乳糖酶突变体库的构建及高活性突变体的快速筛选  

Construction of Saturated Mutation Library for β-Galactosidase and a Rapid Screening Method for Mutants with High Activity

作　　者：秦星[1] 孙宁[2] 张丰华[2] 张宇宏[2] 吴坤[1] 张伟[2] 刘波[2] 

机构地区：[1]河南农业大学生命科学学院,郑州450002 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2014年第2期135-142,共8页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(31101233);农业科技成果转化资金项目(2012GB23260566)资助

摘　　要：构建了亮白曲霉来源的β-半乳糖苷酶活性中心的第219位丝氨酸的毕赤酵母饱和突变体库以筛选水解活性提高的乳糖酶。另外，通过以毕赤酵母发酵基础盐诱导培养基代替传统的BMMY培养基来诱导表达β-半乳糖苷酶和其突变体。结果表明，诱导时间在48～60h且粗酶液中总蛋白量在0．2～0．8mg／mL时，各乳糖酶的活力均高于其在BMMY培养基中的活力；各乳糖酶的比活力在此阶段保持相对稳定，且与纯化后的酶比活力呈正相关，即各乳糖酶的比活力高低可以粗酶液中乳糖酶的比活力来相对定量。在此基础上，利用高通量的48孔培养板创建了一种简便、高通量从B一半乳糖苷酶突变体库中筛选高水解活力乳糖酶的方法，并应用该方法快速地从突变体库中筛选到3个水解活力显著提高的突变体S-38、S-35和S-1，比活力分别提高了10．3％，9．2％和6％，高通量的筛选方法是定向进化技术中的重要环节，为其顺利开展奠定了基础。In this study, a saturated mutation library of Aspergillus candidus β-galactosidase S219 site was firstly constructed to screen lactase with improved hydrolysis activity. Additionally, for eliminating the effects of amino acids and peptides in BMMY medium, which is traditionally used in inducing expression of exogenous genes in pichia, on the purity and specific activity assay of target proteins in crude enzyme, fermentation base salt medium was used instead of BMMY medium at shaking flask and 48-well culture plate level. Under the induction time in the range of 48 h to 60 h and the total protein amount in crude enzyme varying from 0.2 mg/mL to 0.8 mg/mL. It was found that the expression quantity of lactase mutants in fermentation base salt medium was higher compared to BMMY medium. Additionally, the specific activities of lactase mutants in crude enzyme were kept relatively stable and presented positive linear relationship to these of the purified proteins. Using 48-weU culture plate, a simple and high-throughpnt method for screening lactase mutants with improved hydrolysis activity was developed. Furthermore, 3 lactase mutants, S219A, S219V and S219E, with hydrolysis activities improved by 10.3%, 9.2% and 6%, respectively were screened from the mutant library. High-throughput screening method was an important link of directed evolution and laid a solid foundation for its development.

关 键 词：Β-半乳糖苷酶 饱和突变 发酵基础盐培养基 高通量筛选 比活力 

分 类 号：S188[农业科学—农业基础科学]