适用于淀粉同步脱支液化过程的超高温普鲁兰酶的功能改造及高效表达分子基础

Extremely thermostable pullulanase can hydrolyze theα-1,6-glucosidic linkages in starch at high temperature, and can be applied to improve the utilization rate of materials and production efficiency in process for liquefying starch. In the previous work, two pullulanases were heterologous expressed and characterized in our laboratory, the thermostability, catalytic activity and secretion efficiency of pullulanases were enhanced by site-directed mutagenesis or terminal domains truncation. Additionally, we constructed recombinant Brevibacillus sp. WSH2010-06 for the expression and extracellular secretion of pullulanases. However, it was found that the existing pullulanases are difficult to satisfy the requirement of process for liquefying starch. Based on these results, the current project aims to analyses the crystal structure and clarify the molecular mechanism of pullulanases from different enzyme families. And subsequently, reconstruct pullulanases with improved functions, such as extreme thermolability and high activity suitable for synchronous debranching and liquefaction, by the combination of rational design and directed evolution. Furthermore, the project intends to develop approaches for high-efficiency extracellular expression of the extremely thermostable pullulanase in the Bacillus host. These studies will allow us to reveal the structure and function relationship between different pullulanase families, significantly enhance their performance in process for synchronous debranching and liquefying starch, and ultimately improve the utilization rate of starch in fermentation industry.

超高温普鲁兰酶能在高温条件下水解淀粉分子中的α-1,6-糖苷键，应用于淀粉液化过程可有效提高原料利用率和生产效率。在前期工作中，申请人重组表达了两个普鲁兰酶，获得了热稳定性、催化效率和胞外分泌效率显著提高的普鲁兰酶突变体，实现了普鲁兰酶在短小芽孢杆菌中的高效分泌表达。然而，我们发现已有的普鲁兰酶难以同时满足超高温和高活性的双重要求。在此基础上，本项目将解析超高温普鲁兰酶晶体结构，阐明不同家族酶耐热性及催化活性差异的分子机制，开展酶功能定向分子改造，获得满足淀粉同步脱支液化过程要求的超高温、高活性普鲁兰酶，优化超高温普鲁兰酶在芽孢杆菌安全宿主中的高效制备策略。本项目的研究不仅对于阐明不同家族普鲁兰酶结构与功能的关系，显著提高超高温普鲁兰在淀粉同步脱支液化过程中的应用性能具有重要意义，而且对于改善目前发酵工业中淀粉原料综合利用率偏低的现状亦具有积极作用。

(1) 基于高温普鲁兰酶具有极易形成活性蛋白聚集体的特点，筛选了不同类型的表面活性剂；发现Triton X-100的添加，不仅能够有效促进该酶可溶性表达，而且还能增强细胞膜透性，提高重组蛋白的分泌效率；优化了Triton X-100浓度、添加时间等条件，重组酶的胞外活力和分泌效率分别提高至812.4 U/mL和86.0%。在此基础上，构建了N端非必须结构域删除的截短突变体。突变体D437H/D503Y/d1和D437H/D503Y/d2胞外酶所占比例分别为57.3%和60.8%，分泌效率分别是对照的5.3倍和5.6倍，比活力分别是天然酶的1.03倍和1.39倍。(2) 对比了不同类型的表达系统，发现重组酶在重组短小芽孢杆菌中表达时重组酶以胞外分泌形式为主，胞内可溶蛋白和包涵体极少，未经优化初步发酵胞外酶活即可达到50.1 U/mL。考察了重组酶酶学性质，发现短小芽孢杆菌重组酶比活力和半衰期均比大肠杆菌重组酶有不同程度的提高，分别约是后者的1.34倍和1.13倍，说明短小芽孢杆菌对高温普鲁兰酶具有更强的折叠分泌能力。发现了Mg2+具有促进短小芽孢杆菌分泌“高比活”高温普鲁兰酶的独特作用，解析了该酶在短小芽孢杆菌中表达的“折叠主控型”机制：镁离子可以防止HWP从细胞表面脱落，导致P2启动子表达强度降低，HWP和重组酶的合成速度均降低，从而有利于重组酶的正确折叠。在此基础上，在3-L罐中建立了重组短小芽孢杆菌高效发酵制备重组酶的生产工艺。 (3) 对比了不同枯草芽孢杆菌宿主菌株中高温普鲁兰酶表达量的差异，筛选到一株较适合重组酶的表达宿主B. subtilis ATCC6051a；针对该菌株容易形成芽孢、发酵泡沫大、蛋白酶本底表达水平较高的缺点，建立了基于Crispr/Cas9的枯草芽孢杆菌基因组编辑工具载体，敲除了非驯化枯草芽孢杆菌菌株的5个基因，获得了适合高温普鲁兰酶重组表达的宿主菌。