细菌Comamonas serinivorans C35对木质素的降解及其代谢机制初步研究

发布时间：2018-01-09 18:05

本文关键词：细菌Comamonas serinivorans C35对木质素的降解及其代谢机制初步研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：木质素是自然界中最丰富的可再生芳香族化合物,它的生物合成、分解与自然界中的碳循环密切相关。木质素是由三种基本苯丙烷结构单体(松柏醇、芥子醇和对香豆醇),通过碳碳键和碳氧醚键等方式连接形成的一种三维网状无定型酚类聚合物。木质素分子量大,结构复杂且不规则,它作为天然屏障将纤维素和半纤维素紧紧包裹,使得木质纤维素难以被水解发酵,这也是目前生物乙醇生产过程中存在的主要难题。从生物质中提取出的纤维素和半纤维素已广泛应用于乙醇生产、食品和造纸等工业,然而木质素作为储量仅次于纤维素的天然可再生碳源却仍未被合理利用。目前大多数的木质素残渣都是随意堆放或直接燃烧,因此将木质素高效降解并实现高值化利用,对开发以木质纤维素为原料的可再生资源具有非常重要的意义。对木质素的降解方法已进行了多年的研究,但仍存在诸多问题,如物理法面临高能耗挑战,化学法会造成二次污染,而生物法虽对环境友好但效率低。在自然界中真菌是木质素生物降解的先锋者,但由于在基因操纵和蛋白表达等方面存在巨大挑战,目前仍未实现工业化生成。虽然细菌降解能力弱于真菌,但由于其来源广泛、生长迅速、易于大规模应用,近年来在生物质利用方面备受人们关注。因此,筛选能高效降解木质素的细菌,研究其木质素代谢途径及机制,将会为木质素生物降解实现工业化生产提供重要的理论基础。本论文以细菌Comamonas serinivorans C35为研究对象,对其木质素降解性能及代谢机制进行了系统研究。主要研究成果如下:(1)菌株Comamonas serinivorans C35能够在以木质素为唯一碳源的培养基上生长,经过七天的培养,可使碱木质素和磨木木质素培养基中COD的去除率分别达到44.4%、30.8%,表现出较强的木质素降解能力。Comamonas serinivorans C35能使苯胺蓝染料褪色,且在粗酶液中检测到了过氧化物酶和漆酶活性。(2)通过利用傅里叶变换红外光谱(FTIR)和气相色谱/质谱联用(GC-MS)技术检测,发现菌株Comamonas serinivorans C35能够攻击碱木质素和磨木木质素分子结构中的苯环、侧链、醚键以及C=O键等,使其解聚生成多种单环芳香族化合物,包括3-甲基苯甲醛、愈创木酚、香草酸、香草醛、丁香酸、丁香醛、对羟基苯甲酸和阿魏酸等。(3)通过全基因组测序和生物信息学分析,发现菌株Comamonas serinivorans C35的基因组序列中含有编码木质素降解相关酶的基因序列414条,共计15种酶类,包括过氧化物酶、铜氧化酶、漆酶、双加氧酶和脱羧酶等。基于GC-MS和基因组数据分析结果,发现菌株Comamonas serinivorans C35存在至少4条木质素降解途径,分别是苯甲酸盐途径、苯酚途径、对羟基苯乙酮途径和β-酮己二酸途径,其中对羟基苯乙酮途径为首次发现。
[Abstract]:Lignin is one of the most abundant renewable aromatic compounds in nature. Its biosynthesis and decomposition are closely related to the carbon cycle in nature. Lignin is composed of three basic phenylpropane structural monomers (cyperol). A kind of three-dimensional amorphous phenolic polymer formed by carbon-carbon bond and carboxy-ether bond. Lignin has a large molecular weight, complex structure and irregular structure. It acts as a natural barrier to cellulose and hemicellulose tightly wrapped, making lignocellulose difficult to be hydrolyzed fermentation. Cellulose and hemicellulose extracted from biomass have been widely used in ethanol production, food and paper industry. However, lignin as a natural renewable carbon source, second only to cellulose, has not been used properly. At present, most of lignin residues are piled up or burned directly. Therefore, it is very important to degrade lignin efficiently and realize high value utilization, which is very important for the development of renewable resources based on lignocellulose. The degradation methods of lignin have been studied for many years. However, there are still many problems, such as the challenge of high energy consumption in physical method, the secondary pollution caused by chemical method, and the environmental friendly but inefficient biological method. Fungi are pioneers of lignin biodegradation in nature. However, due to the huge challenges in gene manipulation and protein expression, industrialization has not been achieved. Although the ability of bacteria degradation is weaker than fungi, it is easy to be used on a large scale because of its wide source, rapid growth and easy to be used on a large scale. In recent years, people pay more attention to the utilization of biomass. Therefore, screening the bacteria that can efficiently degrade lignin, and study the pathway and mechanism of lignin metabolism. It will provide an important theoretical basis for the realization of industrial production of lignin biodegradation. In this thesis, Comamonas serinivorans C35 is taken as the research object. The degradation performance and metabolic mechanism of lignin were systematically studied. The main results are as follows: 1). Strain Comamonas serinivorans C35 could grow on the medium with lignin as the sole carbon source. After seven days of culture, the removal rate of COD in alkali lignin and lignin grinding medium was 44.4% and 30.8%, respectively. Comamonas serinivorans C35 can make aniline blue dye fade. The activity of peroxidase and laccase was detected in crude enzyme solution by FTIR and GC-MS. It was found that the strain Comamonas serinivorans C35 could attack benzene ring, side chain, ether bond and Cor O bond in alkali-lignin and lignin molecular structure. It depolymerizes to produce a variety of monocyclic aromatic compounds, including 3-methylbenzaldehyde, guaiacol, vanillic acid, vanillin, eugenic acid, eugenaldehyde. P-hydroxybenzoic acid and ferulic acid, etc.) were sequenced and analyzed by bioinformatics. The genomic sequence of the strain Comamonas serinivorans C35 contained 414 genes encoding lignin degradation related enzymes, with a total of 15 enzymes. Including peroxidase, copper oxidase, laccase, dioxygenase and decarboxylase. Based on GC-MS and genomic data analysis results. It was found that there were at least four lignin degradation pathways in strain Comamonas serinivorans C35, namely benzoate pathway and phenol pathway. The p-hydroxyacetophenone pathway and 尾 -ketoadipic acid pathway, of which p-hydroxyacetophenone pathway was first discovered.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172;X505

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