淀粉脱支酶的基因挖掘、功能优化及分泌表达新策略

Starch debranching enzymes can hydrolyze the?alfa-1,6-glucosidic bonds at the branch sites of starch, and can be used to improve raw material utilization rate and production efficiency in starch processing industry. In previous work, we found that the functional diversity of debranching enzymes is required in different starch processes. It was also found that recombinant pullulanases may form active aggregates during the cultivation process, which has not been reported previously. Additionally, we developed a convenient process for the isoamylase to be expressed extracellularly through secretion to the culture medium, even though the enzyme is naturally intracellular. Based on these results, the current project aims to first identify the encoding genes of more starch debranching enzymes by extensively analyzing the small amount of natural enzyme proteins using modern Omics technologies and bioinformatics database mining; and subsequently, to establish the relationship of sequence, structure and function among these enzymes through crystal structural characterization and phylogenetic analysis of the enzyme families. These studies will allow us to understand the enzymes at the most fundamental level and then to reconstruct enzymes with improved functions by the combination of rational design and directed evolution. Furthermore, the project intends to reveal the mechanism for the formation of active pullulanase aggregates, establish the technology for cutinase-mediated secretory expression of isoamylase, and develop novel approaches for high-efficiency extracellular expression of starch debranching enzymes. This project will enable us to thoroughly understand the starch debranching enzyme families, significantly improve their industrial applicability, and ultimately promote further development in the industry of starch sugars.

淀粉脱支酶能水解淀粉分支部位的α-1,6-糖苷键，应用于淀粉糖工业可有效提高淀粉原料利用率和生产效率。前期工作中，我们发现不同的淀粉加工工艺对脱支酶性能需求具有多样性；于世界范围内首次发现普鲁兰酶分泌表达中会形成活性蛋白聚集体现象；探索了将天然定位于细胞浆的异淀粉酶胞外"分泌"技术。在此基础上，本项目结合基于天然微量蛋白的酶基因破译和基于生物信息资源库的酶基因直接挖掘等方法进一步鉴定淀粉脱支酶基因；通过酶家族进化谱及亚家族晶体结构分析比对，阐明脱支酶家族序列-空间结构-功能的关系，结合理性设计和非理性定向进化改造酶功能。此外，通过阐释普鲁兰酶活性蛋白聚集体形成机制，完善角质酶介导的异淀粉酶胞外"分泌"技术，研究脱支酶高效胞外分泌新策略。本项目的研究不仅对深入理解淀粉脱支酶酶学机制，显著提高其工业应用性能具有重要意义，而且对最终推动淀粉糖工业的发展亦具有积极作用。

克隆表达了Bacillus deramificans普鲁兰酶，重组酶最适pH为4.5，最适温度为55 ℃，当与糖化酶协同作用制备葡萄糖，反应52 h，DX值达到92.7%，比对照提高了1.3%；克隆表达了Thermoifida fusca异淀粉酶，重组酶最适 pH为5.5，最适温度为50 ℃，建立了异淀粉酶与α-CGTase双酶复配制备环糊精的同步转化工艺，环糊精总转化率为84.6%，比单酶法提高了24.3%，反应周期是文献报道的1/7-1/5。. 构建了普鲁兰酶双突变体D437H/D503Y，最适温度比天然酶提高了5 ℃，半衰期提高了6倍，结构分析表明，通过引入氢键和大的氨基酸侧链，减少了水分子可及性，提高了酶热稳定性，将突变酶用于糖化反应DX值提高到95.0%，时间缩短12 h；构建了普鲁兰酶N端结构域切除的突变体D437H/D503Y/d1和D437H/D503Y/d2，胞外分泌效率分别是对照的5.3倍和5.6倍，半衰期和催化效率均提高；构建了γ-CGTase突变体(A223K)，建立了γ-环糊精的同步转化工艺，γ-环糊精转化率达到72.5%，提高了21.5%；优化了重组普鲁兰酶制备麦芽糖基-β-环糊精的反应条件，转化率可达48.5%。. 以重组大肠杆菌发酵产普鲁兰酶时，发现了普鲁兰酶极易形成活性蛋白聚集体，当在低温、低IPTG浓度诱导和添加甜菜碱时，可溶性酶活是对照的4.3倍；进而通过3L罐发酵条件优化，胞外酶活达到1567.9 U/mL，为目前资料报道最高；以重组短小芽孢杆菌发酵产普鲁兰酶时，添加镁离子时普鲁兰酶比活比对照提高了2.9倍，二级结构也发生了显著变化，该现象与P2启动子受到阻遏，酶合成速度减慢，更利于正确折叠有关；进而通过3 L罐发酵条件优化，胞外酶活可达1005.8 U/mL，具有高分泌效率、高单位细胞生产强度、耗氧较低等优势。.通过本项目的研究，对淀粉脱支酶酶学机制有了更深入理解，并且获得了工业应用性能优良的酶，对推动淀粉糖工业发展具有积极作用。