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.
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.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2013年第6期997-1002,共6页
Chinese Journal of Applied and Environmental Biology
基金
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)
教育部长江学者和创新团队发展计划项目(IRT1135)资助~~
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
keratinase
Bacillus licheniformis
Escherichia coli
recombinant expression
enzymatic property fermentationoptimization
