同步降解木聚糖主链和支链的高活性双功能酶的定向改造及热稳定性变化的分子机制研究

Xylan is a major component of non-starch polysaccharides, endoxylanase and ɑ-arabinofuranosidase are two important xylan-degrading enzymes which attacks the main chain and arabinosyl side chain of xylan, respectively. Using bifunctional enzymes to simultaneously deconstructs xylan main chain and side chain can expand the scope of feed raw material, improve the utilization efficiency of animal feed, and reduce the production and use cost of feed enzyme. However, there are few researches in the cloning and identification of this kind of bifunctional enzyme, and no publication about the engineering of its function and the molecular mechanism of themostability until now. BiXyn10B/Ara43A is a bifucntional endoxylanase/ɑ-arabinofuranosidase from human gut bacterium Bacteroides intestinalis, it consists of two catalytic domains and exhibits high activity in acidic conditions. However, the enzyme not very stable when subjected to heattreatment. Since the three-dimensional structure of BiXyn10B/Ara43A is unavailable, the traditional rational design is unapplicable for improving its thermostability. In this project, novel rational design method based on the sequence-based prediction of protein stability, directed evolution and site-directed saturation mutagenesis will be applied to improve the thermostability of BiXyn10B/Ara43A. A series of mutations that are beneficial for improving thermostability will be accumulated in BiXyn10B/Ara43A on the basis of maintaining the enzyme’s high catalytic activity, and finally BiXyn10B/Ara43A will be engineered to be a bifunctional xylanase/ɑ-arabinofuranosidase with good thermostability and high catalytic activity. Based on these results, the molecular mechanism of the thermostability will be explored. This study will not only help gain insights into the molecular mechanism of bifunctional xylanase/ɑ-arabinofuranosidase, but also facilitate the exploitation of novel high efficient feed enzyme.

木聚糖是非淀粉多糖的主要组成成分，木聚糖酶和ɑ-阿拉伯糖呋喃糖苷酶分别能降解木聚糖主链和阿拉伯糖支链，采用双功能酶有利于拓宽饲料原料的使用范围、提高动物利用饲料原料的效率、降低酶制剂生产和使用成本等，但目前对此类酶的发掘研究很少，更未见关于其功能改造和热稳定性分子机制的研究报道。人肠道拟杆菌的BiXyn10B/Ara43A是一个双结构域双功能的木聚糖酶/ɑ-阿拉伯糖呋喃糖苷酶，在酸性条件下具有高活性，但热稳定性不够。鉴于该酶的结构信息未知，本项目拟采用基于氨基酸序列预测蛋白质稳定性的理性设计方法、蛋白质定向进化和定点饱和突变的策略，在保持高酶活的基础上逐步累积有利于热稳定性的有益突变，以期获得兼具高活性和良好热稳定性的双功能突变体，并在此基础上探究此双功能酶热稳定性变化的分子机制。该研究不仅能加强同步降解木聚糖主链和支链的双功能酶分子机理的理解，而且有利于高效新型饲料酶的研发。

项目背景：.肠道细菌Bacteroides intestinalis具有优良的木聚糖降解能力，已发现其基因组上存在一个木聚糖利用核心基因簇，其中存在一个双催化域双功能酶BiXyn10B/Ara43A，在酸性条件下能高效水解阿拉伯木聚糖，但热稳定性不佳，限制了其应用开发。.研究内容：.项目以BiXyn10B/Ara43A为模板，先后采用基于氨基酸序列预测蛋白质稳定性的理性设计方法、定点饱和突变和定向进化的策略，在保持高酶活的基础上逐步提高酶的热稳定性，获得兼具高活性和良好热稳定性的突变体，并探究此双功能酶热稳定性变化的分子机制。.重要结果：.研究发现，采用EASE-MM和INPS服务器分析氨基酸突变对蛋白热稳定性的影响，然后开展突变实验验证，获得了7个有利于提高酶热稳定性且保持高活性的阳性突变，通过叠加突变最终获得了活性和热稳定性明显提高的突变体5M。在此基础上，对预测结果中的热点残基开展定点饱和突变，采用“热处理后水解oNPX显色法”进行初步筛选并复验，获得了两个阳性突变位点和突变体7M。最后，以7M为模板构建随机突变体文库，并从中筛选获得两个阳性突变体8M和9M，其在60 ºC下的热稳定性显著提高（由“热处理5 min后完全失活”提升为“热处理25 min后的残余活性分别达42.78%和37.87%”），在国标法条件下对阿拉伯木聚糖的比酶活也提高了3.12和3.89倍。分析结果发现突变发生的区域、氨基酸的极性和侧链大小等均对酶的热稳定性和活性重要影响。项目执行期以通讯作者共发表SCI论文5篇，其中第一标注3篇，第二、三标注各1篇。.关键数据：.项目获得热稳定性和水解活性均有效提高的多个双功能酶突变体，并分析了各突变酶的酶学性质和生物特性，这些结果综合起来为阐明该酶热稳定性变化的分子机制起到关键作用。.科学意义：.该研究通过对双催化域双功能酶的热稳定性进行有效改造，加强了对该类酶热稳定性变化的分子机制的理解，为此类酶的分子改良提供了示范，也有利于高效新型饲料酶的研发。