酸浓度150 mmol/L以及酵母粉浓度60 g/L的条件下可达460.2 U/mL. 研究表明,优化培养条件可以较好地提高角蛋白酶在大肠杆菌中的表达量,结果为利用基因工程菌生产角蛋白酶奠定了基础.
Abstract:
In order to enhance the production of keratinase in Escherichia coli and study the enzymatic properties, the ker gene (1 140 bp) was cloned from a feather-degrading bacterium of Bacillus licheniformis BBE11-1. After the 1 053 bp fragment of signal peptide-truncated ker gene was inserted into the expression vector pET-22b (+), the recombinant plasmid was transformed into E. coli BL21 (DE3). The recombinant keratinase purified by hydrophobic?interaction?chromatography using HiTrap Phenyl-sepharose Fast Flow was a serine protease most active at 50 ℃ and pH 10. The activity of keratinase was enhanced by Mg2+ and K+, and inhibited by Mn2+. The highest keratinase activity was 460.2 U/mL in growth medium containing inducer (0.05 mmol/L IPTG, 150 mmol/L glycine and 60 g/L yeast extract) at 20 ℃. This report focused on the expression and characterization of recombinant keratinase in E. coli. We found that optimizing culture conditions could preferably enhance production of keratinase in E. coli. The results would be helpful in producing keratinase by gene engineering strain.

参考文献/References:

1 Brandelli A, Daroit DJ. Riffel A. Biochemical features of microbial keratinases and their production and applications [J]. Appl Microbiol Biotechnol, 2010, 85 (6): 1735-1750 2 Gupta R, Ramnani P. Microbial keratinases and their prospective applications: an overview [J]. Appl Microbiol Biot, 2006, 70: 21-33 3 Gupta R, Sharma R, Beg QK. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology [J]. Crit Rev Biotechnol, 2013, 33 (2): 216-228 4 Hu H, He J, Yu B, Zheng P, Huang Z, Mao X, Yu J, Han G, Chen D. Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes [J]. Biotechnol Lett, 2013, 35 (2): 239-244 5 Lin X, Wong SL, Miller ES, Shih JC. Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis [J]. J Ind Microbiol Biotechnol, 1997, 19 (2): 134-138 6 Porres JM, Benito MJ, Lei XG. Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris [J]. Biotechnol Lett, 2002, 24 (8): 631-636 7 Radha S, Gunasekaran P. Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain [J]. J Appl Microbiol, 2007, 103 (4): 1301-1310 8 Radha S, Gunasekaran P. Sustained expression of keratinase gene under PxylA and PamyL promoters in the recombinant Bacillus megaterium MS941 [J]. Bioresour Technol, 2008, 99 (13): 5528-5537 9 Lin HH, Yin LJ, Jiang ST. Cloning, expression, and purification of Pseudomonas aeruginosa keratinase in Escherichia coli AD494(DE3)pLysS expression system [J]. J Agric Food Chem, 2009, 57 (9): 3506-3511 10 Lin HH, Yin LJ, Jiang ST. Expression and purification of Pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system [J]. J Agric Food Chem, 2009, 57 (17): 7779-7784 11 Lin HH, Yin LJ, Jiang ST. Functional expression and characterization of keratinase from Pseudomonas aeruginosa in Pichia pastoris [J]. J Agric Food Chem, 2009, 57 (12): 5321-5325 12 Yamamura S, Morita Y, Hasan Q, Rao SR, Murakami Y, Yokoyama K, Tamiya E. Characterization of a new keratin-degrading bacterium isolated from deer fur [J]. J Biosci Bioeng, 2002, 93 (6): 595-600 13 Tiwary E, Gupta R. Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli [J]. J Agric Food Chem, 2010, 58 (14): 8380-8385 14 Lin X, Lee CG, Casale ES, Shih JC. Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain [J]. Appl Environ Microbiol, 1992, 58 (10): 3271-3275 15 Mergulhao FJ, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli [J]. Biotechnol Adv, 2005, 23 (3): 177-202 16 李兆丰. 软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D]. 无锡: 江南大学, 2009 [Li ZF. Expression of α-cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and analysis of its product specificity [D]. Wuxi: Jiangnan University, 2009] 17 Radha S, Gunasekaran P. Purification and characterization of keratinase from recombinant Pichia and Bacillus strains [J]. Protein Express Purif, 2009, 64 (1): 24-31 18 李飞, 李迅, 孙静, 王飞, 赵林果. 木聚糖酶基因xynB在不同大肠杆菌表达系统中的表达比较[J]. 应用与环境生物学报, 2011, 17 (1): 100-103 [Li F, Li X, Sun J, Wang F, Zhao LG. Expression of xylanase gene xynB in Escherichia coli [J]. Chin J Appl Environ Biol, 2011, 17 (1): 100-103] 19 Sorensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli [J]. Microb Cell Fact, 2005, 4: 1 doi:10.1186/1475-2859-4-1 20 Tang JB, Yang HM, Song SL, Zhu P, J AG. Effect of glycine and Triton X-100 on secretion and expression of ZZ-EGFP fusion protein [J]. Food Chem, 2008, 108 (2): 657-662 21 Kaderbhai N, Karim A, Hankey W, Jenkins G, Venning J, Kaderbhai MA. Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli [J]. Biotechnol Appl Biochem, 1997, 25 (1): 53-61 22 Yang Y, Zhang D, Liu S, Jia D, Du G, Chen J. Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli [J]. J Ind Microbiol Biotechnol, 2012, 39 (1): 99-104

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备注/Memo

备注/Memo:
国家自然科学基金项目(30900013)、国家高技术研究发展计划(863计划)项目(2011AA100905,2011AA100901)、国家“十二五”关键技术研究发展计划项目(2011BAK10B03)及教育部长江学者和创新团队发展计划项目(IRT1135)资助
更新日期/Last Update: 2014-01-03