Expanding the selectivity and activity of galactose oxidase towards bioactive saccharide

本项目旨在以半乳糖氧化酶亲本酶为基础，构建具有新型和高度特异性底物选择性的糖氧化酶文库。我们将结合基因组数据库的同源性搜索（Prozomix Limited），定向进化（曼彻斯特大学），细菌宿主中的高通量酶表达（Prozomix）和快速高分辨率分析筛选技术（Bio-Shape Ltd和曼彻斯特大学）进行酶发现。生物催化氧化反应因其优越的区域选择性和立体选择性以及低环境影响，在制药和精细化工领域具有取代许多化学催化氧化反应的潜力此外，蛋白质的氧化酶交联是使食品功能化的重要方法这种生物工程方法的特别有吸引力的工具是碳水化合物修饰酶，如氧化酶，它可以高度选择性，如广泛使用的葡萄糖和半乳糖氧化酶所证明的那样。尽管氧化酶的应用范围非常广泛，但就底物特异性而言，目前可用的氧化酶范围非常窄，这突出了对具有更广泛底物范围的碳水化合物修饰酶的需求该项目旨在通过开发一系列对常见聚糖（包括半乳糖苷、甘露糖苷、乳糖、果糖、唾液酸和氨基糖）具有不同底物特异性的氧化酶来解决可用生物催化剂的这一空白。我们将选择能够在细菌表达系统中容易地以克量和更大数量生产的生物催化剂，从而可以在生物技术中找到广泛的应用，包括低聚和多糖（乳糖，淀粉，纤维素），糖脂和糖蛋白的修饰和重塑。本项目旨在结合项目p提供的关键方法，生成一个具有活性、健壮性和更多样化的氧化酶设计库

This project aims to generate a library of sugar oxidases with novel and highly specific substrate selectivity based on parent galactose oxidase enzymes. We will use a combination of homology searches of genomic databases (Prozomix Limited),directed evolution (The University of Manchester), high-throughput enzyme expression in bacterial hosts (Prozomix) and fast high-resolution analytical screening techniques (Bio-Shape Ltd and The University of Manchester) for enzyme discovery. Background Biocatalytic oxidation reactions have the potential to substitute many chemically catalysed oxidations in the pharmaceutical and fine chemical industry due to their superior regio- and stereoselectivity and low environmental impact.[1] Furthermore, oxidative enzymatic cross-linking of proteins is an important method to functionalising foods.[2] Particularly attractive tools for such bioengineering methods are carbohydrate modifying enzymes such as oxidases, which can be highly selective as demonstrated by the widely used glucose and galactose oxidases. Despite their plethora of applications, a surprisingly narrow range of oxidases in terms of substrate specificity is currently available, which highlights the need for carbohydrates-modifying enzymes with broader substrate scopes.[3] This project aims to address this gap in available biocatalysts by developing a range of oxidases with distinct and diverse substrates specificity towards common glycans including galactosides, mannosides, lactose, fructose, sialic acids and amino sugars. We will select for biocatalysts that can be easily produced in gram quantities and larger in bacterial expression systems and thus could find wide applications in biotechnology including modification and remodelling of oligo- and polysaccharides (lactose, starch, cellulose), glycolipids and glycoproteins. Aims This project aims to now generate a designer library of active, robust and more diverse oxidases using a combination of key methods provided by project p