【作者】 顾榕；

【导师】 丁重阳；

【作者基本信息】 江南大学 ， 发酵工程， 2022， 硕士

【摘要】 UDP-葡萄糖4-差向异构酶（UDP-galactose 4-epimerase,GALE,EC 5.1.3.2）是灵芝多糖合成途径中糖供体合成的重要酶,它参与UDP-葡萄糖与UDP-半乳糖的相互转化,与多糖中半乳糖残基含量密切相关。本文对来源于灵芝的GALE基因进行异源表达,进而解析其酶学性质和催化特征。同时,通过构建灵芝GALE过表达重组菌株,分析其液态发酵过程中多糖产量、单糖组成等性质的变化,探究GALE对灵芝多糖合成的影响规律。主要研究内容及结果如下:（1）克隆得到灵芝CGMCC5.26菌株编码UDP-葡萄糖4-差向异构酶的基因片段,导入大肠杆菌异源表达后进行酶学性质、底物转化率等方面的分析。酶学性质研究表明重组酶最适反应p H为6,在p H 7-9范围内有较好的稳定性;最适反应温度为30℃,温度在40℃时稳定性最好;低浓度的Fe²⁺和Mg²⁺对GALE有激活作用;以UDP-葡萄糖为底物时,K_m为0.824 mmol·L^-1,k_cat为1.333 s^-1;该酶对游离单糖表现出催化活性,可催化D-葡萄糖、半乳糖醛酸、N-乙酰葡萄糖胺;最佳反应条件p H 8.0、温度30℃,添加0.05 mmol·L^-1 Fe²⁺,底物与酶浓度比为1.8时UDP-葡萄糖的转化率从16.0%升至39.4%。（2）构建灵芝过表达的重组质粒exp-GL30389-e GFP,使用聚乙二醇（PEG）转化法将过表达质粒转化至灵芝原生质体,通过潮霉素筛选得到转化子;q RT-PCR结果显示转化菌株T9、T20、T24中目的基因的转录水平明显提升,分别提高了分别提高了7.68倍、2.02倍、2.42倍;转化子在激光共聚焦显微镜下观察检测到e GFP荧光信号。以上结果表明过表达质粒成功转入灵芝的原生质体,且融合蛋白GL30389-e GFP在菌丝体内成功表达。（3）将上述三株灵芝过表达菌株与野生型菌株进行液体发酵,探究过表达GALE对灵芝多糖发酵的影响,结果显示过表达菌株的最大生物量和碳源消耗速率明显低于野生型。三株过表达菌株的最大胞外多糖（EPS）产量均高于野生型,其中T20、T24的胞外多糖产量分别提高了9.9%和19.6%;T9的最大胞内多糖含量（IPS）相比野生型提高了59.8%。胞外多糖、胞内多糖组分中的半乳糖和甘露糖的比例增加,葡萄糖的比例减少。菌体形态方面,过表达菌株形状更为规则,趋于圆球状,菌体表面粗糙度更低。在不同的发酵过程中,过表达菌株的平均当量直径都小于野生型菌株的平均当量直径,且S和M型菌球比例较野生型更高。更多还原

【Abstract】 UDP-galactose 4-epimerase（GALE,EC 5.1.3.2）is an essential enzyme in the polysaccharide synthesis pathway of Ganoderma lucidum that generates sugar donors.It participates in the interconversion of UDP-glucose to UDP-galactose and is intimately connected to the galactose residue content of polysaccharides.The GALE gene from Ganoderma lucidum was heterologously expressed in this study,and its enzymatic and catalytic characteristics were determined.Meawhile,the influence regulation of GALE on monosaccharide composition ratio were investigated by constructing a recombinant strain of Ganoderma lucidum GALE overexpression and analyzing the changes in polysaccharide yield and monosaccharide composition during liquid fermentation process.The main research contents and results are as follows:（1）The gene fragment encoding GALE was cloned from Ganoderma lucidum strain CGMCC 5.26 for heterologous expression.The recombinant enzyme were analyzed for enzymatic propertiesand conversion rate.The study of enzymatic properties showed that,the optimum reaction p H was 6,with good stability in the range of p H 7-9;the optimum temperature was 30°C,with the greatest stability at 40°C;Fe²⁺and Mg²⁺ions could activate GALE;When UDP-glucose was used as the substrate,the K_m was 0.824 mmol·L^-1,the k_catwas1.333 s^-1;The catalytic activity was observed for D-glucose,galacturonic acid and N-acetylglucosamine.The conversion was increased from 16.0%to 39.4%under the optimal reaction conditions,p H 8.0,temperature 30℃,adding 0.05 mmol·L^-1 Fe²⁺,substrate/enzyme is1.8.（2）The recombinant plasmid exp-GL30389-e GFP was constructed and overexpressed in Ganoderma lucidum.The overexpression plasmid was transformed into Ganoderma lucidum protoplasts using polyethylene glycol（PEG）transformation method.Transformants were obtained by hygromycin selection.The results of q RT-PCR showed that the transcription levels of the target genes in the transformed strains T9,T20 and T24 were significantly increased by7.68 times,2.02 times,and 2.42 times.The e GFP fluorescence signal was detected in the observation of transformants by confocal microscopy.The above results indicated that the overexpression plasmid was successfully transferred into the protoplast of Ganoderma lucidum,and the fusion protein GL30389-e GFP was successfully expressed in the mycelium.（3）The above three Ganoderma lucidum overexpression strains and wild-type strains were subjected to liquid fermentation to investigate the effects of GALE overexpression on Ganoderma lucidum polysaccharide fermentation.The results revealed that the maximal biomass and carbon source consumption rate of recombinant strains was lower than that of the wild type.The maximum EPS yield of T20 and T24 was significantly higher than that of the wild type,increasing by 9.9%and 19.6%,respectively.The maximum IPS content of T9 was59.8%higher than that of the wild type.The proportion of galactose and mannose in the exopolysaccharide and intracellular polysaccharide fraction increased and the proportion of glucose decreased.In terms of cell morphology,the shape of the overexpression strain was more regular,tending to be spherical,the surface roughness of the cell body was lower.In different fermentation processes,the average equivalent diameter of the overexpression strain was smaller than that of the wild-type strain and the ratio of S and M type bacteria balls was higher than that of the wild type,which was more conducive to the production of Ganoderma lucidum polysaccharide.更多还原