基于结构分析的嗜热CBM拆分-易位-组合融合对木聚糖酶稳定性-活性影响

Because of degrading xylan,the main non-starch polysaccride in creal, xylanase has become an important enzyme product in bakery industry, but high temperature and insoluble substrate limit xylanase usage.Thermophilic carbohydrate binding module (CBM) relates to enzyme stability and activity, however, CBM is usually big, and research is limited in deleting or fusing big CBM at GH10 xylanase C-terminus. Based on structural analysis, the project intends to use Aspergillus niger xylanase (Xyn) as a model molecule of GH11 family. Through disassembling thermophilic Thermotota maritima CBM into C1 and C2 modules and fusing to the Xyn, different functions of smaller modules C1 and C2 are investigated in enhancing thermostability and activity. Moreover, effects are investigated by swapping the CBM, C1, and C2 to the Xyn N-terminus and compared with their effects at C-terminus on xylanase. Furthermore, additive effects are investigated by fusing the C1 and/or C2 in four different combinations at the Xyn bi-termini. The project disasembles the CBM, swaps the modules to the GH11 xylanase N-terminus, and fuses small modules at the Xyn bi-termini, with which to investigate "effect of different functions of smaller structure contained in big CMB on its thermostabilizing effect" and effect of N- and bi-terminal domain-fusion on evolution of multi-domain enzyme. Additionally, thermostable and high active chimeric enzymes are created for practical usage, with which to efficiently improve bakery food quality and creal utilization efficiency. Thereby, the project has theoretical and practical importance.

木聚糖酶能降解非淀粉多糖从而成为重要的焙烤用酶制剂，但高温和不溶性底物严重影响其广泛应用。嗜热碳水化合物结合结构域(CBM)与酶活性-稳定性有关，但是CBM结构较大、且研究局限于GH10家族木聚糖酶C端去除/融合。基于结构域分析，项目组拟以黑曲霉Xyn为GH11家族木聚糖酶模式分子，将嗜热海栖热袍菌CBM拆分为C1/C2两个结构融合Xyn，解析C1/C2对酶稳定性、活性影响有何不同；而后将CBM/C1/C2结构域易位至N端，与C端比较对酶性质影响有何不同；进而以四种方式将C1/C2组合融合在酶两端，探讨组合融合对稳定性、活性的叠加效应。通过CBM拆分、易位、组合融合，探讨嗜热CBM所含较小结构的不同功能对其稳定性影响、深入解析嗜热结构域稳定性作用、探讨结构域易位、双端融合对多结构域酶分子进化作用；获得稳定性、不溶性底物降解活性增强的融合酶，更有效提高焙烤品质量，有理论和实际应用双重意义。

本研究通过对嗜高温海栖热袍菌(Thermotaga maritima)GH10家族木聚糖酶C端CBM9结构域分析，将CBM拆分为C1和C2亚结构域分别融合在黑曲霉(Aspergillus niger)GH11家族木聚糖Xyn C端构建Xyn-C1和Xyn-C2，将稳定性结构域C2易位至Xyn N端构建C2-Xyn，进而将C1、C2组合双端融合构建C2-Xyn-C2，C2-Xyn-C1，C1-Xyn-C1，C1-Xyn-C2；得到稳定性酶分子Xyn-C2，C2-Xyn，C2-Xyn-C2，C1-Xyn-C2，其中C2-Xyn-C2稳定性最好，50ºC时半下半失活时间（t1/250）为8.69 h，分别是野生酶Xyn和单端融合酶Xyn-C2，C2-Xyn的24.8、7.5-、-7.1倍，特别是在80 ºC 时t1/2为2.85 h，而Xyn、Xyn-C2、C2-Xyn t1/250为17.6 min, 1.0 h, 1.16 h，园二色光谱研究显示C2-Xyn-C2酶分子变性温度（Tm）比Xyn提高1.8 ºC，熵变（ΔS）降低、自由能（ΔG）降低，三个结构域间产生堆积效应，具有稳定性结构。双端融合酶C2-Xyn-C2对山毛榉木聚糖和燕麦木聚糖催化效率显著提高，比野生酶和单端融合酶催化效率（kcat/km）提高7.9-1.2倍。本研究提供了同时提高酶分子稳定性和活性的双端融合结构域方法，阐明了C2-Xyn-C2稳定性机理。同时开发了双退火PCR方法扩增高特异性DNA、长片断反向PCR构建含有重复DNA序列的质粒构建方法、开发了分子内平端连接、分子内同源重组重组质粒构建方法、探讨了线性DNA体外三步连接。