基于理性化设计的β-甘露聚糖酶分子定向改造

项目名称： 基于理性化设计的β-甘露聚糖酶分子定向改造

项目编号： No.31271811

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 李剑芳

作者单位： 江南大学

项目金额： 77万元

中文摘要： 目前国内已商品化的β-甘露聚糖酶在酶学特性、酶活性等方面均存在一定的缺陷，例如较弱的底物亲和力、较低的比酶活以及对各种极端环境较差的耐受性等，已成为限制我国β-甘露聚糖酶产业发展的瓶颈。为获得底物亲和力强、热稳定性好的优良β-甘露聚糖酶，本项目拟采用三个步骤对宇佐美曲霉β-甘露聚糖酶进行定向改造：首先，采用分子对接、分子动力学模拟等手段进行理性设计并借助融合蛋白及定点突变技术获得底物亲和力强的嵌合酶和突变酶。其次，采用分子动力学模拟、与耐热β-甘露聚糖酶的多序列比对等手段，以蛋白质的整体能量值和RMSD值为指标进行理性设计并实施点突变或肽段替换，获得热稳定性好的突变酶。最后，将得到的较理想的实验结果进行理性化组合设计，拟获得底物亲和力强/热稳定性高的工程酶。本研究工作有望在基于理性设计的分子定向改造领域进入国际先进行列，为获取优良β-甘露聚糖酶提供新方法、新途径和新策略。

中文关键词： β-甘露聚糖酶；分子定向改造；底物亲和力；热稳定性；理性化设计

英文摘要： Currently, the commercialized β-mannanases in our country have some shortcomings in enzymatic properties and enzymatic activity, such as the low substrate affinity and spcific activity, the poor tolerance to extreme surroundings and so on, which have limited the development of our country's β-mannanases industry. To gain the excellent β-mannanases that have strong substrate affinity or/and good thermostability, the directed molecular modification will be taken to the Aspergillus usamii β-mannanase by three steps. Firstly, rational design based on moleculer docking and molecular dynamics simulation, protein fussion and fixed-point mutation will be used for acquireing the chimera-enzyme and mutant-enzyme, which have strong substrate affinity. Secondly, using methods such as molecular dynamics simulation and multiple sequences alignment with thermostable β-mannanases, making rational design taking the overall energy value and the RMSD value of the protein as indexes and fixed-point mutation or peptide replacement to gain the mutant-enzyme with good thermostability. Lastly, making rational combination design based on the ideal experimental results to gain the engineered enzyme with strong substrate affinity and good thermostability. This project is expected to cross the world advanced cavalcade in the field of direc

英文关键词： β-mannanase；Directed molecular modification；Substrate affinity；Thermostability；Rational design