乳糖酶突变体库的构建及高活性突变体的快速筛选

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...

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Bibliographic Details
Published in中国农业科技导报 no. 2; pp. 135 - 142
Main Author 秦星 孙宁 张丰华 张宇宏 吴坤 张伟 刘波
Format Journal Article
LanguageChinese
Published 2014
Subjects
β-半乳糖苷酶
发酵基础盐培养基
比活力
饱和突变
高通量筛选
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ISSN1008-0864

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Summary: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。
Bibliography:β-galactosidase ; aturated mutation ; ermentation base salt medium; high-throughput screening ; specific activity
QIN Xing , SUN Ning , ZHANG Feng-hua, ZHANG Yu-hong, WU Kun, ZHANG Wei, LIU Bo （ 1.College of Life Sciences, Henan Agricultural University, Zhengzhou 450002; 2.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China）
11-3900/S
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
ISSN:1008-0864