Study On Enzymatic Extraction Of Resveratrol From Polygonum Cuspidatum And Synthesis Of Resveratrol Esters

Resveratrol is a natural polyphenol with significant antioxidant activity,which plays an important role in preventing cancer,protecting cardiovascular and nervous systems,improving immunity and prolonging life.It is now of great application value in foods,medicines,dietary supplements,health care products and cosmetics.Due to the low bioavailability of resveratrol and its strong metabolism and excretion ability in vivo,the esterification of resveratrol and carboxylic acid to resveratrol ester can help to increase the lipophilicity,cell penetration and bioavailability of resveratrol,and there are synergistic effecst between them.In this study,a strain with high yield of beta-glucosidase(BGL,EC3.2.1.21)was selected,which was used to efficiently hydrolyze polydatin in Polygonum cuspidatum to obtain resveratrol.Then,it was reacted with organic acids to form various resveratrol esters.By analyzing the enzyme-producing ability of 14 strains of different fungi(including 4unidentified)with the potential of producing beta-glucosidase deposited in the laboratory,Aspergillus niger SK34.002 was identified as the strain with the highest enzyme productivity,and its extracellular BGL enzyme activity was 6.68 U/mL.The ITS sequence data from A.niger SK34.002 was submitted to the NCBI GenBank database under accession No.MH032751.BGL production was slower than the growth of A.niger SK34.002,and the enzyme was produced from the 3rd day.The maximum enzyme production period was about8 days.The produced enzyme solution was used for hydrolyzing P.cuspidatum powder,and the hydrolysis effect to polydatin was remarkable.Experiments showed that the strain had good genetic stability.The fermentation broth of A.niger SK34.002 was collected as a crude enzyme solution.The purified BGL was obtained by activated carbon decolorization,ammonium sulfate fractionation,HiTrap Q HP ion exchange chromatography and Superdex200 10/300 GL gel filtration chromatography.The specific enzyme activity was 76.4 U/mg,and the purification ratio was 45 times.The purity and molecular weight of the enzyme were determined.The results indicated that BGL is a single subunit enzyme with molecular weight of 128 kDa.Through the investigation of the enzymatic properties of BGL,the optimum pH of BGL was 4.5,and the enzyme activity was almost lost when the pH exceeded 7.0.After 24 hours in citric acid buffer of pH 3.0~4.5,the enzyme activity still remained above 90%.The enzyme exhibited high activity between 60~70°C,and its optimum reaction temperature was 70°C,but it was unstable at this temperature.The enzyme kept high stability below 55°C.Owing to its excellent thermal stability and storage stability,it had obvious advantages in industrial production.Common metal ions and EDTA had no effect on enzyme activity,indicating that BGL was a non-metal dependent enzyme.The substrate specificity study of the pure enzyme revealed that the enzyme could hydrolyze all of the selected 11 substrates.Three of the best-performings were cellobiose(209.9 U/mg protein),4-nitrophenyl-?-D-glucoside(pNPG,173.1 U/mg protein)and polydatin(75.9 U/mg protein).The inhibitory effect of glucose on BGL was studied.The results indicated that glucose showed competitive inhibition to BGL when pNPG was used as substrate,and the inhibition constant K_i was 3.8 mM.The pure enzyme was analyzed by SDS-PAGE.After in-gel digestion,the peptide spectrum and amino acid information of each peptide segment were analyzed by MALDI-TOF/TOF-MS.The MS data were used to query the Mascot protemics database,resulting in one significant match,beta-glucosidase[Aspergillus niger],with a Mascot score of 183 and the amino acid coverage of 5%.BGL was identified as a beta-glucosidase,but only two effective matching peptides suggested that the enzyme might be a new protein.The enzyme was used to enzymatically hydrolyze polydatin in P.cuspidatum powder.The optimum conditions of enzymatic hydrolysis were obtained by single factor experiments:?-glucosidase dosage 2.7 U/(g P.cuspidatum),pH 4.5,60°C for 2.5 h.Under these conditions,the conversion rate of polydatin was over 98%,and the content of resveratrol was 6.2 times higher than that without enzymatic hydrolysis.With 21%ethanol(w/w)/22%ammonium sulfate(w/w)aqueous two-phase system as solvent,resveratrol was extracted directly from the hydrolysate of P.cuspidatum by ultrasonic-assisted aqueous two-phase extraction(UAATPE).Resveratrol was purified by polyamide column chromatography.The purity of resveratrol was 80.8%and the yield was 73.6%.Six resveratrol esters were synthesized from resveratrol and organic acids in two steps:resveratrol triacetylsalicylate,resveratrol tricinnamate,linolenic acid-3-resveratrol ester,linolenic acid-4'-resveratrol ester,resveratrol triarachidonic acid esters and resveratrol triDHA esters.The structures of these compounds were identified by MS,IR,1H NMR and 13C NMR.