κ-卡拉胶酶CgkX催化区的重组表达菌株筛选和发酵优化  

Recombinant Expression and Fermentation Optimization of the Catalytic Module of κ-carrageenase CgkX

作　　者：张兰 苏平安 杨雪梅 于文功[1] 韩峰[1] 

机构地区：[1]海洋药物教育部重点实验室山东省糖科学与糖工程重点实验室中国海洋大学医药学院,山东青岛266003

出　　处：《现代生物医学进展》2016年第6期1008-1011,1127,共5页Progress in Modern Biomedicine

基　　金：海洋公益性行业科研专项(201105027-3;201005024);国家自然科学基金项目(31070712;41376144);国家科技支撑计划项目(2013BAB01B02)

摘　　要：目的:CgkX是来自Pseudoalteromonas sp.QY203的一种高活性、高稳定性的κ-卡拉胶酶,本文旨在构建CgkX催化结构域(CgkX-CM)重组表达菌株,并对发酵条件进行优化,提高CgkX-CM的产量。方法:在分析κ-卡拉胶酶CgkX-CM基因序列的基础上,构建多种CgkX-CM表达载体,在大肠杆菌BL21(DE3)中进行重组表达,通过酶活测定筛选其中产量最高的表达菌株,并优化发酵起始pH、诱导温度、IPTG浓度、装液量以及甘氨酸浓度等因素。结果:构建了5种重组表达菌株,其中E.coli BL21(DE3)/p ET22b-CgkX-CM酶产量是其他重组菌株的7.4-11.6倍。确定了该菌株最佳发酵条件为:培养基含1 g·L^(-1)甘氨酸(起始pH 7.5),装液量为75 m L,0.1 mmol·L^(-1) IPTG 20℃诱导28 h,酶产量是优化前的7.3倍。结论:经过重组表达载体筛选和发酵优化,CgkX-CM产量大幅度提高,为今后比较CgkX全长及其催化区域的酶学性质,确定C端Big_2结构域对CgkX的作用奠定了基础。Objective：CgkX-CM is the catalytic module of κ-carrageenase CgkX from Pseudoalteromonas sp.QY203.The purpose of this study was to construct and screen several recombinant expression strains followed by the optimization of the fermentation to get a high yield of CgkX-CM.Methods：A variety of expression vectors were constructed and transformed into E.coli BL21（DE3）.These strains were induced by IPTG to screen the most efficient strain.Initial p H,induction temperature,concentration of IPTG,culture volume and concentration of glycine were optimized.Results：Five recombinant expression vectors were constructed and screened,and E.coli BL21（DE3）/p ET22b-CgkX-CM exhibited the highest enzymatic activity,which was 7.4-11.6 times of other strains.The optimal fermentation conditions of this strain were conformed as follows：initial p H of 7.5,induction temperature of 20℃,concentration of IPTG of 0.1 mmol·L^（-1）,culture volume of 75 m L and concentration of glycine of 1 g·L^（-1）.The optimized enzyme production was 7.3-fold compared to the initial condition and reached 42.6 U/m L.Conclusions：The production of CgkX-CM reached 42.6 U/m L through recombinant expression vectors construction and fermentation optimization.CgkX-CM could be used for the structure-function relationship study of κ-carrageenase CgkX and κ-carrageenan oligosaccharide preparation.

关 键 词：κ-卡拉胶酶 催化结构域 重组表达 发酵优化 寡糖制备 

分 类 号：R93[医药卫生—生药学] Q78[医药卫生—药学]