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:
- 金额:$ 42.96万
- 依托单位:
- 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:
- 关键词:
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循环的关键一环,在甲烷释放到环境中之前,它们在湿地、稻田土壤和垃圾填埋场等许多环境中氧化甲烷。因此,它们减轻了这种强有力的温室气体的影响,减少了全球变暖。甲烷单加氧酶(MMO)是一种细菌酶,催化细菌氧化甲烷的第一步。它引起了化学家的极大兴趣,因为它在常温常压下将甲烷氧化成甲醇,这一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的衍生物,因此它在生物转化和生物修复方面具有很大的潜力,比传统的化学路线污染更少。由于具有广泛的底物特异性,MMO的结构一直是生物学家感兴趣的主题,其中一个目标是试图了解MMO的结构是如何允许催化如此广泛的化合物的,以及通过突变、强制进化或构建突变酶和杂交酶来改变其结构将如何改变其催化作用。这是世界上MMO分子生物学和生物化学专家提出的一项雄心勃勃的拨款提案。我们建议在MMO的活性部位构建关键突变体,检测关键底物对催化作用的影响,并操纵这种酶,以便能够确定底物进入活性部位的途径,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成部分是如何相互作用的,以及底物进入机制和电子转移途径是如何在MMO中氧化的。在一种新的方法中,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。这将涉及使用一种名为DNA稳定同位素探测(DNA-SIP)的技术,这是我们最初开发的,目的是能够定义环境中活性甲烷氧化细菌的种群结构。这涉及将甲烷等13C底物喂给土壤等环境样本中包含的细菌。只有带有MMO的活性甲烷氧化菌才会被这种重的稳定的同位素标记。然后,我们可以通过密度梯度离心法从土壤中存在的数千个非甲烷营养细菌的所有DNA中分离出编码MMO及其近亲的重DNA(包含甲烷氧化菌和相关细菌的整个基因组)。通过使用聚合酶链式反应(PCR),我们可以从以前未培养的细菌中分离出新的MMO序列,这些序列随后可以缝合成我们开发的质粒,使我们能够重建具有不寻常的生物催化特性的新的MMO。对这些重组酶的分析将有助于阐明MMO的作用机理,也将产生具有工业应用潜力的新型生物催化剂,用于无污染的生物转化反应。
项目成果
期刊论文数量(10)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Aerobic Utilization of Hydrocarbons, Oils and Lipids
碳氢化合物、油类和脂类的有氧利用
- DOI:10.1007/978-3-319-39782-5_4-1
- 发表时间:2018
- 期刊:
- 影响因子:0
- 作者:Khmelenina V
- 通讯作者:Khmelenina V
Microbial biotechnology meets environmental microbiology.
- DOI:10.1111/j.1751-7915.2009.00090_11.x
- 发表时间:2009-03
- 期刊:
- 影响因子:5.7
- 作者:Murrell JC;Smith TJ
- 通讯作者:Smith TJ
Molecular dynamics simulation to rationalize regioselective hydroxylation of aromatic substrates by soluble methane monooxygenase.
分子动力学模拟合理化可溶性甲烷单加氧酶对芳香族底物的区域选择性羟基化。
- DOI:10.1016/j.bmcl.2015.01.069
- 发表时间:2015
- 期刊:
- 影响因子:2.7
- 作者:Sigdel S
- 通讯作者:Sigdel S
Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.
- DOI:10.1093/femsle/fnx137
- 发表时间:2017-07-06
- 期刊:
- 影响因子:2.1
- 作者:Lock M;Nichol T;Murrell JC;Smith TJ
- 通讯作者:Smith TJ
Encyclopedia of industrial biotechnology : bioprocess, bioseparation, and cell technology
- DOI:10.5860/choice.48-1218
- 发表时间:2010
- 期刊:
- 影响因子:0
- 作者:M. Flickinger
- 通讯作者:M. Flickinger
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
John Murrell其他文献
Ethical considerations in clinical veterinary research.
临床兽医研究中的伦理考虑。
- DOI:
10.1016/j.tvjl.2023.106026 - 发表时间:
2023 - 期刊:
- 影响因子:2.2
- 作者:
C. Adami;John Murrell;P. Fordyce - 通讯作者:
P. Fordyce
John Murrell的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('John Murrell', 18)}}的其他基金
Microbial degradation of isoprene in the terrestrial environment
陆地环境中异戊二烯的微生物降解
- 批准号:
NE/J009725/1 - 财政年份:2012
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Microbial food webs in Movile Cave
莫维尔洞穴的微生物食物网
- 批准号:
NE/G017956/2 - 财政年份:2012
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Microbial food webs in Movile Cave
莫维尔洞穴的微生物食物网
- 批准号:
NE/G017956/1 - 财政年份:2009
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Joint SOLAS Bergen Mesocosm Experiment
联合 SOLAS 卑尔根中宇宙实验
- 批准号:
NE/E011446/1 - 财政年份:2008
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Joint SOLAS Bergen Mesocosm Experiment
联合 SOLAS 卑尔根中宇宙实验
- 批准号:
NE/E011527/1 - 财政年份:2008
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Joint SOLAS Bergen Mesocosm Experiment
联合 SOLAS 卑尔根中宇宙实验
- 批准号:
NE/E011438/1 - 财政年份:2008
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Facultative methanotrophs and environmental regulation of methane oxidation
兼性甲烷氧化菌与甲烷氧化的环境调控
- 批准号:
NE/E016855/1 - 财政年份:2007
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
相似海外基金
Role of intestinal serotonin transporter in post traumatic stress disorder
肠道血清素转运蛋白在创伤后应激障碍中的作用
- 批准号:
10590033 - 财政年份:2024
- 资助金额:
$ 42.96万 - 项目类别:
Functional analysis of alkylglycerol monooxygenase; an unexpected modulator of Wnt signalling and embryogenesis
烷基甘油单加氧酶的功能分析;
- 批准号:
BB/W017032/1 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
Research Grant
Sustained eIF5A hypusination at the core of brain metabolic dysfunction in TDP-43 proteinopathies
持续的 eIF5A 抑制是 TDP-43 蛋白病脑代谢功能障碍的核心
- 批准号:
10557547 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
Modulation of Sympatho-Adrenal Function by Tissue Resident Macrophages
组织驻留巨噬细胞对交感肾上腺功能的调节
- 批准号:
10726938 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
Pathogenesis, prevention and treatment of corticosteroid-resistant gut GVHD
皮质类固醇耐药性肠道GVHD的发病机制及防治
- 批准号:
10585851 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
The Underlying Mechanisms of Visual Impairment and Myopia in Prematurity
早产儿视力障碍和近视的潜在机制
- 批准号:
10584723 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
A novel brain-to-pancreatic islet neural circuit regulates glucose homeostasis
一种新型脑-胰岛神经回路调节葡萄糖稳态
- 批准号:
10886883 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
Alpha-Synuclein-Specific T cells in Parkinson's Disease Pathogenesis
帕金森病发病机制中的α-突触核蛋白特异性 T 细胞
- 批准号:
10752172 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别:
Development of approaches to apply CRISPR/Cas9-mediated gene conversion to model complex genetic traits in mice
开发应用 CRISPR/Cas9 介导的基因转换来模拟小鼠复杂遗传性状的方法
- 批准号:
10565297 - 财政年份:2023
- 资助金额:
$ 42.96万 - 项目类别: