Characterization Of The UDP-Galactose 4-epimerase And Its Effect On Polysaccharide Synthesis Of Ganoderma Lucidum

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.