摘要
[目的]为降低L-天冬酰胺酶Ⅱ的生产成本提供依据。[方法]通过PCR反应扩增大肠杆菌JM109成熟区的L-天冬酰胺酶II基因(ansB),将扩增片段与pMD18-T载体连接,构建重组质粒pMD18-T-asp。对pMD18-T-asp与质粒pET-22b进行双酶切后,将它们连接起来,转化感受态细胞,筛选重组表达载体pET-22b-asp。利用重组表达载体pET-22b-asp转化BL21(DE3),并对其发酵条件以及分离、纯化工艺进行了初步研究。[结果]成功克隆长度为960 bp的L-天冬酰胺酶II基因。重组表达载体pET-22b-asp也构建成功,并在BL21中得到表达。在重组菌接入TB培养基后2 h加入IPTG、培养基的初始pH值为7.2、装液量为12%,L-天冬酰胺酶Ⅱ酶活最高。重组天冬酰胺酶的纯化收率为82%,比活力达196 U/mg。[结论]重组天冬酰胺酶的胞外表达大大简化了其分离步骤。
[Objective] The aim of the research was to provide the basis for reducing the production cost of L asparaginase Ⅱ. [Method] L-asparaginase Ⅱ gene（ansB） in JM109 maturation zone of Escherichia coli was amplified through PCR reaction. The amplified fragment was connected with pMD18-T vector to construct the recombinant plasmid pMD18-T-asp. After double-enzyme digestion of pMD18-T-asp and plasmid pET-22b, they were connected to transform the competent cells and screen out the recombinant expression vector pET-22b-asp which was used to transform BL21（DE3）. And its fermentation conditions, the separation and purification process were preliminarily studied. [Result] L-asparaginase Ⅱ gene with the length of 960 bp was successfully cloned, The recombinant expression vector pET-22b-asp was also successfully constructed and it was expressed in BL21, When IPTG was added after inoculating the recombinant bacteria on TB medium for 2 h with the initial medium pH value of 7,2 and liquid volume in flask of 12%, the enzyme activity of L-asparaginase Ⅱ was highest.. The purification yield of the recombinant asparaginase was 82% and the specific activity reached 196 U/mg, [Conclusion] The extracellular expression of the recombinant asparaginase simplified its separation steps greatly.
出处
《安徽农业科学》
CAS
北大核心
2008年第16期6653-6654,6659,共3页
Journal of Anhui Agricultural Sciences
基金
江苏工业学院资助项目(ZMF04020105)
常州市科技局资助项目(KYZ0603017C)
镇江市科技局资助项目(KYZ0-603023C)
关键词
L-天冬酰胺酶Ⅱ
胞外表达
大肠杆菌
L-asparaginase Ⅱ
Extracellular expression
Escherichia coli
