第10家族高比活木聚糖酶XYL10C高效催化机制研究

：The xylanases of GH 10 are commonly more resistant to stress than those from GH 11. However, GH 10 xylanases have much lower catalytic efficiency, which limits their widespread use in industrial applications. The objective of this study is to probe the high catalytic efficiency mechanisms of GH 10 xylanases and to provide theoretical basis for their molecular improvement. Two highly identical GH 10 xylanases are selected. XYL10C with the specific activity of 1,4236U/mg has higher catalytic efficiency (kcat/Km > 11,000 ml/mgos) than all known xylanases.The crystal structure of XYL10C has been solved. XylE has a known crystal strucutre but is much lower in catalytic efficiency. By analysis of the pre-steady state enzyme kinetics, molecualr docking, molecualr dynamics simulation and calculations with CAVER, the key amino acids related with catalytic efficiency will be identified, including the residues that are involved in determination of catalytic rate, the residues composed of the active pockets or in close proximity to it, and the non-active site "bottleneck" residues. The functions of these residues will be verfiried by site-directed mutagenesis. Moreover, a library of hybrid enzymes will be constructed by using SCHEMA and screened on a large-scale to identify the module strucutre related to the high catalytic efficiency of XYL10C. The data will be analyzed in combination, experiments will be conducted for verification, and the key module structure and residues will be identified. As a result, the catalytic mechanism of XYL10C for high efficiency will be revealed.

10家族木聚糖酶较11家族木聚糖酶普遍具有更好的抗逆性，但较低的催化效率是其应用的瓶颈，开展其高效催化机制研究将为木聚糖酶的分子改良提供依据。我们获得了一个催化效率最高(kcat/Km>1.1万ml/mgos,比活>1.4万U/mg)的10家族木聚糖酶XYL10C，并解析了其晶体结构，其与另一个已解析结构、但催化效率只有53 ml/mgos的木聚糖酶 XylE 序列一致性很高。以这两种酶为核心材料，通过非稳态动力学分析、分子对接及动力学模拟、CAVER 计算等方法，确定与催化效率相关的关键氨基酸，包括与二者催化限速步骤相关的残基、活性口袋及附近的关键残基、非活性位点的"瓶颈"残基等，并通过突变研究确认。同时，通过SCHEMA构建杂合文库进行高通量筛选，确定与 XYL10C 高效催化特性相关的结构模块。整合定点突变和模块重组研究结果并开展组合突变研究，揭示第 10家族木聚糖酶的高效催化机制。

热稳定性好同时具有高催化活性的木聚糖酶具有重要的工业应用前景。本项目运用实验研究及计算机辅助设计相结合的方法，以第10家族木聚糖酶XYL10C和4F8X为研究对象，进行模块的拆分和重组，将XYL10C的重点研究模块替换到4F8X所对应的模块上，总共构建了10个突变体，通过酵母表达和蛋白纯化以及酶学性质的测定我们发现，7个能检测到木聚糖酶活力，模块2，模块5以及模块8的替换大大提高了4F8X的催化效率，与野生酶4f8x相比分别提高了2.0倍，3.3倍和1.7倍，从而锁定了在XYL10C的高效催化中起关键作用的三个模块。通过多序列比对、通道分析和分子对接，我们发现模块2中175位的E位点在底物结合产物释放方面发挥很大的作用，通过E175位点饱和突变的方法来研究该位点在XYL10C的各项性质中所起的重要作用，突变体E175H和E175N 的比活性有大幅度提高，比野生酶分别提高了4.1倍和3.7倍。突变体4F8X-Q99E比活较野生型酶4F8X提高8.8倍，催化效率提高了3.7倍，与XYL10C突变结果相符合。因此E175位点（Q99E）与酶的高效催化相关。我们对XYL10C α5螺旋两端loop区N185G、N208G两个位点的突变研究，单点突变后发现，突变体 N185G和N208G催化效率较野生酶XYL10C分别提高了 40%和50%。同时，实验证明loop2上的53/54位点与酶在低温下的酶活力密切相关。项目研究进一步提高了第10家族木聚糖酶XYL10C的催化活性，同时为进一步的指导设计和改良第10家族木聚糖酶及其它糖苷水解酶提供了依据。利用项目的研究方法对TlXyn10A、XylE 和 XynE2木聚糖酶和甘露聚糖酶PMan5A进行了改良研究。