源于Streptomyces sp. L10904的G/11家族木聚糖酶诱导特异产物生成分子机制研究

A large number of hemicellulosic agricultural waste rich in xylan has been produced in China each year and its recycling rate is very low. It is the effective value-added way to hydrolyze hemicelluloses waste using enzyme, which has the great economic and environmental significance. The yield of functional components in xylooligosaccharides are very low and latter purification process are complex, high cost, difficult to achieve large scale production so far. To clarify the molecular mechanism of how specific products in different degree of polymerization formed by enzymatic hydrolysis, especially the binding mode and way of enzyme-substrate, is the key to solve this problem. In this study, the molecular mechanism of the key substrate binding site in enzyme molecule take as the breakthrough point for analyze the molecular mechanism of specific products. Using the protein homology modeling and molecular docking method to preliminary determinate the binding site. Furthermore different binding modes, binding sites and tendency of side chain binding are analyzed to elucidate the molecular mechanism of specific produces. Basis on this, the key binding sites are reconstructed with mutation to verify the molecular mechanism of specific produces. The scientific problem for molecular mechanism of specific products generated has been studied to improve basic molecular mechanism of xylanase catalysis, which increase the effective use of agricultural waste, reduce environmental pollution and lay a theoretical foundation for realizing the industrialized preparation of the high purity xylooligosaccharides.

我国每年产生大量富含木聚糖的半纤维素类农业废弃物，其资源再生利用率极低。利用酶法制取高附加值低聚木糖是其增值利用有效途径，经济效益和环保意义巨大。目前，酶法生产低聚木糖的功能性成分产率低，后期提纯复杂，成本高，难以实现大规模生产。解决这一难题的关键是阐明酶水解底物如何趋向生成不同聚合度特异性产物的分子机制，尤其需明确酶与底物的结合方式及途径。研究以酶分子关键特定结合位点为突破口来解析木聚糖酶水解底物生成特异性产物的分子机制。采用酶蛋白同源建模、分子对接等手段初步锁定与产物生成相关的底物结合位点，进一步解析不同结合方式、结合位点数量及侧链结合趋向性导致的酶选择催化生成不同特异性产物的分子机制。在此基础上对关键结合位点进行突变重构，对产物生成分子机制验证。探讨酶催化特异性产物生成分子机制这一科学问题完善了酶分子催化理论，为有效利用农业废弃物，减轻环境污染，实现工业化制备高纯低聚木糖奠定理论基。

利用酶法处理我国每年大量产生的农业废弃半纤维素类物质来制备高附加值低聚木糖是农业废弃物资源再生和增值利用的有效途径，其经济效益和环保意义巨大。而目前酶法生产低聚木糖存在酶解处理后产物功能性成分得率低，后期提纯操作复杂，成本高，难以大规模生产等系列问题。项目研究针对目前酶法处理存在功能性成分得率低这一主要问题，以提高酶对底物水解的特异性产物生成效率为研究重点，通过研究木聚糖酶分子与底物酶结合时的结合位点为突破口来解析酶水解底物高效生成特异性产物的分子机制。.研究采用酶蛋白同源建模、酶与底物分子对接等手段锚定酶分子上与功能产物生成相关的底物结合位点。并采用定点突变法对锚定的关键位点的氨基酸残基进行了替换，同时在原核系统中对突变酶进行了重组表达。研究选择木糖酶10（GH10-xyn）及11（GH11-xyn）家族进行改造，并去除xyn-GH10的结构域以消除其对水解的影响。研究发现木聚糖酶催化模型中±2结合位点对酶的水解特性有密切联系，对其的分子改造成功消除了水解产物中的木糖，显著提高了水解产物中功能低聚木糖的含量，同时酶活较改造前提高了25.1%。研究在此基础上对关键结合位点进行突变重构，对产物生成分子机制验证。研究的相关结果完善了酶分子特异性催化理论的分子机制，获得的改造酶具有应用于功能低聚木糖实际生产的潜力。研究为有效利用我国大量农业废弃物资源，减轻环境污染，实现工业化制备高纯低聚木糖奠定理论及技术应用基础。