基于定向进化的木聚糖酶热稳定性和催化活性研究

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 experiment will be carried out to evaluate the feasibility of feed application.

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。

本课题采用定向进化技术，以提高木聚糖酶热稳定性和催化活性为主要目的，获得多个突变体，分析了突变序列和酶学性质，阐明部分木聚糖酶结构-功能关系,为获得理想的木聚糖酶提供新思路和方法。.1..以杂合木聚糖酶ATXX为基础，采用易错PCR技术，构建表达文库，采用96孔板法筛选文库，其中突变体ATXX113的催化活性、热稳定均有获得提高，其突变位点为：D20V-L49P-R122Q-T129A。突变位点氨基酸与相互作用的氨基酸在之间氢键键长发生显著变化。该杂合木聚糖酶具备了TfxA结合水解不溶性底物的能力。为了适用于低聚木糖制备，对获得的两种木聚糖酶进行了固定化研究。.2..采用易错PCR技术对tfx基因进行突变，构建突变文库，采用96孔板法筛选获得5个活性和热稳定性显著提高的突变体。对其中一个突变体的氨基酸突变位点为R129Q-H256L；其催化活性和在70℃下的热稳定性较野生型酶分别提高了23.2%和14.1%。.3..克隆到解淀粉芽孢杆菌木聚糖酶A基因（baxa, GenBank: KM624029），在E. coli BL21和P. pastoris GS115获得分泌表达，表达产物reBaxA-BL21水解桦木木聚糖、燕麦木聚糖和山毛榉木聚糖的主要产物分别为木五糖、木四糖和木三糖。.4..以baxa为模板，采用易错PCR技术，构建表达文库（1463个单菌落），采用96孔板法筛选文库，获得2株突变体（pCbaxA50、pCbaxA186），其催化活性和热稳定有显著提高。测序结果表明，pCbaxA50表达产物reBaxA50的氨基酸突变位点为：S138T。reBaxA50得比活为9.38 U•mg−1，是其亲本的3.5倍。reBaxA50在60°C的半衰期为9.74 min，TAG-DSC分析结果表明reBaxA和reBaxA50的Tm分别为88.95 °C和89.15 °C。以bax和tfx为模板，采用DNA shuffling技术构建突变文库（pCold TF为载体），在筛选平板上获得了约2400个单菌落。对其中的122个菌落进行了测序，并对其中的10个催化活性有所提高的突变体进行酶学性质分析。采用DNA shuffling技术的碱基突变率远高于易错PCR。