【作者】 韩冰；

【导师】 邢新会；

【作者基本信息】 清华大学 ， 化学工程与技术， 2008， 博士

【摘要】 甲烷氧化菌及甲烷单加氧酶(MMO)在生物转化、生物修复和温室气体CH4的减排等方面具有重要的应用价值。本论文围绕甲烷氧化菌及MMO在应用过程中存在的问题:1)催化氧化过程受辅酶NADH再生的限制;2)甲烷氧化菌生长速度慢、细胞密度低,难以满足工业生物催化的需要;3)可以工业应用的甲烷氧化菌资源有限,MMO外源表达技术不成熟,利用微生物分子生态学、基因工程、生物工程等手段对甲烷氧化菌在我国煤矿土壤中的分布、MMO蛋白的异源表达、甲烷氧化菌的高密度培养及其催化氧化丙烯的过程进行了研究。利用稳定性同位素探针、生物芯片等多种分子生态学方法解析我国典型瓦斯煤矿土壤中甲烷氧化菌群落的组成和活性。?型与II型甲烷氧化菌均存在于该碱性煤矿土壤中。首次报道了煤矿中甲烷氧化菌的组成,为进一步分离具有特殊功能的甲烷氧化菌和建立用于吸收煤矿瓦斯气的生物过滤器提供帮助。利用Trial andError的方式对颗粒性甲烷单加氧酶(pMMO)异源表达技术进行了系统的研究,通过对启动子、表达载体及表达宿主的比较和选择,初步建立了异源表达pMMO的研究平台和方法。构建了由烷烃单加氧酶启动子调控pmoCAB的pCompmo质粒,在重组产气肠杆菌中实现了pMMO的转录、翻译,但由于pMMO在折叠过程受到其它因素影响,不能稳定得到正确组装的蛋白。利用pMMO原始启动子构建了含有pmo完整基因簇的pCHP质粒,并转入大肠杆菌中,首次实现了pmo完整基因簇在大肠杆菌中的克隆。以甲烷氧化菌Methylosinus trichosporium OB3b为对象,石蜡油作为甲烷传递体可以极大的缩短细胞生长的迟滞期,促进细胞快速、高密度生长。在5L发酵罐中,石蜡油浓度为5%时,干重高于9.89gL-1,为无石蜡油添加时的4.5倍。以M. trichosporium OB3b整细胞催化丙烯制备环氧丙烷为例,对甲烷氧化菌应用于工业催化进行了基础的研究。当以OD660为8的细胞进行催化氧化时,反应22小时,产物环氧丙烷最高浓度可达到12.2mM。同时发现发酵液直接用于催化反应,可以减少传统两步法的工序,缩短“生长与催化”周期。更多还原

【Abstract】 Methanotrophs are capable of utilizing methane as the sole carbon and energysource, and contain the key enzymes of methane monooxygenases (MMO) whichcatalyze the oxidation of methane and short-chain alkanes and alkenes.Methanotrophs and MMOs play significant roles in biotransformation,bioremediation and greenhouse gas assimilation. Whereas many problems ofmethanotrophs have limited their applications in industrial biotechnology, includinglack of efficient NADH regeneration system, low growth rate and low cell density,lack of strains which are suitable for applications under various conditions, and lackof genetic engineering tools for MMOs. In order to overcome these deficiencies,molecular ecology, gene engineering and biotechnology methods were used toinvestigate the diversity of methanotrophs in coal mine, hetero-expression of MMO,high cell density culture of methanotroph and optimization of oxidation of propenecatalyzedbyMMO.We successfully elucidated the methanotroph community and activity from atypical Chinese alkaline coal mine soil using several complementary molecularecologytechniques, including microarrayand stable isotope probing. Both type I andtype II methanotrophs were present and active. To our knowledge this is the firstdetailed molecular analysis of methanotrophs in the coal mine environment and thedata obtained from the molecular ecology study will provide essential information toisolate and establish biofilters using the most appropriate methanotrophs to mitigateexplosionsandreleaseofmethanegasfromcoalmines.Heterogenous expression of particulate methane monooxygenase (pMMO) wassystemicallystudiedby“TrialandError”approach.Byconstructionofdifferentkindsof promoters and vectors combined with different host cells, hetero-expressionsystem of pMMO was established. Expression plasmid pCompmo was constructed inwhich pmoCAB gene was under the promoter of alkane monooxygenase. It wasproved that the recombinant Enterobacter aerogenes pCompmo showed transcription and translation activityunder certain cultivation conditions. Unfortunately, due to theimproperpMMOrefoldingorassembly,theenzymeactivityinrecombinant cells wasnot as stabled as that in wild strain. Furtherly, another plasmid pCHP containing thewhole pmo gene cluster including the native promoter was constructed andtransformed into Escherichia coli. This was the first report on successful cloning ofpmo genecluster,althoughpreviousresearchersbelievedpMMOwastoxicto E.coli.Paraffin as the methane vector, which can shorten the lag phase and helpMethylosinus trichosporium OB3b to reach high cell density, was selected to enhancethe growth rate of OB3b. Pilot study was carried out in 5L fermenter. With theaddition of 5% (v/v) paraffin, the highest densityof cultivated cells was 9.89gdrywtL-1,whichwas4.5timesofthecontrol.Optimization of epoxypropane biosynthesis from propene using the whole cellsof M. trichosporium OB3b was investigated. The cells suspended in phosphate buffer(pH 7) (OD660=8) accumulated 12.2 mM epoxypropane over 22 h. Meanwhile it wasfound that the cell culture broth could directlycatalyze the biotransformation withoutany pretreatments, which benefit for simplifying the traditional two-stagegrowth-catalysisprocess,andshortenthewholeprocesstime.更多还原