粗糙脉孢菌糖转运蛋白功能及产酶条件优化研究

发布时间：2018-11-27 15:55

【摘要】：本论文对粗糙脉孢菌(Neurospora crassa)进行了两方面的研究工作。一部分是三个纤维二糖转运蛋白基因的研究,另一部分是对产酶培养基优化的研究。微生物将生物质降解为各种糖类物质,这些糖类物质通过糖转运蛋白运送到细胞中,并在细胞中进行进一步的代谢。在自然界中,糖转运蛋白对于碳水化合物的代谢起着至关重要的作用。在非葡萄糖转运蛋白的研究中,纤维二糖转运蛋白的研究非常少见。粗糙脉胞菌具有纤维素降解,乙醇发酵,木糖应用等杰出特点,是一个非常有工业应用潜力的丝状真菌。2010年,有科学家首次从粗糙脉孢菌中克隆鉴定了纤维二糖转运蛋白基因CDT1、CDT2。但是对其生理生化功能、基因表达调控等分子机理还不很了解。本论文以三个纤维二糖转运蛋白为研究对象,分别为cDT1、CDT2和CDT3。实验中构建并获得了三个基因的单敲除体菌株,双敲除体菌株及三敲除体菌株。并且在不同碳源培养基中生长这些菌株,通过纤维素内切酶酶活,木聚糖酶酶活等指标反映各个菌株之间的生长差异。结果表明,在以纤维二糖为碳源的培养基中生长的三敲除体菌株T123生长较双突变菌株D12差,说明CDT3基因与纤维二糖转运相关。并通过绿色荧光蛋白GFP对糖转运蛋白进行了定位。定位结果显示,糖转运蛋白均在各种膜上进行表达。本论文第二部分是以粗糙脉孢菌基因组测序菌株FGSC2,489为对象,采用响应面分析法对粗糙脉孢菌摇瓶发酵产纤维素酶的培养基进行优化。在初始培养基的基础上,采用该优化培养基,最大纤维素酶活可达：1.27FPU/mL,是优化前纤维素酶活的2.02倍；CMC酶活14.15IU/mL是优化前的1.88倍,木聚糖酶活24,13IU/mL是优化前的1.86倍,葡萄糖苷酶酶活1.22IU/mL是优化前的2.08倍。
[Abstract]:In this paper, two aspects of research on (Neurospora crassa) were carried out. One is the study of three cellular-disaccharide transporter genes, the other is the optimization of enzyme production medium. Microorganisms degrade biomass into various carbohydrates which are transported to cells by sugar transporters and further metabolized in cells. Sugar transporters play an important role in carbohydrate metabolism in nature. In the study of non-glucose transporter, the study of fibronectin is very rare. With outstanding characteristics of cellulose degradation, ethanol fermentation, xylose application and so on, C. crassica is a filamentous fungus with great potential for industrial application. Some scientists first cloned and identified the cellular-disaccharide transporter gene (CDT1,CDT2.) from C. crassa. However, its physiological and biochemical functions, gene expression regulation and other molecular mechanisms are not well understood. In this thesis, three cellular-disaccharide transporters (cDT1,CDT2 and CDT3.) were studied. Three single knockout strains, double knockout strains and triple knockout strains were constructed and obtained. These strains were grown in different carbon source media, and the growth differences were reflected by cellulose endonuclease activity and xylanase activity. The results showed that the growth of triple-knockout strain T123 was worse than that of double mutant strain D12 in the medium with cellulose disaccharide as carbon source, indicating that the CDT3 gene was related to fiber disaccharide transport. The sugar transporter was located by green fluorescent protein (GFP). The localization results showed that glucose transporters were expressed on various membranes. In the second part of this paper, the response surface analysis was used to optimize the medium for cellulase production by using response surface analysis (RSM), which was based on the genomic sequencing strain FGSC2489 of C. crassa. On the basis of the initial medium, the maximum cellulase activity was 1.27 FPU / mL, 2.02 times of that before optimization. The enzyme activity of CMC was 1.88 times of that before optimization, the activity of xylanase was 1.86 times of that before optimization, and the activity of glucosidase 1.22IU/mL was 2.08 times of that before optimization.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3411

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