重组L-门冬酰胺酶工程菌的表达和PEG的化学修饰  

Expression of recombinant L-asparaginase engineering bacteria and polyethylene glycol chemical modification

作　　者：曹荣月[1] 申克宇[1] 王学军[1] 凤姣[1] 刘景晶[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《生物学杂志》2005年第3期8-10,共3页Journal of Biology

基　　金：江苏省自然科学基金(BK2001078);江苏省社会发展基金(BJ2000033)

摘　　要：目的提高重组L-门冬酰胺酶(rL-ASP)工程菌的表达量,分离纯化rL-ASP并对之进行PEG化学修饰。方法将带有编码rL-ASP的基因的质粒(pKA)导入不同的宿主菌中,挑出高表达菌株,同时优化发酵培养基,分离纯化获得的高纯度rL-ASP再用PEG进行化学修饰,SDS-PAGE检测修饰效果。结果在pH7.0的条件下,宿主菌为JMl09的工程菌pKA/JMl09酶活力最高,三角瓶振摇培养的酶活力可达216×103IU/L;发酵罐发酵培养,酶活力达312×103IU/L。纯化后的rL-ASP比活力为220IU/mg,rL-ASP经过PEG化学修饰生成rL-ASP-PEG,分子量发生改变。结论改变目标蛋白表达的宿主菌和优化发酵工艺,提高了rL-ASP的表达量,纯化的rL-ASP经过PEG化学修饰后分子量增大。Polyethylene glycol(PEG) modified recombinant L-asparaginase (rL-ASP) was made by pKA/JM109.Plasma with L-asparaginase gene (pKA) was induced into various host bacterium such as JM105、JM109、DH5α and the highest active strain pKA/JM109 was obtained.Fermentation process on engineering bacterium was improved and the product was purified and modified by PEG.Engineering bacterium(pKA/JM109) had a higher enzyme activity.It was 216×10~3IU/L in flasks and 312×10~3IU/L in a ferment tank.rL_ASP was modified by PEG to form rL_ASP_PEG.The result showed that Changing host bacteria and composition of media ould increase enzyme activity and enzyme products.Molecular of rL-ASP-PEG was larger than that of rL-ASP's on SDS-PAGE.

关 键 词：发酵 重组L-门冬酰胺酶 聚乙二醇 化学修饰 

分 类 号：Q51[生物学—生物化学] TQ92[轻工技术与工程—发酵工程]