LPMO-inspired artificial metalloenzymes for selective oxidation

生物催化正在迅速成为一个强大的和广泛的工具，用于一系列化学工业-从复杂和高度功能化的药物合成到生物燃料的批量生产。生物催化的普及源于设计和制造定制催化剂的能力不断提高，这些催化剂能够进行一系列具有现有化学方法无法比拟的反应性和选择性的理想转化。其中一种转化是有机分子的选择性氧化-用于生产数百万吨醇的反应，羰基和环氧化物前体每年都在化学工业的所有领域，但仍然远远没有优化。作为日益增长的环境问题的结果，对催化方法的需求从未如此之大，生物催化可能是一个最佳的解决方案。化学方法不断发展，但仍然需要苛刻的条件、高温，并且经常存在杂质过度氧化的问题，而酶可以在环境条件下在水中进行相同的反应，并且对所需化合物具有完全的选择性。裂解多糖单加氧酶（LPMO）是一类基于Cu的金属酶，于2010-2011年发现，其能够进行多糖中发现的C-H键的选择性氧化。其独特的活性位点由两个组氨酸残基配位的铜离子形成，其中一个是蛋白质的氨基末端，通常被称为“组氨酸支架”。这些酶不仅彻底改变了生物燃料生产领域，还改变了自然界中铜催化氧化的概念。铜的活性位点位于蛋白质的溶剂暴露表面，这对于多糖活性是完美的，但阻止这些酶有效地选择性氧化小分子。本项目的目标是生产和纯化LPMO启发的人工金属酶，并评估它们对小分子底物的氧化能力。在添加Cu离子之前或之后，将合成能够模拟His支架排列的配体，表征并偶联至工程化的球状蛋白。如此形成的人工金属蛋白将通过各种技术来表征，例如X射线晶体学，EPR光谱学和质谱学。酶活性和氧化能力将通过对一系列可能的底物进行测定来评价。进一步的工程和表征周期将被执行，以调整金属酶的性质和底物范围。成功的候选人将获得蛋白质生产，纯化和工程，光谱学方面的技能（例如EPR，NMR），结构生物学和生化分析。该项目的候选人将具有化学，生物化学或自然科学学位，对生物无机/生物化学有浓厚的兴趣，将从事多学科项目，获得各种化学和生物化学技术的经验。

Biocatalysis is rapidly emerging as a powerful and widespread tool for a range of chemical industries - from the synthesis of complex and highly functionalised pharmaceuticals to the bulk production of biofuels. The popularity of biocatalysis stems from an ever-increasing ability to design and manufacture bespoke catalysts which are able to carry out a range of desirable transformations with reactivity and selectivity which are unrivalled by existing chemical methods.One such transformation is the selective oxidation of organic molecules - a reaction which is used to produce millions of tons of alcohols, carbonyl- and epoxide precursors each year in all areas of chemical industry, but is still far from optimised. As a consequence of growing environmental concerns, the need for a catalytic methodology has never been greater and biocatalysis may present an optimal solution. Chemical methods are evolving but still require harsh conditions, high temperatures and often present the problem of over-oxidised impurities, whereas enzymes can perform the same reactions in water, under ambient conditions and with complete selectivity to the desired compound.Lytic polysaccharide monooxygenases (LPMOs) are a class of Cu-based metalloenzymes, discovered in 2010-2011, which are able to perform the selective oxidation of C-H bond found in polysaccharides. Their unique active site is formed by a copper ion coordinated by two histidine residues, one of which being the amino-terminus of the protein, in an arrangement commonly referred to as the "Histidine brace". These enzymes have not only revolutionised the field of biofuel production, but also transformed the notion of how Cu-catalysed oxidations occur in Nature. The copper active site is found on a very solvent exposed surface of the protein, which is perfect for polysaccharide activity, but prevents these enzymes to be effective in the selective oxidation of small molecules.The objectives of this project will be to produce and characterise LPMO-inspired artificial metalloenzymes, and to evaluate their oxidative abilities towards small molecule substrates. Ligands able to mimic the His brace arrangement will be synthesised, characterised and coupled to an engineered globular protein, before or after addition of Cu ions. The so-formed artificial metalloprotein will be characterised by a variety of techniques, such as X-ray crystallography, EPR spectroscopy and mass spectrometry. The enzymatic activity and oxidative abilities will be evaluated by assays on an array of possible substrates. Further cycles of engineering and characterisation will then be performed to tune the properties and the substrate scope of the metalloenzyme.The successful candidate will gain skills in protein production, purification and engineering, spectroscopy (e.g. EPR, NMR), structural biology and biochemical assays.Candidates for this project will have a Chemistry, Biochemistry or Natural Sciences degree with a strong interest in bioinorganic/biological chemistry and will be working on a multidisciplinary project, gaining experience in a wide variety of Chemistry and Biochemistry techniques.