Heterologous Expression And Enzymatic Properties Of Key Enzymes Involved In Ganoderma Lucidum Polysaccharides Synthesis In Escherichia Coli

Ganoderma lucidum polysaccharide?GLP?is the main active product during in G.lucidum fermentation process,which has many biological activities such as anti-tumor,blood sugar lowering and blood lipids lowering.At present,the synthetic pathways for GLP are not completely clear,and the number of key enzymes involved is numerous and their properties are unclear.Based on the previous study of nucleoside sugars biosynthetic pathway of G.lucidum,the paper explored the properties and catalytic characteristics of key enzymes,phosphoglucomutase?PGM?,UDP-glucosepyrophosphorylase?UGPG?and phosphomannose isomerase?PMI?,to supplement information for related research in G.lucidum.It also could provide an effective basis for the development of the synthetic fermentation strategy of GLP.The main research contents and results are as follows:?1?The key enzyme genes,gl-pgm,gl-ugpg and gl-pmi,derived from G.lucidum were constructed with expression vector pET-28a?+?and transformed the recombinant vectors into host cell E.coli BL21?DE3?,who lost the related gene,then three recombinant strains E.coli-QCpgm/pET28a?+?-gl-pgm,E.coli-QCugpg/pET28a?+?-gl-ugpgandE.coli-QCpmi/pET28a?+?-gl-pmi were obtained.The recombinant strains were induced to express recombinant enzymes,and the productions were isolated with Co-NTA resin.Thus,the purified recombinant enzymes GLpgm,GLugpg and GLpmi were obtained.The specific activity of GLpgm,GLugpg and GLpmi were 4.75 U·mg^-1,6.26 U·mg^-11 and 13.68 U·mg^-1,respectively.?2?Enzymatic properties studies showed that the optimum pH of the purified GLpgm was 8.5,and it was 7.5 for both GLugpg and GLpmi;the optimum reaction temperatures of GLpgm,GLugpg and GLpmi were 35,40 and 30?,respectively.Both pH and temperatures are similar to the properties of them derived from plants and fungi.For the three recombinant enzymes,Ag⁺,Cu²⁺and SDS had strong inhibitory effects;while Mn²⁺and Mg²⁺could improve the enzymatical activities.The K_m and k_cat/K_m of GLpgm were 0.68 mmol·L^-1,196.08mmol·L^-1·s^-1;GLugpg were 0.25 mmol·L^-1,818.60 mmol·L^-1·s^-1;and GLpmi were 0.50mmol·L^-1,1105.22 mmol·L^-1·s^-1,respectively.The results indicated that the catalytic efficiency of the key enzymes of G.lucidum on the substrate were higher relatively compared with relevant enzymes from other sources.?3?The regulation of metal ions,Mg²⁺and Mn²⁺,during the liquid fermentation of G.lucidum was preliminary studied.At the fermentation level,the results showed that the addition of Mg²⁺and Mn²⁺promoted the biomass,the productions of exopolysaccharides?EPS?and intracellular polysaccharides?IPS?,the activities of PGM,UGPG and PMI significantly.In the group with additional Mg²⁺and Mn²⁺,the biomass increased by 23.26%and 42.04%;the EPS yield increased by 34.04%and 25.53%,and the IPS yield increased by 31.20%and15.67%,respectively.At the transcriptional level,the addition of Mg²⁺and Mn²⁺increased the transcription of three key enzyme genes.Among them,Mg²⁺promoted the transcriptions of the gene of PGM and UGPG greatly,which were 4.20 times and 2.68 times that of the control group,respectively.Mn²⁺improved the transcription of the gene of PMI significantly,which was 5.66 times that of the control group.All above illustated Mg²⁺and Mn²⁺could enhance the synthesis of polysaccharides in quantity by increasing the transcription of the gene of key enzymes in the pathway of GLP.