Processing Of Glutamic Acid Decarboxylase Conversing Of γ-Aminobutyric Acid

The purpose of this study is to build glutamate decarboxylase engineered bacteria using recombinant DNA technology, to study the reorganization of glutamate decarboxylase into the preparation of y-aminobutyric acid process. In this paper, determination of y-aminobutyric acid induced conditions, inducing conditions, culture conditions, the enzymatic conversion conditions and the separation and purification, has done the appropriate research, and results as following:1. Cloning the gene of glutamate decarboxylase, identification the nucleotide sequence; build of the expression vector then induction and expression in E. coli.In this study, preliminary screening of almost20strains of genetically engineered bacteria, early-five strains and the initial measured activity of three higher B3C1, B3C3, BI2.2. Establish a suitable method for the determination of the enzymatic synthesis of y-aminobutyric acid, the use of orthogonal experiments, draw a standard curve.(1) Determination method:Substrate (0.2mol/L acetic acid sodium acetate buffer) pH6.0, the dilution factor of70times, boric acid buffer solution plus1.0ml, phenol plus0.5ml, sodium hypochlorite amount of5.0ml.(2) Standard curve:A=1.2447C+0.0149,Correlation coefficient: R2=0.9924.3. To optimize the induction conditions and culture conditions, determine the maximum amount of medium of producing bacteria composition:(1) The optimal conditions were:inducer isopropyl-β-D-galactose thiol (IPTG) to add the best time for four hours after inoculation, the concentration is0.8mmol/L, induction time of four hours and temperature of35℃.(2) The optimal culture conditions were:the yeast extract1.01%,0.6%sodium chloride,1.5%peptone, incubation time11hours15minutes, shaking speed250r/min, the initial pH value of6.5, inoculum size of3.0%, liquid volume to11.70ml.In accordance with the optimal conditions for fermentation culture, three sets of activity data measured the average of1227.33μmol/(5ml·h), which was increased by12.29%compared with the original1093.00 μmol/(5ml·h).4. Response surface methodology to optimize the conversion of glutamic acid decarboxylase enzyme reaction conditions, the SAS software analysis results are as follows:Enzymatic conversion conditions are0.290mol/L、pH4.99acetic acid-sodium acetate buffer system as the substrate, PLP addition of1.0mmol/L,60min transformation time,6.00mL enzyme added amount and52.10℃transformation temperature. The activity reached1461.84μmol/(5ml·h) by using the optimal culture, which was increased by19.11%compared with the original1227.33μmol/(5ml·h).And the test for capacity of B3C1cell transformation was used to calculate the y-aminobutyric acid molar yield of85.27%5. Preliminary exploration of the y-aminobutyric acid separationand and purification process route2%active carbon(2%of the γ-aminobutyric acid crude solution volume)was added at first, then concentrated under vacuum was used, concentrated volume was1/5of the original system.85%ethanol was added and the addition of5:4(concentrated volume:85%ethanol). Filtered while hot and waited to the room temperature, then cooling and crystallization in the0～4℃in an ice bath.Finally we got the crystal and the shape was slender cylindrical.The purity was calculated to86.00%and the final total yield of75.87%.