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

基本信息

批准号：
BB/F012713/1
负责人：
John Murrell
金额：
$ 42.96万
依托单位：
University of Warwick
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%2FF012713%2F1
关键词：
Novel monooxygenase biocatalysts environment laboratory

项目摘要

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.

甲烷氧化细菌在环境中非常重要，因为它们是全球生物地球化学甲烷周期的关键联系，并且在许多环境中，例如湿地，稻田土壤和垃圾填埋场，在将这种甲烷释放到环境中之前。他们因此减轻了这种有效的温室气体的影响并减少全球变暖。甲烷单加氧酶（MMO）是一种细菌酶，可催化细菌甲烷氧化的第一步。化学家引起了极大的兴趣，因为它在环境温度和压力下将甲烷氧化为甲醇，这通常需要高温和压力以及昂贵的催化剂。 MMO非常不寻常，因为它还将氧化许多其他烷烃，烷烃和芳香族化合物及其取代的衍生物，因此它在“绿色化学”反应中具有巨大的生物转化和生物修复的生物催化剂，比传统的化学途径更少。由于这种广泛的底物特异性，MMO的结构一直是生物学家引起了极大兴趣的主题，并且一个目的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，强迫进化或构建突变和杂种酶改变其结构，这会改变其催化性。这是MMO分子生物学和生物化学方面的世界专家的雄心勃勃的赠款。我们建议在MMO的活性位点构造关键突变体，并检查对关键底物催化的影响并操纵该酶，以便能够定义底物进入活性位点的进入途径并产生新型的重组酶，并能够氧化新的substrates。我们还旨在定义如何相互相互作用的不同成分，以及如何进行底物进入和电子转移途径的机制，以在MMO中进行氧化位点。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或相关的Di-Iron Center单加速酶的DNA序列，以便能够构建新的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，我们最初开发了该技术，以便能够定义环境中活性甲烷氧化细菌的种群结构。这涉及将13C毛的喂食（例如甲烷）喂入环境样品（例如土壤）中包含的细菌。只有具有MMO的活性甲烷营养物才能用这种重稳定同位素标记。然后，我们可以将编码MMO及其亲属的重型DNA（包含甲烷营养的整个基因组和相关细菌）分离，这些DNA与数以千计的非甲烷营养细菌中的所有DNA与所有DNA中的所有DNA都通过密度梯度梯度离心分解。通过使用聚合酶链反应（PCR），我们可以将新型的MMO序列与以前未经培养的细菌分离出来，随后可以将其缝合到我们开发的质粒中，这些质粒使我们能够以不寻常的生物催化特性来重现新型MMO。对这些重组酶的分析将阐明MMO的作用机理，并产生新的和新颖的生物催化剂，并有可能在行业中用于非污染生物转化反应。