The Biosynthesis Of Uridine Diphosphate Glycosyl Ligand And Its Glycosylation Of Quercetin

Quercetin is a flavonoid compound that exists widely in nature,and it is commonly found in fruits and vegetables,such as onions,apples,and tea.Quercetin has anti-cancer,antibacterial,anti-oxidant,anti-free radical,anti-aging and other pharmacological effects.Quercetin is not only an antioxidant,but also a medical complex.However,like other types of flavonoids,quercetin is extremely unstable in the water phase and has poor water solubility,resulting in low bioavailability.In order to solve this problem,more researchers have invested in the study of quercetin.Compared with the chemical method,the glycosylation modification of quercetin by enzymatic method is more widely used,which can change its physical and chemical properties.Therefore,this article used glycosyltransferases from different sources to glycosylate quercetin,which were BTGT-1 from Bacillus thuringiensis,FGT from grape fruit and GT-2 from Escherichia coli.Using UDP-glucuronic acid as the glycosyl donor and quercetin as the glycosyl acceptor,the synthesis of the reaction was detected by HPLC.The molecular weight was determined by mass spectrometry and structure of the glycoside product was speculated by 1H-NMR.The specific research content was as follows:(1)Utilize immobilized enzyme to prepare UDP-glucose.The three key enzymes Nah K,Glm U,and PPA that synthesized UDP-glucose were respectively fixed.In addition,the amount of fixation temperature,resin required for fixation,and fixation time were explored to obtain the optimal fixation conditions:When fixed at 20°C for 1 h,the required amount of resin was1.0 g,and the Nah K enzyme immobilization rate was 56.8%;when fixed at 25°C for 1 h,the required amount of resin was 0.8 g,and the immobilization rates of Glm U and PPA enzyme were 96.5%and 92.0%respectively.In view of the poor immobilization effect of Nah K enzyme,free enzyme was still used for reaction,while Glm U and PPA enzyme used immobilized enzyme for reaction.The reaction effect was detected by HPLC,and the results showed that the yield of UDP-glucose synthesized by immobilized enzyme was 9.71 mg/m L,and the yield of UDP-glucose synthesized by free enzyme was 9.16 mg/m L.The effect of the two was not much different.Purification was carried out with a strong basic anion adsorption column Q,the purity was above 90%,and it was dialyzed and lyophilized for use.(2)Synthesis of UDP-glucuronic acid by double enzyme coupling.Using UGD from Streptococcus pyogenes and LDH from pigs,the two enzymes were coupled to oxidize UDP-glucose to generate UDP-glucuronic acid.The reaction would generate a high-energy by-product NADH,which would have a feedback inhibition effect on the reaction.Therefore,in this experiment,LDH was added to realize the cyclic regeneration of NAD~+/NADH to weaken the inhibitory effect of the reaction.The production rate of the product is about 60.17%.Purification was carried out by the strong basic anion adsorption column Q.The purity was more than 90%.The samples were dialyzed and lyophilized for later use.It was confirmed that the product produced was UDP-glucuronic acid by mass spectrometry and 1H-NMR.(3)Glycosylation modification of quercetin.Glycosyltransferases BTGT-1,FGT,GT-2from different sources were used for glycosylation modification of quercetin.The UDP-glucuronic acid prepared in the laboratory was used as the glycosyl donor,quercetin was the glycosyl acceptor,and glycosyltransferases from different sources were used as catalysts for glycosylation reactions.HPLC was used to detect the synthesis of the reaction.The results showed that,under the catalysis of FGT enzyme,a new glycoside product was formed in the system.The other two glycosyltransferases could not catalyze the glycosylation reaction of quercetin.And explored the optimal reaction conditions as:the reaction was carried out at 35℃and p H=8.0 for 36 h,and the reaction rate was 11.68%.Sephadex LH-20 column was used to separate and purify glycoside products.The purity was more than 90%.Detected by mass spectrometry and 1H-NMR,the molecular weight of the glycoside product is 478,and the glycoside structure is inferred to be quercetin-3-O-β-D-glucuronide.(4)With vitamin C as a positive control,the antioxidant activities of quercetin,quercetin-3-O-β-D-glucoside,and quercetin-3-O-β-D-glucuronide were compared respectively.The four experiments of reducing power,DPPH free radical scavenging,hydroxyl free radical scavenging and superoxide anion free radical scavenging analysis were carried out respectively.In addition to superoxide anion free radical scavenging ability,the results showed that the scavenging ability of the two glycosides was better than that of quercetin.Within a certain concentration range,the antioxidant activity of quercetin-3-O-β-D-glucuronide was better than that of quercetin-3-O-β-D-glucoside.