甘蔗渣降解菌的应用研究及其功能基因的克隆表达

发布时间：2018-03-24 18:02

  本文选题：海洋细菌　切入点：原位富集　出处：《集美大学》2015年硕士论文

【摘要】：海洋环境中含有丰富的微生物资源,基于技术等原因,只有少部分的海洋微生物被分离、研究及应用。由于生活环境不同于陆地,多数海洋微生物具有独特的生理生化及代谢功能,具有耐盐、嗜热、嗜冷等特征,可以说海洋环境比陆地蕴含更多极端微生物。深入研究海洋微生物,能够为人类获得更多的生物资源。甘蔗渣是甘蔗制糖后的残留物,是一种生物质资源,主要成分为纤维素、半纤维素和木质素,本研究采用原位富集方法,于近海及深海环境中富集甘蔗渣降解菌。首先利用分子生态学方法研究了两个富集位点下未培养甘蔗渣降解菌的菌群组成情况。结果显示两个文库均有各自较为明显的优势菌群:深海文库优势细菌类群为变形菌门,其中γ-变形菌纲在深海文库中的丰度最大;近海文库优势细菌类群为拟杆菌门。同时,从两个富集样品分离得到29株细菌,主要属于变形菌门与厚壁菌门两个类群,与未培养得到的优势菌群一致。对从深海环境中分离到的两株芽孢杆菌GZ03、GZ04降解甘蔗渣的情况进行了分析,结果发现两株菌均能在甘蔗渣降解上具有应用价值:两个菌株能够降解甘蔗渣产较高浓度还原糖,离子色谱结果显示降解产物为葡萄糖、果糖混合液;通过GZ03菌株产还原糖单因素条件优化实验发现,菌株产还原糖最适培养基为2%甘蔗渣(60目)、3%Na Cl、蒸馏水配置,产还原糖最适条件为:4%接种量、35℃摇床培养60 h;GZ04菌株采用固体发酵方法可降解甘蔗渣半纤维素,于28℃恒温发酵5 d,半纤维素质量减少20%。本研究也针对这两株菌一些参与甘蔗渣降解的酶类进行了研究。对菌株GZ03进行全基因组测序,分析测序结果,对其中编码果聚糖蔗糖酶基因GH5进行克隆表达。果聚糖蔗糖酶的最适水解温度为30℃,最适p H为6.0,最适蔗糖浓度为400 m M。果聚糖最适合成条件为:30℃、p H7.8、蔗糖浓度400 m M,并且在36 h合成量最大。对GZ04菌株产的木聚糖酶进行酶学性质研究,发现该酶的最适温度为40℃,最适p H值为5.0,在40℃环境下具有良好的稳定性,并且在不同p H环境下均能维持较高酶活力。合理使用甘蔗渣,减少生产废料,实现蔗渣资源的可循环、可发展利用日益受到关注,而开发海洋来源的细菌降解甘蔗渣制糖是当前高效利用甘蔗渣的新途径。综上所述,本研究从海洋环境中分离得到两株芽孢杆菌均有潜在的应用价值,能够为合理使用甘蔗渣提供新思路,在利用海洋微生物降解甘蔗渣制糖应用上提供了理论依据,发掘了海洋微生物功能基因,为木质纤维的合理利用提供了更为广阔的研究及应用前景。
[Abstract]:The marine environment is rich in microbial resources. For reasons such as technology, only a small number of marine microbes are separated, studied and applied. Most marine microbes have unique physiological, biochemical and metabolic functions, and have the characteristics of salt tolerance, thermophilic, cold, etc. It can be said that the marine environment contains more extreme microbes than on land. Bagasse is a kind of biomass resource, which is mainly composed of cellulose, hemicellulose and lignin. Bagasse degrading bacteria were enriched in offshore and deep sea environment. Firstly, the microflora composition of uncultured bagasse degrading bacteria at two enrichment sites was studied by molecular ecology method. The results showed that the two libraries had their own characteristics. Obvious dominant flora: the dominant bacteria group in deep sea library is Proteus. Among them, 纬 -Proteus is the most abundant in the deep sea library, and the dominant bacteria group is Bacteroid phylum in the offshore library. At the same time, 29 strains of bacteria are isolated from two enriched samples, which mainly belong to two groups: Proteus and phylum thuringiensis. The degradation of bagasse by two strains of Bacillus sp. GZ03GZ04 isolated from deep-sea environment was analyzed. The results showed that the two strains could be used in the degradation of bagasse. Two strains could degrade the high concentration of reducing sugar from bagasse. Ion chromatography showed that the degradation product was glucose and fructose mixture. By optimizing the single factor condition of reducing sugar production of GZ03 strain, it was found that the optimum medium for reducing sugar production of the strain was 2% bagasse, 60 mesh, 3% NaCl3, and distilled water, and the optimum medium for reducing sugar production was 2% bagasse. The optimum condition for reducing sugar production was that the strain GZ04 could degrade hemicellulose from bagasse by solid fermentation at 35 鈩，

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