Study On The Biosynthesis Of ?-Aminobutyric Acid By Lactobacillus Brevis

?-Aminobutyric acid?GABA?is a non-protein amino acid distributed in mammals,plants and microorganisms.It has several physiological protperties such as reducing blood pressure,accommodating hormone secretion,and controlling asthma.It is widely used in chemical,food,pharmaceutical,agricultural and other industries.In this paper,the screening and identification of GABA-producing strains,optimization of fermentation medium and culture conditions,establishment of fermentation process and whole-cell catalytic synthesis of GABA were carried out.The main results are as follows:1.More than 90 strains were isolated from samples including pickled Chinese cabbage,goat's milk,yogurt,pickled mustard tuber and kimchi.The strain DLF-19076 with high-yield of GABA production was screened from pickled mustard,and GABA was analyzed by paper chromatography and high performance liquid chromatography to determine the yield of 4.23g/L.The morphological characteristics,physiological and biochemical test and 16S rDNA sequencing analysis of the strain showed that the homology of 16S rDNA sequences of strain DLF-19076 and Lactobacillus brevis KLDS 1.0726 was as high as 99%,which was consistent with the physiological and biochemical test results.This strain was identified as belonging to Lactobacillus brevis,named Lactobacillus brevis DLF-19076.2.The fermentation medium components and culture conditions were optimized,and the results were as follows:carbon source was 30 g/L glucose,nitrogen source was 20 g/L cottonseed meal powder,substrate was 60 g/L,MgSO₄?7H₂O was 0.4 g/L,MnSO₄?H₂O was0.2 g/L,temperature was 35°C,initial pH was 5.0,and inoculum amount was 6%.After 72 h of culture,GABA yield was 30.34 g/L,which was 6.17 times higher than that before optimization.A high-efficiency synthetic GABA fermentation process was established.After fermentation for 72 h,L-MSG and glucose were almost exhausted.GABA yield was 44.73g/L,and molar conversion was 91.67%.Subsequently,constant pH fed-batch fermentation was carried out,and final molar conversion rate reached 81.74%,production rate was up to1.05 g/?L?h?,GABA concentration was 64.81 g/L,compared to uncontrolled pH and constant pH 5.0,the yield increased by 47.43%and 113.61%,respectively.The cheap cottonseed meal powder was used as nitrogen source to produce GABA,which could reduce the fermentation cost,increase the added value of cottonseed meal powder,and realize the comprehensive utilization of agricultural by-products.3.The whole cell catalyzed synthesis of GABA was optimized,and the results were as follows:Buffer system was 0.2 M citric acid-Na₂HPO₄,temperature was 35?,pH was 4.5,PLP concentration was 10?M,cell concentration was 100 g/L?wet weight?,and substrate concentration was 60 g/L.The effects of different cell permeabilization treatments on the catalytic synthesis of GABA were investigated,and the optimal treatment condition was determined by sonification at 40 KHz power for 40 min.After 7 h catalytic reaction,GABA yield was 44.78 g/L,molar conversion was 91.80%,and production intensity was up to 13.65g/?L?h?.The whole cell fed-batch conversion experiment showed that after three feedings,GABA yield in the reaction system was 85.71 g/L,molar conversion was 87.84%,and the maximum production intensity was 10.96 g/?L?h?.Compared with batch-catalyzed,the yield increased by 91.40%.