Study On Production And Purification Of γ-aminobutyric Acid By Biotransformation Using Rice Bran Endogenous Enzyme

Subject, Specialty: Cereals, Oils and Plant Protein Engineer Time of enrollment: 01-09-2008 Master graduate student: Nana Guan Time of oral defense:14-11-2010 Faculty adviser: Hui Zhang, Prof. Time of conferring degree:γ-Amino butyrate(GABA)is a kind of important central nervous system inhibitor, which has multiple functions such as tranquilize mind, enhance memory, lower blood pressure, treat epilepsy and adjust hormone secretion. In rice bran, it contains glutamic acid decarboxylation enzyme (GAD) which can catalyze the decarboxylation of glutanic acid to enrich GABA. In this paper, glutamic acid decarboxylation enzyme (GAD) was used to catalyze the decarboxylation of glutamic acid to enrich GABA was studied. The process of directly enrichment of GABA was studied first. Optimal technological conditions were obtained through the single factor and the orthogonal test,and conditions obtained were: 0.02mol/L Na2HPO4- citric acid buffer (pH5.6) was added, control the ratio of material to liquid to 1∶8, the concentration of substrate glutamic acid was 0.04mol/L, react for 14h in the water bath of 40℃.In this condition, the purification of GABA enriched was 10.50%.Ion exchange was carried out on enrichment fluid after ultra filtration to extract the GABA in it. First Ultra filtration process was carried out on rice bran enrichment. GABA recovery ratio and interception rate of macro-molecule protein and carbohydrate was chose as targets to select the optimal conditions .with the use of MWCO 10KDa ultra filter film, The purity of GABA in ultimate ultra filter penetrative fluid increased to 17.33% from the original of 10.50%. Then Various factors that influence ion exchange capacity were investigated, and then D001 pocket strong acid cation exchange resin was chose to carry on the experiment finally. The optimum technology conditions were as follows: adjust the loading fluid to pH2.0, load at the concentration of 2mg/mL, after absorbed completely, 2mol/L ammonia water was used to elute it. In this condition, the concentration of GABA in the solution is 79.27% and 82.28%GABA can be recovered.Reverse micelle extraction was used to extract the GABA in ultra filtration penetrative fluid. Two-film theory was used to characterize GABA mass transfer coefficient in reverse-micellar and reverse-micellar dynamic experiments were carried out on different factors in order to determine the control type of mass transfer in reversed micellar extraction. The experiment of forward- and backward-extraction of reversed micellar were optimized, the optimal conditions of forward-extraction were as follows: concentration of AOT is 50mmol/L,concentration of GABA is 2.5mg/mL,pH 2.0,concentration of NaCl is 100mmol/L,temperature is 25℃, extraction time is 20min. The optimal conditions of backward-extraction were as follows: temperature is 55℃, extracting time for 30min, and the pH of aqueous phase is 9.0. Under this condition, we can get the concentration of GABA was 80.95%,and recover yield of GABA was 64.49%..