普鲁兰酶的理性设计及其在枯草芽孢杆菌中的分泌表达研究

The majority of starches used for the production of sugars in industry contain 60-90% of amylopectin. This is a branched component in which linear chains of α-1,4-glucose residues are interlinked by α-1,6-glucosidic linkages. The debranching enzyme (pullulanase) is necessary to increase the efficiency of the hydrolysis of starch by cutting the α-1,6 glucosidic linkages, and can be widely used in starch processing, bio-based chemicals, and fuel fields. Currently, pullulanase in China is mainly dependent on imports, which is not conducive to the development of related industries. In our previous study work a modified pullulanase was obtained by using the error-prone PCR and sequence deletion. On the basis of our previous study, in this research project the 3D homology structure of the modified pullulanase will be built and analyzed, and the key sites of the functional residues will be selected based on the pKa values and B-factors, which have important influence to the enzyme bioactivity. The enzyme will be engineered through iterative saturation mutagenesis at these key residue sites. Then the engineered enzyme will be planted into the Bacillus subtilis, and to be multiplied efficiently. The structures of the wild enzyme and the mutants will be compared carefully, to explore the structural factors that affect the enzymatic . Then the modified pullulanase will be transformed into Bacillus subtilis for secretory expression. These general conclusions will provide fundamental information for the rational protein design of pullulanase.

淀粉原料含有60-90%支链淀粉，普鲁兰酶能高效水解支链淀粉的α-1,6-糖苷键，加速糖化过程，提高淀粉的利用率和水解效率，在淀粉加工、生物能源与生物基化学品领域有着重要的用途。目前我国所用的普鲁兰酶主要依赖进口，不利于相关产业的发展。我们前期利用常规易错PCR和序列切除技术获得了长野芽孢杆菌普鲁兰酶突变体，本项目在此基础上通过同源建模对其蛋白质三维结构进行分析，选取对酶学性质具有重要影响的关键位点，并利用迭代饱和突变技术对关键位点进行改造，获得适用于工业生产的突变体酶，最后将其导入枯草芽孢杆菌宿主中进行高效分泌表达。通过对野生酶与系列突变体酶的结构与功能比对研究，分析关键氨基酸残基的改变对普鲁兰酶性质产生的影响，为普鲁兰酶的理性设计和改造研究提供有意义的指导。

本项目建立了自动诱导和两相筛选相结合的高通量筛选方法，采用易错PCR对普鲁兰酶进行改造，通过构建和筛选突变库文库，获得了具有较好的耐酸和耐热性的突变体PulA12。首次提出了两种全新的方法来指导酶分子理性改造，包括活性氢键网络方法、蛋白质模拟热探测法。这些理论方法的进展具有很高的创新性和学术价值。利用上述两种方法指导，理性设计构建了突变体N680D，与野生型长野普鲁兰酶相比，其pH适应性和耐酸性有所提高；利用迭代饱和突变获得了10个酶学性质改良的单点或多点突变体，其中两个突变体同时在温度适应性、比酶活、热稳定性三个特征上均显著优于野生型普鲁兰酶，符合工业淀粉糖化要求。将长野普鲁兰酶突变体插入穿梭载体并导入蛋白酶缺陷的枯草芽孢杆菌宿主中，进一步筛选廉价的碳源和氮源作为发酵的主要原料，通过系统优化发酵培养基和培养条件，降低了生产成本，提高了转化率和产率，发酵80 h普鲁兰酶的酶活达到414.48 U/mL。综上所述，本项目的研究成果具有较高的理论和应用价值。