Mechanistic Enzymology of Bacterial Lignin Degradation

用于制造塑料、药品、家用化学品和工业溶剂的化学品主要来自石油，因此，21世纪最大的科学挑战之一是我们如何为这些重要化学品找到可再生资源。一个显而易见的研究对象是植物木质纤维素，它是植物细胞壁的主要成分，存在于农业废弃物等生物质中。植物木质纤维素的一种成分是木质素：一种天然难以分解的芳香聚合物，因为它是由C-O醚键和C-C键结合在一起的，不会被酸或碱处理分解。我们已经从土壤中分离出能够分解木质素的细菌，我们已经详细研究了第一个细菌降解木质素的酶，一种叫做过氧化物酶的酶，来自约氏红球菌（Rhodococcus jostii）的RHA1，我们开始了解细菌分解木质素的生化途径。我们最近发现了第二种可以分解木质素的酶，一种来自Sphingobacterium sp. T2的锰依赖性超氧化物歧化酶，这是第一次发现这种酶对木质素有活性。我们希望详细研究这种酶的反应机制，以确定到底是什么反应物质负责攻击木质素。我们还从荧光假单胞菌中发现了另一种可以攻击木质素的细菌dyp过氧化物酶，因此我们希望确定它释放的产物的结构，并确定这种反应的机制。我们最近还确定了来自fusca Thermobifida的Dyp过氧化物酶的三维结构；我们希望利用这种结构对这种酶进行“定向进化”，以提高其对木质素的活性。我们认为，除了Dyp过氧化物酶和Mn超氧化物歧化酶等木质素氧化酶外，还需要其他辅助酶和蛋白质来进行细菌的木质素分解。在这个项目中，我们将尝试确定一种酯酶，它可以打破木质素和半纤维素之间的酯键；以及一种脱氢酶，通过淬灭木质素分解过程中产生的自由基中间体来帮助木质素分解。然后，我们将用来自植物生物量或工业来源的木质素样品测试这些辅助酶和木质素降解酶的组合，以测试它们是否有效地在体外分解木质素，以及我们是否可以使用这些酶来生产芳香化学品。

The chemicals that are used to make plastics, drugs, household chemicals & industrial solvents come primarily from oil, so one of the greatest scientific challenges for the 21st century is how we can find renewable sources for these important chemicals. One obvious place to look is plant lignocellulose, the major component of plant cell walls, found in biomass such as agricultural waste. One component of plant lignocellulose is lignin: an aromatic polymer that is naturally hard to break down, since it is held together by C-O ether linkages and C-C bonds that are not broken down by acid or base treatment. We have isolated bacteria from soil that are able to break down lignin, we have studied in detail the first bacterial lignin-degrading enzyme, an enzyme called peroxidase DypB from Rhodococcus jostii RHA1, and we are beginning to understand the biochemical pathways used by bacteria to break down lignin.We have recently identified a second type of enzyme that can break down lignin,a manganese-dependent superoxide dismutase from Sphingobacterium sp. T2, This is the first time that an enzyme of this kind has been shown to have activity towards lignin. We wish to study in detail the reaction mechanism of this enzyme, to determine exactly what is the reactive species that is responsible for attacking lignin. We have also found another bacterial dyp peroxidase from Pseudomonas fluorescens that can attack lignin, and so we wish to determine the structure of the product that it releases, and determine the mechanism of this reaction. We have also recently determined the three-dimensional structure of a Dyp peroxidase enzyme from Thermobifida fusca; we wish to use this structure to carry out "directed evolution" on this enzyme, to improve its activity towards lignin.We believe that, as well as the lignin-oxidising enzymes such as Dyp peroxidases and Mn superoxide dismutase, there must be other accessory enzymes and proteins that are needed for bacterial lignin breakdown. In this project, we will try to identify an esterase enzyme that breaks an ester linkage between lignin and hemi-cellulose; and a dehydrogenase enzyme that assists lignin breakdown by quenching radical intermediates generated during lignin breakdown. We will then test combinations of these accessory enzymes and lignin-degrading enzymes with samples of lignin either from plant biomass, or from industrial sources, to test whether they are effective for lignin breakdown in vitro, and whether we can use these enzymes to produce aromatic chemicals.