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-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。

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.