N-糖基化提高第11家族木聚糖酶催化效率的分子机理研究

N-glycosylation is one of the most common and yet complex forms of post-translational modification, and affects enzyme secretion, activity, thermostability, binding affinity, substrate specificity, etc. In our previous study, an endo-1,4-β-xylanase (Xyn11D2) of glycosyl hydrolase family 11 from Penicillium sp. D2 was successfully expressed in Pichia pastoris and simultaneously secreted glycosylated and non-glycosylated proteins. The proteins had similar enzymatic properties, including optimal pH and temperature, pH stability, and thermostability, but had distinct variance in catalytic efficiency and specific activity. The protein with an N-glycosylation at N32 showed significantly increased catalytic efficiency and specific activity. The objective of this study is to undermine the molecular mechanisms of N-glycosylation that improve xylanase catalytic efficiency. The N-glycosylated mutants, which have different N-Glycosylation sites on the protein surface, will be constructed based on sequence analysis, rational design, and site-directed mutagenesis. Bioinformatical and experimental approaches will be employed, determination of enzymatic characterization and the parameters of secondary structure, substrate binding and thermostability, analysis of the hydrolysis products and cleavage modes of xylooligosaccharides, and construction of a enzyme-substrate binding model by computer simulation. To clarify the molecular mechanisms of N-glycosylation of the regulation of the structure and function of the family 11 xylanase, and thereby for later use of the N-glycosylation on xylanase molecules modified to provide a theoretical basis.

N-糖基化影响酶的一系列功能，例如分泌能力、酶活力、热稳定性、亲和力和底物特异性等。我们发现来源于青霉Penicillium sp. D2的第11家族木聚糖酶Xyn11D2的N32位点上的N-糖基化明显提高酶的催化效率和比活力。本项目结合酶学和生物信息学，通过序列比对、理性设计和定点突变构建位于蛋白表面上的不同位点的N-糖基化突变体，测定不同突变体的酶促动力学参数和比活力,筛选出提高酶的催化效率的突变体。通过分析各突变体的二级结构，与底物的结合参数，稳定性相关的热力学参数，产物分析和木寡糖的切割方式，并利用计算机模拟构建酶与底物的结合模型，分析N-糖基化修饰后酶与底物相互作用的变化。解析N-糖基化提高酶的催化效率的分子动力学机理，阐明N-糖基化调控第11家族木聚糖酶结构和功能的分子机制，从而为以后利用N-糖基化对木聚糖酶进行分子改良提供理论基础。

在自然界中，微生物分泌多种糖苷水解酶用于降解植物多糖。这些酶通常发生N-和O-糖基化修饰。糖基化是翻译后修饰最常见也是最复杂的形式之一，影响酶的分泌，活性，热稳定性，底物特异性，催化效率等，但这些作用机制尚不完全清楚。在本研究取得主要结论如下：1）来自Penicillium sp.D2的GH11的内切-1,4-β-木聚糖酶Xyn11D2在毕赤酵母中成功表达，同时分泌糖基化和非糖基化两种类型的蛋白。相比非糖基化酶，N32位发生N-糖基化酶的催化效率和比活性均提高近1.8倍。分子模拟结构分析发现N32位的N-糖基化的作用可能参与在亚位点+2或+3处的酶与底物相互作用。因此，N32的N-糖基化可以提高木六糖和木五糖的催化效率，而不能提高木四糖。 2）在毕赤酵母中异源表达具有三个潜在N-糖基化位点（N87，N124和N335）的烟曲霉GH10的木聚糖酶（Af-XYNA）。 N-糖基化的Af-XYNA（WT）表现出良好的温度和pH最适度以及良好的热稳定性。为揭示N-糖基化对Af-XYNA的作用，该酶通过EndoH去糖基化或在N124处通过定点突变去糖基化修饰。去糖基化酶和突变体N124T显示处相同的较窄的pH适应范围，较低的比活性以及较差的pH和热稳定性。说明N124位点糖基化是影响酶的性质的最主要的糖基化位点。 3）来自Talaromyces leycettanus的GH3的β-葡糖苷酶Bgl3A在毕赤酵母中高效表达。纯化的酶具有优异的酶学性质，催化性能较高。然而，Bgl3A在pH 4.0-5.0下的稳定性较差，限制了其下游的工业应用。进一步的定点突变验证了过度的O-糖基化在pH稳定性上作用。通过突变消除多个潜在的O-糖基化位点，两个突变体在更宽的pH范围下稳定，因此，消除过量O糖基化可以改善此酶的pH稳定性。 4）定点诱变构建三个N-糖基化去除突变体T44A，S228A和S299A。与野生型Bgl3A相比，Bgl3A不同位点的N-糖基化修饰对酶的分泌和酶学性质的影响具有明显差异。其中，N226位的N-糖基化在维持酶的表达和功能方面至关重要，而去除N297位点的N-糖基化可以提高对纤维二糖的催化效率。总的来说，这项研究揭示了N-和O-糖基化的关键作用，可以广泛适用于其他植物细胞壁降解酶。