基于定向进化的木聚糖酶热稳定性和催化活性研究

项目名称： 基于定向进化的木聚糖酶热稳定性和催化活性研究

项目编号： No.31201831

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 畜牧学与草地科学、兽医学、水产学

项目作者： 刘明启

作者单位： 中国计量学院

项目金额： 22万元

中文摘要： 木聚糖酶作为安全、高效的饲料添加剂，可大幅度提高单胃动物对植物性饲料的利用率。现有的大多数木聚糖酶催化活性较低，且在高温条件和动物消化道中稳定性较差，这直接影响其应用效果。因此，提高木聚糖酶的热稳定性和催化活性是当前急需解决的重大难题。 本研究在前期木聚糖酶改性的基础上，以6种不同来源嗜温性和嗜热性木聚糖酶为亲本，拟采用定向进化中的易错PCR和DNA shuffling技术构建木聚糖酶基因突变文库，以温度为选择压力，同时参考酶活指标，建立96微孔板分光光度法的高通量表达文库筛选方法，获得耐高温、高酶活的木聚糖酶突变体。分析突变酶及其亲本酶的热稳定性、催化活性和水解模式特点；探明突变酶与其亲本在结构上的差异，阐明与酶热稳定性相关的结构特征及突变酶热稳定性机制；研究突变酶在模拟制粒过程中和动物消化道环境中的稳定性，为其应用提供科学依据。

中文关键词： 木聚糖酶；定向进化；文库；热稳定性；催化活性

英文摘要： Xylanase used as safely and high efficient feed additive can improve the utilization rate of phto-feed by monogastric domestic animals. In feed application, xylanases have to be stable in high temperature during feed preparation, but the catalytic activity is needed at physical conditions of domestic animals. Although most of existing wild-type xylanases have their own advantages, they still cannot satisfy the demand of being applied in feed industries. Hence, attentions are focused on discovery of new xylanases or improvement of existing ones to meet the requirements of feed industries. Directed evolution, which mimics Darwinian evolution in nature and does not require three-dimensional structure, is a powerful tool for protein engineering and enzyme modification. In the study, the mutational library was constructed using 6 xylanases as parents (including mesophilic and thermophilic xylanases) by directed evolution (Error-prone PCR and DNA shuffling). The thermostable and high catalytic activity mutants were screened in 96-well format under the control of temperature. This research is conducted to study the relationships between enzyme properties and amino acid substitutions by assaying catalytic activity, substrate affinity and hydrolysis pattern of wild type and mutants. Furthermore, thermostability experime

英文关键词： Xylanase；Directed evolution；Library；thermostability；Catalytic activity