基于理性化设计的β-甘露聚糖酶分子定向改造

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 directed molecular modification based on the rational design, and supply the new method, new approach and new strategy to gain excellent β-mannanases.

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。

β-甘露聚糖酶在食品、饲料、生物能源等多个领域具有较广泛的应用前景，但目前已商品化的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。相比于从自然界中筛选优良产酶菌株和对现有菌株诱变育种等传统方法，借助各种生物数据库和生物计算软件，采用理性化设计的方法对现有酶分子定向改造以获得性状优良的酶品种更加快捷有效。本课题以宇佐美曲霉 (Aspergillus usamii) 来源的β-甘露聚糖酶为研究模板，通过理性化设计并采用融合碳水化合物结构域（CBM）、定点突变、结构替换等手段对β-甘露聚糖酶分子进行定向改造。主要研究成果如下：完成了来源于A. usamii 两个糖苷水解酶家族（GH 5和26）的β-甘露聚糖酶基因（Auman5A和Auman26A）的克隆、生物信息学分析、异源表达以及重组酶的酶学性质分析等研究；成功构建了分别连接1家族和27家族CBM的融合酶（AuMan5A-CBM和AuMan5A-R-C），与原酶相比，两种融合酶的热稳定性和对底物的亲和力有显著提高；成功构建并获得了底物亲和力提高的突变酶（Y115F和Y111F/Y115F），探讨了酶与底物亲和力改变的机理；成功构建并获得了热稳定性和催化效率显著提高的loop结构替换突变酶AuMan5A-Af，阐述了酶热稳定性和催化效率改变的机理。研究成果为在工业领域中广泛和有效的应用提供了优良的工程酶品种，同时为其它蛋白质/酶分子的定向改造提供技术平台和理论依据。