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

了解和开发用于糖生物学工业的口服唾液酸酶。研究设计和方法1- PG0352和NanH的结构理解（1-2年级）考虑到PG0352和NanH唾液酸酶的新活性和CBM结构域，需要了解它们的结构信息，以了解它们的作用模式和指导工程。-使用GS和JR建立的方案，我们将以载子和配体结合形式（例如3/6- s -乳糖，SLeA/X, S-Tn， DANA）纯化和结晶完整的NanH和PG0352及其分离的CBM结构域（其中唾液偶联物不会被切割）。我们已经有了纳米CBM （GS， JR）的结晶条件，这表明其他结构具有良好的潜力。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（trip突变）来促进配体结合。与此同时，我们将根据pdb命中信息对NanH和PG0352进行深入的生物信息学和建模研究，以预测可能提高特异性的残基。人类口腔微生物组数据库资源的初步筛选揭示了大量具有新潜力的唾液酸酶。-我们将挖掘该资源和一系列口服元基因组数据集，用于新型唾液酸酶，我们将生产密码子优化（新型IP）克隆，我们将使用我们建立的糖偶联物（GS）和糖蛋白（DS）去氮化分析，使用标准生化方法（硫代巴比妥酸测定（GS））或衍生的HPLC或MS方法（DS-将在Ludger进行）纯化和评估活性。这些也将进入结晶试验，以增加成功的结构解决方案的机会。基于建模、结构信息(1)以及催化精氨酸三联体和特征trip结构域和aspbox的保守性，我们将针对活性位点周围的残基（例如T. cruzi酶的Trp212和Tyr119）和CBM接触配体的配体结合残基。通过我们分离检测CBM结构域的新方法，以及使用仍应结合配体的失活突变体，我们能够实现这一目标的可能性增加了。

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.