Biotransformation Of Geniposide In Gardenia Jasminoides By Trichoderma Harzianum And Separation Of The Target Product

Genipin is a kind of iridoids, which was used for the treatment of inflammation, jaundice, lipid peroxidation because of its obvous effects. Genipin is also used as a kind of excellent natural biological crosslinking agent and widely used in the field of biological materials. The content of genipin in gardenia fruits is very low(about0.005%-0.01%), while geniposide presents richly(about3%-8%). At present, β-glucosidases are usually used to transform geniposide to produce genipin. Geniposide were extracted from gardenia fruits firstly, and then hydro lyzed by immobilized β-glucosidases. However, the cost of the method is high and the technology is complicated, but the yield of genipin is not necessary high. It’s well known that biotransformation of raw herb by microorganism directly has many advantages, such as high specificity, mild reaction conditions and clean production as well as low cost. In this paper, a microbial transformation method was developed to convert geniposide in DFA to genipin, and the separation of biotransformed products and the key enzyme were studied.Firstly, a specific strain with high activity of β-glucosidase named Trichoderma harzianum CGMCC2979was selected by preliminary and secondary screening. The transformation conditions were optimized for maximum genipin production. The optimum fermentation temperature was30℃, the optimum concentration of DFA in the medium was80g/L, the optimum pH was7.0, and the fermentation time was48h. Under the optimal conditions, the conversion rate of geniposide into genipin could reach97.7%. Fermentation in a5L bioreactor was carried out based on the shake flask cultivation, and the operation conditions were optimized. After40h, fermentation at30℃,250rpm and air flow rate of3.0vvm, the conversion rate of geniposide reached52%.Secondly, geniposide-glycosidase from T.harizinum was isolated and purified to homogeneity through DEAE sepharoseTM and Superdex200column chromatography. The enzyme hydrolyzed the β-glucoside of geniposide to produce genipin. It also showed P-nitrophenyl β-D-glucoside (pNPG) activity, but couldn’t hydrolyze the glycosides of steriodal saponins in Dioscorea zingiberensis including trillin. The molecular weight of the enzyme was estimated to be76kDa based on its mobility in SDS-PAGE and was further determined as74.4kDa based on MALDI/TOF. Using geniposide as substrate, the enzyme activity showed pH and temperature optima of4.0-5.0and50℃, respectively. The Km for geniposide was4.02mM and the Vmax was813μmol(h mg protein)-1.Thirdly, the technology for purificating of genipin from fermentation broths was studied. The aqueous two-phase system consisting of28%(w/w)1-butanol and15%(w/w) ammonium sulphate was selected because of its highest yield of genipin (92.4μg/g). Static adsorption experiments showed that the absorption rate of XAD16N is90.6%when the pH of broth was4.0, and the dynamic adsorptive capacities of genipin on macroporous resin was3. lmg/g. The elution condition of silica gel column chromatography was optimized for87%chloroform,17%methanol. After aqueous two-phase extraction (ATPE), macroporous resin and silica gel column chromatography, the purity of genipin reached98%, with recovery of57%. NMR and LC/Q-T of MS methods were used to identify the product.