Novel monooxygenase biocatalysts from the environment and the laboratory

来自环境和实验室的新型单加氧酶生物催化剂

• 批准号: BB/F01449X/1
• 负责人: Thomas Smith
• 金额: $ 37.45万
• 依托单位: Sheffield Hallam University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF01449X%2F1
• 关键词: Novel; monooxygenase; biocatalysts; environment; laboratory

Methane oxidising bacteria are very important in the environment since they are a key link in the global biogeochemical methane cycle and oxidise methane in many environments such as wetlands, paddy field soils and landfills before this methane is released into the environment. They thereby mitigating the effects of this potent greenhouse gas and reduce global warming. Methane monooxygenase (MMO) is a bacterial enzyme that catalyses the first step in methane oxidation by bacteria. It is of great interest to chemists because it oxidises methane to methanol at ambient temperatures and pressures, a reaction normally requiring high temperatures and pressures and expensive catalysts. MMO is very unusual in that it will also oxidise very many other alkanes, alkenes and aromatic compounds and their substituted derivatives and therefore it has great potential for use as a biocatalyst in biotransformations and bioremediation in 'green chemistry' reactions that are less polluting than traditional chemical routes. The structure of MMO has been the subject of considerable interest for biologists because of this broad substrate specificity and one aim has been to try to understand how the structure of the enzyme allows the catalysis of such a wide range of compounds and how changing its structure by mutagenesis, forced evolution or construction of mutant and hybrid enzymes will alter its catalytic utility. This is an ambitious grant proposal from world experts in the molecular biology and biochemistry of MMO. We propose to construct key mutants in the active site of MMO and to examine the effects on catalysis of key substrates and to manipulate this enzyme in order to be able to define the pathway of entry of substrates into the active site and to generate novel recombinant enzymes which are able to oxidise new substrates. We also aim to define how the different components of the enzyme interact with each other and how the mechanisms of substrate entry and electron transfer pathways to the site of oxidation in MMO are carried out. In a novel approach, we also wish to carry out 'gene mining' from the environment to capture DNA sequences that encode MMO or related di-iron centre monooxygenases in order to be able to construct new and exciting biocatalysts. This will involve the use of a technique called DNA-Stable Isotope Probing (DNA-SIP) which we originally developed in order to be able to define the population structure of active methane oxidising bacteria in the environment. This involves feeding 13C-substrates such as methane to bacteria contained within environmental samples such as soils. Only the active methanotrophs with MMO will be labelled with this heavy stable isotope. We can then isolate the heavy DNA (containing the whole genomes of methanotrophs and related bacteria) encoding MMO and its relatives from all of the DNA from the thousands of non-methanotrophic bacteria present in soil by density gradient centrifugation. By use of the polymerase chain reaction (PCR), we can then isolate novel MMO sequences from previously uncultivated bacteria which can subsequently be stitched into plasmids that we have developed which allow us to recreate novel MMOs with unusual biocatalytic properties. Analysis of these recombinant enzymes will shed light on the mechanism of action of MMO and also generate new and novel biocatalysts with potential for use in industry in non-polluting biotransformation reactions.

甲烷氧化细菌是全球甲烷生物地球化学循环的关键环节，在湿地、水田土壤和垃圾填埋场等许多环境中氧化甲烷，使甲烷释放到环境中，因此甲烷氧化细菌在环境中非常重要。因此，它们减轻了这种强效温室气体的影响，并减缓了全球变暖。甲烷单加氧酶（MMO）是一种细菌酶，它催化细菌氧化甲烷的第一步。化学家们对此非常感兴趣，因为它在室温和常压下将甲烷氧化成甲醇，而这个反应通常需要高温高压和昂贵的催化剂。MMO非常不寻常，因为它还会氧化许多其他烷烃、烯烃和芳香族化合物及其取代衍生物，因此它在“绿色化学”反应中作为生物催化剂和生物修复具有很大的潜力，这些反应比传统化学途径污染更少。由于这种广泛的底物特异性，MMO的结构一直是生物学家非常感兴趣的主题，其中一个目标是试图了解酶的结构如何允许催化如此广泛的化合物，以及如何通过诱变，强制进化或突变和杂交酶的构建改变其结构将改变其催化效用。这是一项雄心勃勃的资助提案，来自MMO分子生物学和生物化学领域的世界专家。我们建议在MMO的活性位点构建关键突变体，并检查对关键底物的催化作用的影响，并操纵该酶，以便能够确定底物进入活性位点的途径，并产生能够氧化新底物的新型重组酶。我们还旨在确定酶的不同组分如何相互作用，以及在MMO中底物进入和电子转移途径如何进行氧化位点的机制。在一种新颖的方法中，我们还希望从环境中进行“基因挖掘”，以捕获编码MMO或相关双铁中心单加氧酶的DNA序列，以便能够构建新的和令人兴奋的生物催化剂。这将涉及使用一种称为dna稳定同位素探测（DNA-SIP）的技术，我们最初开发这种技术是为了能够确定环境中活性甲烷氧化细菌的种群结构。这包括将13c基质（如甲烷）喂给环境样本（如土壤）中含有的细菌。只有含有MMO的活性甲烷氧化菌才会被标记为这种重稳定同位素。然后，我们可以通过密度梯度离心从土壤中存在的数千种非甲烷营养细菌的所有DNA中分离出编码MMO及其亲属的重DNA（包含甲烷营养菌和相关细菌的整个基因组）。通过使用聚合酶链反应（PCR），我们可以从以前未培养的细菌中分离出新的MMO序列，然后将其缝合到我们开发的质粒中，从而使我们能够重建具有不同寻常生物催化特性的新型MMO。对这些重组酶的分析将有助于揭示MMO的作用机制，并产生具有应用于无污染生物转化反应的新型生物催化剂。

项目成果

Metal(loid) speciation and transformation by aerobic methanotrophs.

• DOI: 10.1186/s40168-021-01112-y
• 发表时间: 2021-07-06
• 期刊: Microbiome
• 影响因子: 15.5
• 作者: Karthikeyan OP;Smith TJ;Dandare SU;Parwin KS;Singh H;Loh HX;Cunningham MR;Williams PN;Nichol T;Subramanian A;Ramasamy K;Kumaresan D
• 通讯作者: Kumaresan D

Microbial biotechnology meets environmental microbiology.

• DOI: 10.1111/j.1751-7915.2009.00090_11.x
• 发表时间: 2009-03
• 期刊: Microbial biotechnology
• 影响因子: 5.7
• 作者: Murrell JC;Smith TJ
• 通讯作者: Smith TJ