超高温淀粉加工酶在枯草芽孢杆菌中高效制备的分子基础

Hyperthermophilic starch processing enzymes show extremely temperature stability and have important application value in starch processing. At present, the recombinant expression levels of hyperthermophilic starch processing enzymes are generally low, and that may be caused by the low protein folding levels of hyperthermophilic starch processing enzymes in host strains, which in turn affect the translocation and extracellular expression. Bacillus subtilis is an important host strain for the expression of food enzymes, and owns potential and advantage for high-level expressions of hyperthermophilic starch processing enzymes. In previous work, we constructed a B. subtilis expression system with independent intellectual property rights, and studied the effects of promoter, chaperone protein, secretory element, signal peptide and cell wall negative charge on the expressions of heterologous proteins. This project was designed to explore the key factors that affect the intracellular folding of hyperthermophilic starch processing enzyme precursors in B. subtilis and optimize the intracellular folding environment of the host strain; enhance the extracellular folding of hyperthermophilic starch processing enzymes in host strain and reduce the level of extracellular degradation; coupling coordinate the balance among protein synthesis, translocation and extracellular folding to achieve the high-level expressions of hyperthermophilic starch processing enzymes. This project will not only provides theoretical foundation and key technology for the high-level expressions of hyperthermophilic starch processing enzymes in B. subtilis, but also has positive significance for promoting high-efficient liquefaction of starch to improve starch utilization.

超高温淀粉加工酶具有极高的耐热性，在淀粉加工过程中具有重要的作用。目前超高温淀粉加工酶的重组表达水平普遍低，造成此现象的根本原因是其在异源表达宿主菌中的蛋白折叠水平差，进而影响了其跨膜分泌和胞外表达。枯草芽孢杆菌作为重要的食品酶制剂表达宿主菌，具有高效表达超高温淀粉加工酶的潜力和优势。前期工作中我们构建了具有自主知识产权的枯草芽孢杆菌表达系统，研究了启动子、伴侣蛋白、分泌元件、信号肽、细胞壁电荷等对异源蛋白表达的影响。在此基础上，本项目拟分析影响超高温淀粉加工酶前体在枯草芽孢杆菌中胞内折叠的制约因素并优化其折叠环境；强化重组酶在宿主菌中的胞外折叠并降低胞外降解水平；偶联协调蛋白合成、跨膜分泌和胞外折叠三者之间的平衡以实现超高温淀粉加工酶的高效制备。本项目的实施不仅为超高温淀粉加工酶在枯草芽孢杆菌中的高效表达提供理论基础和关键技术，而且对于推动淀粉高效液化提升淀粉利用率具有积极意义。

超高温淀粉加工酶具有极高的耐热性，在淀粉加工过程中具有重要的应用价值。枯草芽孢杆菌具有食品安全和蛋白分泌能力强的特点，是重要的工业酶制剂表达宿主菌。为了解决超高温淀粉加工酶重组表达水平低的问题，本项目将不同来源超高温α-淀粉酶和超高温普鲁兰酶在枯草芽孢杆菌中进行了重组表达；通过不同类型宿主菌优化，信号肽筛选提升分泌效率，过表达嗜热古菌伴侣蛋白改善胞内折叠水平，过表达分泌途径蛋白强化宿主分泌能力，挖掘利用新型蛋白表达增强因子，加热处理提高可溶性酶活，发酵优化平衡菌体生长与产酶等组合策略，有效提高超高温α-淀粉酶和超高温普鲁兰酶在枯草芽孢杆菌中的重组表达水平，其中超高温α-淀粉酶胞外酶活可达3806.69 U•mL-1，为目前资料报道的最高水平，同时鉴定获得枯草芽孢杆菌新型重组蛋白表达增强因子PonA；利用CRISPR/Cas9基因编辑系统敲除了枯草芽孢杆菌中的丙氨酸消旋酶基因dal，构建了D-丙氨酸营养缺陷型枯草芽孢杆菌宿主菌，实现超高温α-淀粉酶无抗性标记食品安全高效表达。相关研究成果发表论文8篇（其中SCI论文6篇），授权发明专利2件，申请发明专利3件。培养博士研究生1名，硕士研究生1名。本项目对于超高温淀粉加工酶规模化制备和嗜热古菌来源蛋白重组表达具有积极的推进作用。

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