Study On Purification And Characterization Of Rice Bran GAD

γ-aminobutyric acid (GABA) is a non-protein amino acid with several important physiological functions such as neurotransmission, induction of hypotensive effects, and tranquilizes effects. GABA is produced primarily by the decarboxylation of L-glutamic acid (Glu) catalyzed by glutamate decarboxylase (GAD) [EC4.1.1.15]. Rice bran was an effective catalyst for GABA production with relative high GAD activities. In this paper, GAD and calmodulin (CaM) were extracted and purified from rice bran, and some researches on their structure and characters were taken. The purpose was to know something about the regulate mechanism of rice bran GAD and to give some help for GABA accumulation.First, a validated, selective, and sensitive chromatographic method for determination of GAD activity in plant tissues by determination of GABA was developed using a gradient system. Determination was obtained by pre-column derivation of the sample with 2,4-dinitrophenylhydrazine (FDNB) and separating the corresponding derivatives with RP-HPLC on a C18 column and UV detection (360nm). The calibration curve was linear over a range of 0.2-5 mg/mL GABA with a correlation coefficient of 0.9954. The precision of this method was high (RSD=0.061%). This method also possesses high recovery rate (>99%) and good stability in a month. We detected the GAD activity of several plants tissues and got a conclusion that rice bran had relatively high GAD activity, it is a good source of GAD activity for GABA production.Rice bran GAD was purified through chromotograpy method (DEAE-Sephrose FF,HW-55F and Superdex 200) and Native-PAGE method. Two isoforms of GAD: RbGADL1 and RbGADL3 were got from rice bran. The molecular weights studied by SDS-PAGE were: 73000and 47000 respectively. The molecular weights of RbGADL1 and RbGADL3 studied by mass spectrum were: 139113.97 and 46985.65. This indicated that RbGADL1 was composed by two subunits of about 70000, and RbGADL3 have no subunite. As the recoverate of RbGADL3 was much higher, RbGADL3 was chosen as main study object.RbGADL3 was identified by peptide mass fingerprinting (PMF) and peptide sequence tag (PSI). The peptide mass fingerprinting of RbGADL3 was quite similar with Os01g0926300. PSI-BLAST homology result indicated that RbGADL3 had relatively high homology with Os03g0113700 (Oryza sativa (japonica cultivar-group))and OsI₀9713 (Oryza sativa Indica Group). The secondary structure of RbGADL3 was studied by the circular dichroistic spectra, Raman spectra, Fourier transform infrared spectra, and bioinformatics prediction. The results were summarized asα-helix of 30%-45%,β-sheet of 10%-20%,β-turn of 10%-30% and random coil of 30%-60%. Then,"Homology Modeling"and"Fold Recognition"were taken to predict the third structure of RbGADL3.Rice bran CaM (RbCaM) was purified by Phenyl Sepharose CL-4B with the molecular weight of 17221.89 tested by mass spectrum. RbCaM was identified by PMF and PSI, and got the same protein: Os03g0699000, which was an storage protein from Oryza sativa (oleosin, japonica cultivar-group). The UV-visible spectra,fluorescence spectra and circular dichroistic spectra were taken to study the structure change of RbCaM with or without Ca2+. The secondary structure of RbCaM was summarized by the results of circular dichroistic spectra and bioinformatics prediction:α-helix of 30%-50%,β-sheet of 0%-10%,β-turn of 0%-30% and random coil of 27%-44%."Homology Modeling"study of RbCaM structure failed for no templet was found. But several fold regions were recognized by"Fold Recognition". And the prediction result of PredictProtein server showed that RbCaM appears as compact, as a globular domain and may have Protein kinase C phosphorylation site,N-myristoylation site and Oleosins signature in it.The enzymatic characters of RbGADL3 and the effect of RbCaM on RbGADL3 were also studied. The results showed that RbGADL3 was a kind of Ca2+ dependent CaM binding protein. The activity of RbGADL3 was regulated by Ca2+/RbCaM. 6μg RbCaM was needed to half-active 1mg of RbGADL3 at the Ca2+ concentration of 0.23mmol/L.The condition of GABA production by rice bran was optimized and got the output of 2.3g GABA/100g rice bran. Then, a method for continuousγ-aminobutyric acid (GABA) production using immobilized rice bran (IRB) was developed. Calcium alginate was chosen to entrap rice bran directly. The operation and storage stability were improved by immobilization of rice bran. And the GABA production was 12.95% higher than the free rice bran method after reaction time of 60 hours. The high output and purity of the product permit its biotechnological potential to be exploited in food.