Immobilization Of β-Glucosidase And Its Application In Production Of Genipin

Genipin is the aglycone of geniposide,which presents in the fruit of Gardenia jasminoides Ellis or Eucommia ulmoides Oliver.This iridoid compound has various pharmacological effects,such as cholagogue, anti-inflammatory,liver protection and anticancer.Genipin can be prepared by hydrolyzing geniposide withβ-glucosidase,β-glucosidase is a biocatalyst with poor stability,activity and availability.Therefore,it has to be immobilized to increase its service life.The purpose of this article is to prepare genipin byβ-glucosidase immobilized with sodium alginate, which has little toxic and low price.The main content of this dissertation is as follows:1、Study of the preparation conditions of immobilized enzymeUsing sodium alginate as the carrier and glutaraldehyde as the cross-linking agent,the best immobilization method of theβ-glucosidase has been studied by single factor experiments.Moreover,the optical immobilization condition has been improved by orthogonal experiment with the four most influence factors.In the end,the best immobilization method is cross-linking-entrapment-cross-linking.In first step,200mgβ-glucosidase and 3%sodium alginate are cross-linked in 2.5% glutaraldehyde solution at 22℃for 2h.Second,it is solidified at 30℃for 1h in 4%CaCl₂ solution.At last,it is cross-linked again at 4℃for 2h in 0.1%glutaraldehyde solution.Enzyme activity and fixed ratio reach to 38.68U/g and 70.48%,respectively.2、Study of the zymologic qualities of immobilized and free enzymes.The optimum pH of free and immobilized enzymes is 5.0 and 4.0. Acid stability is increased by immobilizing.Optimum temperature of free and immobilized enzymes is 55℃and 60℃,respectively.The thermal stability of the immobilized enzyme has changed,that is,the K_m of immobilized enzyme is 4.118mmol/L while the K_m of free enzyme is 0.153mmol/L.The affinity of immobilized enzyme with substrate is lower than that of the free enzyme.The activity of immobilized enzyme reaches to the top after using 6 times and decreases 30%after using 15 times, which has achieved good operation stability.Storing stability of immobilized enzyme is also improved.3、Study of the hydrolysis conditions of immobilized enzyme by continuous reactorThe optimum condition of immobilized enzyme by continuous reactor is:the proportion of immobilized enzyme and substrate 40:1, concentration of substrate 10mg/ml,at 55-60℃for 7-9h,and buffer solution 0.2M pH4.0 acetic acid.4、Study of the hydrolysis conditions of immobilized enzyme by successive reactorThe optimum condition of successive reaction by immobilized enzymes is:concentration of substrate 10mg/ml,temperature 50℃, reaction time 3h,the flow rate of peristaltic pump 30ml/min.The yield under the optimum condition is 60.7%.5、Separation,purification and structure identification of genipinAfter purifying by macroporous resins（D101）,silica gel column chromatography and recrystallization,the purity of the product is above 99%.Meantime,the obtained product is proved to be genipin according to LC-MS and ¹H-NMR.