堆囊菌木聚糖酶Xyn11B特征序列对其膜定位和酶学性质的影响

Microbial genome sequences directly reveal inherent genetic code. They show us lots of information and features that have long been there in the microbial world, but unrevealed at present. Based on genome sequence analysis of cellulolytic bacteria, the topic of three microbial strategies for plant cell wall degradation was suggested, facilitate the understanding of the potential diversity of strategy used by microorganism in cellulose and hemicellulose degradation. Sorangia in order Myxococcales enable to grow on crystalline cellulose filter paper as the sole carbon source, and this characteristic is unique among cellulolytic bacteria. Analysis of genome sequence of Sorangia revealed some characteristic sequences, such as lipoprotein signal peptide and polyserine linkers (PSL), among its cellulases or hemicellulases. These characteristic sequences are relatively rare among polysaccharide degradating bacteria and overlooked before. They are emergence during the genome-wide analysis and research. What is important is that these characteristic sequences, to some extent, are universal among the enzymes involved in insoluble polysaccharide degradating. Interesting and fortunately, one of the sorangia xylanases, named as Xyn11B, encode a GH11 catalytic domain and the characteristic sequences, both lipoprotein signal peptide and polyserine linkers. The low molecular weight and detectable activity to xylane makes Xyn11B an excellent model to investigate the effect of characteristic sequences on its biochemical properties. This project aims to focus on three aspects according to the 2 characteristic sequences and a GH11 catalytic domain of Xyn11B. Firstly, we intend to determine the effect of sorting property of lipoprotein signal peptide on subcellular location of Xyn11B at two levels, both in situ and component separation, with the help of labeling antibody. Secondly, we intend to reveal the function of polyserine linker (PSL) in Xyn11B. The different mutants of PSL will be constructed and the effect of mutation on the biochemical properties of Xyn11B will be probed. The last one is associated with catalytic domain (CD). Different mutants of CD will be constructed and the effect of mutation on the biochemical properties of Xyn11B will be investigated in-depth. The crystal structure will be further analyzed for significant mutation to reveal the relationship of sequence information, enzymic properties and the structure. In conclusion, all the information will help to demonstrate the function of the characteristic sequences.

微生物基因组序列直接揭示固有遗传密码，由此展示出很多微生物世界早已存在，目前尚缺乏认知的信息和特征，产生了如微生物降解纤维素"第三种策略"等新内容，促进了对降解策略多样性的认识。粘细菌目中纤维堆囊菌具有直接生长于结晶纤维素滤纸上的能力，基因组分析中发现其（半）纤维素酶中一些鲜见的特征序列，如脂蛋白信号肽和聚丝氨酸区。它们在微生物多糖降解酶中的存在具有普遍性。Xyn11B同时具有脂蛋白信号肽、聚丝氨酸区和木聚糖酶催化域，是研究这些特征序列功能的良好模型。本研究拟利用免疫标记法，通过细胞组分分离和细胞原位定位，揭示脂蛋白信号肽特征与膜定位的关系；构建和研究聚丝氨酸区突变体对Xyn11B生化性质的影响，明确聚丝氨酸区的功能特征；构建和研究催化域突变体对Xyn11B酶学性质的影响，并通过蛋白晶体结构解析，建立序列、性质和结构之间的关系。由此全面解析Xyn11B中特征序列对膜定位和酶学性质的影响。

微生物基因组分析显示微生物多糖降解酶中独特序列是降解（半）纤维素策略多样性的基础。本研究以纤维堆囊菌木聚糖酶Xyn11B中鲜有研究但又具有存在普遍性的特征序列如脂蛋白信号肽和聚丝氨酸区为切入点，解析特征序列对酶组分的定位和酶学性质影响，促进对降解策略多样性的认识。.实验证明，纤维堆囊菌Xyn11B脂蛋白信号肽可以在大肠杆菌和黄色粘球菌表达系统中被识别和发挥作用。细胞组分分离后的酶活性和Western Blot检测，以及细胞超薄切片的免疫金标记实验证明：不同于大肠杆菌中的脂蛋白信号肽的作用是将目的蛋白分拣定位于细胞内外膜上，Xyn11B中脂蛋白信号肽不仅通过信号分拣引导Xyn11B定位于内外膜，还进一步以一定的方式将Xyn11B “输送”到细胞外。膜上的与胞外的蛋白之间转变调控的机制是一个有意义的科学问题。.异源表达中发现Xyn11B可规律地降解为20、25、27 kDa三个片段。Xyn11B聚丝氨酸区是其不稳定的原因，其脂蛋白信号肽的存在对维持Xyn11B的完整性具有一定作用，融合表达方式可保持Xyn11B的稳定性。未检测到大肠杆菌和酵母菌中表达的Xyn11B存在磷酸化，但酵母表达Xyn11B中具有N-糖基化和可能的O-糖基化，并能够抵抗蛋白酶的降解。聚丝氨酸（PSL）区域截短突变体研究证明PSL区域抗击蛋白酶的稳定和非稳定的分界点为S55-S56之间，PSL区域长短影响着对蛋白酶的易感性，不影响酶产物谱。截短体研究中发现111-131位区域影响蛋白的热稳定性。.底物标记法证明Xyn11B催化域从寡聚糖的非还原端释放木二糖，蛋白晶体解析证明Xyn11B的loop区L1和L2是只产生木二糖的原因。L1删除突变体完全失去活性，L2删除突变体主产物为木二糖和木三糖，证明L2影响产物谱。对111-131区域芳香族氨基酸替换突变结果证明123Y、126 Y 和130Y三个位点，以及区域外的346W与酶的热稳定性高度相关。嫁接Y123-T131区域序列至Xyn11C能够提高其最适反应温度。这些突变体展现的有意义的特性，其机制既具有理论意义又具有指导蛋白改造的应用潜力。