Heterologous Expression And Enzymatic Properties Of UDP-glucose Dehydrogenase From Ganoderma Lucidum

Ganoderma lucidum polysaccharide is a secondary metabolite in the fermentation of G.lucidum.It has a variety of biological activities such as immune regulation,anti neuropathy,anti-tumor and so on.At present,the synthetic route of G.lucidum polysaccharides has not been completely analyzed,and the involved enzymes are numerous and their properties are unknown.On the basis of the sugar donor synthesis pathway of G.lucidum,this topic analyzed the related enzyme UDP glucose dehydrogenase（UGD）,studied its properties and other characteristics,in order to improve the enzymology information of edible and medicinal fungi and G.lucidum UGD and provide reference for future applications.The main research contents and results are as follows:（1）First,the ugd gene in E.coli BL21（DE3）was knocked out by CRISPR/Cas9technology.PCR identification was carried out by designing primers at the upstream and downstream of the knock out site（each 500 bp）,which showed that only nucleic acid bands consistent with the total size of homologous arm（1000 bp）were obtained,proving that the target fragment had been knocked out of the genome.Then the gene fragment encoding UDP glucose dehydrogenase of G.lucidum CGMCC5.26 strain was cloned and introduced into E.coli BL21（DE3）with ugd knocked out for heterologous expression and purification.The specific activity of G.lucidum UGD was 3.6 U·mg^-1.（2）The optimal fermentation conditions were obtained by optimizing the fermentation of the above constructed strains:the initial p H was 7,IPTG was added at 2 h of growth,the IPTG final concentration was 0.5 mmol·L^-1,and the total enzyme activity of E.coli expressing heterologous UGD was increased by 25.4%compared with the original conditions after induction at 200 r·min^-1 and 16℃for 24 h.（3）Enzymatic properties of G.lucidum UGD were analyzed.The study of enzymatic properties showed that the optimum reaction p H was 9,and the stability was high when p H was 6-8.The optimum reaction temperature of UGD is 30℃.G.lucidum UGD will rapidly inactivate,when the temperature is greater than or equal to 35℃.0.5 mmol·L^-1 Mg²⁺and Ca²⁺can obviously promote the UGD activity of G.lucidum,Cu²⁺,Zn²⁺,Ba²⁺,Ni²⁺can inhibit the enzyme activity at high concentrations,the surfactant SDS has a significant inhibitory effect on G.lucidum UGD.The K_m of UGD is 0.14 mmol·L^-1,and the maximum reaction rate V_maxis 1428.6μmol·min^-1·L^-1,the catalytic constant k_cat is 2.5 s^-1.The affinity with substrate is higher than that of most bacteria and lower than that of most plants.（4）The enzyme modification of G.lucidum UGD showed that 310 Met and 144 Met were mutated into Cys and Leu at the same time,and the activity of UGD increased by 55.2%to 5.6 U·mg^-1.The optimum reaction temperature of the mutant is 30℃.When the temperature is more than 20℃but less than 40℃,the remaining enzyme activity of the mutant after one hour of storage is more than 80%,and the remaining enzyme activity reaches43.4%after one hour of storage at 45℃.The K_m of the mutant was 0.12 mmol·L^-1,and the maximum reaction rate V_max was 1724μmol·min^-1·L^-1.Compared with the wild type,the K_mvalue of the combined mutant decreased,indicating that its affinity with the substrate increased.The specific enzyme activity and efficiency after immobilization were improved compared with those of wild type.It was found through molecular dynamics simulation that the combined mutant had a relatively stable state,and the RMSD was lower than that of the wild type as a whole,maintained at about 1.5?.The catalytic reaction of the combined mutant and wild type reached equilibrium when the reaction time was 12 h.The conversion of UDP glucuronic acid was 58.4%and 38.7%,respectively.（5）To improve the stability and catalytic efficiency of the enzyme,chitosan was used as the carrier for the immobilization of UGD.The optimal conditions for the immobilization were as follows:the concentration of glutaraldehyde crosslinking chitosan carrier is 0.5%,and the crosslinking time is 1 h,the time for the carrier to adsorb the pure enzyme is 12 hours.Under these conditions,the immobilization efficiency was 19.9%,the recovery rate of enzyme activity was 7.3%,and the specific enzyme activity was 1.3 U·mg^-1.The optimal reaction temperature of the immobilized UGD is 30℃,the remaining enzyme activity after being placed at 40℃for one hour is 70.2%.Therefore,immobilized UGD is more stable than the free enzyme,and can improve the stability of the UGD in alkaline environment.Under the optimal conditions,the efficiency of immobilized mutant enzyme was 26.8%,the recovery rate of enzyme activity was 11.8%,and the specific enzyme activity was 1.8 U·mg^-1.