Engineering improved specificity and activity into oral bacterial sialidases for glycan biotechnology applications

改进口腔细菌唾液酸酶的特异性和活性，用于聚糖生物技术应用

• 批准号: 1711801
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1711801
• 关键词: Engineering; improved; specificity; activity; into

To understand and develop orally derived sialidases for the glycobiology industry.Study design and methodology1-Structural understanding of PG0352 and NanH (Yr 1-2)Structural information on PG0352 and NanH sialidases is needed given their novel activity and CBM domains to understand mode of action and direct engineering. -Using protocols established by GS and JR we will purify and crystallise full NanH and PG0352 and their isolated CBM domains (where sialloconjugates will not be cleaved) in apo- and ligand-bound forms (e.g. 3/6- S-lactose, SLeA/X, S-Tn, DANA). We have conditions for crystallization of the NanH CBM (GS, JR) suggesting good potential for other constructs. To increase chances of novel ligand bound information we will produce inactive versions (FRIP mutations) to promote ligand binding.-In parallel we will undertake in-depth bioinformatic and modeling studies of NanH and PG0352 based on pdb-hit information and that gained here to predict residues that might improve specificity.2-Mining of the oral microbiome for novel sialidases (Yr 1-2)Preliminary screening of the Human Oral Microbiome Database resource reveals a plethora of sialidases with novel potential. - We will mine this resource and a range of oral metagenome datasets for novel sialidases that we will produce as codon optimized (novel IP) clones that we will purify and assess for activity using our established glyconjugate (GS) and glycoprotein (DS) desialylation assays using standard biochemical methods (Thiobarbiturate assay (GS)) or derivatised HPLC or MS based methods (DS- to be performed at Ludger). These will also enter crystallisation trials to increase the opportunities for successful structure solution.3- Engineering sialidases for specificity and activity improvements (Yr 2-3)-Based on modeling, structural information (1) and on the conservation of the catalytic Arginine triad and characteristic FRIP domain and Asp-boxes we will target residues surrounding the active site (e.g. Trp212 and Tyr119 of the T. cruzi enzyme) and ligand-binding residues of the CBM contacting ligands. The likelihood that we will be able to achieve this is increased by our novel approach examining the CBM domains in isolation and also in the use of inactive mutants that should still bind ligands.

研究设计和方法学1-了解PG0352和NanH(yr1-2)的结构信息，因为它们具有新的活性和CBM结构域，以了解作用模式和指导工程。-使用GS和JR建立的方案，我们将以脱氧核糖核酸和配体结合的形式(例如3/6-S-乳糖、SLEA/X、S-TN、DANA)纯化和结晶完整的NaH和PG0352及其分离的CBM结构域(其中唾液酸偶联物将不被切割)。我们有条件结晶NanH煤层气(GS，JR)，这表明它有很好的其他结构的潜力。为了增加新的配体结合信息的机会，我们将产生非活性版本(FRIP突变)来促进配体结合。-同时，我们将根据PDB-HIT信息和在这里获得的预测残基来预测可能提高特异性的残基，对NanH和PG0352进行深入的生物信息学和建模研究。2-挖掘口腔微生物组寻找新的唾液酸酶(yr1-2)对人类口腔微生物组数据库资源的初步筛选显示出大量具有新潜力的唾液酸酶。-我们将挖掘这一资源和一系列新型唾液酸酶的口服元基因组数据集，我们将产生密码子优化(新型IP)克隆，我们将使用标准生化方法(硫代巴比妥酸分析(GS))或衍生高效液相或质谱分析法(DS-将在Ludger进行)来纯化和评估我们建立的糖共轭(GS)和糖蛋白(DS)去催化分析方法的活性。这些也将进入结晶试验，以增加成功结构解决方案的机会。3-改进特异性和活性的工程唾液酸酶(yr2-3)-基于建模、结构信息(1)以及催化精氨酸三联体和特征Frip结构域和Asp-box的保守，我们将针对活性部位周围的残基(例如T.ruzi酶的Trp212和Tyr119)和CBM接触配体的配体结合残基。通过我们的新方法分离地检查CBM结构域，以及使用仍然应该结合配体的非活性突变体，我们能够实现这一点的可能性增加了。