人工构建几丁质酶复合体及其高效降解几丁质的调控研究

Enzymatic hydrolysis of chitin is a hot spot due to its green process, mild conditions, and strong specificity. However, the existing chitinase components cannot reduce crystallinity of chitin effectively. Meanwhile, diacetyl chitobiose is easy produced in the hydrolysis process，which will severely inhibits the activity of exo-chitinase. Thus, the enzymatic hydrolysis of chitin is inefficient, which restricts its large-scale application. In this project, the artificially constructed chitinase complex was used to efficiently degrade chitin for N-acetylglucosamine (GlcNAc) production. Firstly, polysaccharide cleavage monooxygenase is introduced in existing chitinase components. The action mode of oxidative cleavage of chitin glycosidic bonds was analyzed. Through regulating its synergistic effect with endo-chitinase, the crystallinity of chitin was greatly decreased. Meanwhile, polysaccharide cleavage monooxygenase, Endo-chitinase, Exo-chitinase and N-acetylglucosaminidase were assembled into a multi-enzyme complex using the protein scaffold extracellular. Efficacy effect of multi-enzymes is gathered in space, which can reduce accumulation of intermediate metabolites such as diacetyl chitobiose. Improve the catalytic efficiency of multi-enzyme complex by regulating protein scaffold structure and optimization of catalytic reaction conditions. Finally, efficient conversion of chitin to GlcNAc was reached. The research will provide reference for the efficient assembly of artificially multi-enzymes and provide new means for chitin efficient utilization.

酶法降解几丁质因过程绿色、条件温和及专一性强等优点，一直是几丁质资源化利用的热点。然而，现有几丁质酶系对几丁质结晶度破坏有限，且几丁质酶解过程中易产生几丁二糖，严重抑制外切几丁质酶活性，造成酶法降解几丁质效率低下，制约了其规模化应用。本项目以人工构建几丁质酶复合体高效降解几丁质合成N-乙酰氨基葡萄糖（GlcNAc）为目标，通过在现有几丁质酶系中引入多糖裂解单加氧酶，解析其氧化断裂几丁质糖苷键的作用方式，调控其与内切几丁质酶的协同作用，大幅降低几丁质的结晶度；以蛋白支架介导多糖裂解单加氧酶、内切几丁质酶、外切几丁质酶及N-乙酰氨基葡萄糖苷酶胞外自组装为多酶复合体，在空间上聚集多酶的协同效应，降低几丁二糖等中间代谢物积累；通过调节蛋白支架结构和催化反应条件变换，提高多酶复合体的催化效率，实现几丁质向GlcNAc的高效转化。研究成果将为人工组装多酶提供借鉴，同时为几丁质资源的高效利用提供新手段。

几丁质作为自然界中第二大可再生资源，可降解为高附加的N-乙酰氨基葡萄糖产品，相比传统的酸水解法，酶法降解因过程绿色、条件温和及专一性强等优点，一直是研究的热点。然而，现有酶法降解难以降解结晶型几丁质，且需要多种酶的协同作用，造成酶法降解几丁质整体效率低下，制约了其规模化应用。本项目通过蛋白支架介导多糖裂解单加氧酶、内切几丁质酶、外切几丁质酶及N-乙酰氨基葡萄糖苷酶胞外自组装，构建多酶复合体，以期实现结晶型几丁质向N-乙酰氨基葡萄糖的高效转化。首先通过异源表达，水解活性和模式研究，筛选得到了高活性的多糖裂解单加氧酶，其可通过打断几丁质分子表面结构，高效降低其结晶度。同时，结合全基因组和肽指纹图谱分析，从高效几丁质降解菌中获得6个几丁质降解关键酶，经过异源表达和活性测定筛选获得了内切几丁质酶、外切几丁质酶及N-乙酰氨基葡萄糖苷酶；把获得的四种酶与锚定蛋白进行融合表达，同时将带有能与锚定蛋白结合的粘连蛋白基因融合表达得到支架蛋白，接着将纯化得到的支架蛋白及含有对应锚定蛋白的几丁质酶进行复配、自组装为多酶组装体。非变性聚丙烯酰胺凝胶电泳以及凝胶排阻色谱证明了多酶组装体的成功形成。将几丁质酶复合体应用于结晶型几丁质底物的降解，在最优的组装体系下，多酶组装体对于结晶型几丁质的降解效率，相较于游离几丁质酶提升4.6倍，表明通过组装成功提高了酶之间的协同作用，实现了结晶型几丁质向N-乙酰氨基葡萄糖的高效制备。此外，通过酶吸附分离与水解过程的耦合，多酶组装体可多次重复使用，制备出来的N-乙酰氨基葡萄糖产品纯度达98%，表现出较好的应用前景。本项目的研究成果为几丁质资源的炼制提供了新的理论基础，为也为其它多酶协同体系的组装提供了思路与借鉴。项目共发表SCI论文12篇，授权中国发明专利2项，申请中国发明专利4项，参加国内外会议15人次，引进博士后1名，培养博士研究生生1名，硕士研究生4名。

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