Screening Of Lacotbacillus High-efficiency Producing γ-aminobutyric Acid From L-glutamate Acid And Optimization Of Biotransformation Conditions

Gamma-aminobutyric acid (GABA), serves as a major inhibitory neurotransmitter in mammalian nervous systems. GABA has several positive physiological functions to human health Therefore, it can be widely used as a new functional factor.This dissertation includes breeding of GABA-producing mutant strain, the optimization of culture and transformation conditions in shake flask and fermenter. The main research content and results were as follows:First, a GABA-yielding strain was isolated from samples. Morphology analysis, physiological and biochemical properties and phylogenetic analysis based on the 16S rDNA sequence indicated that the strain belonged to the genus Lactobacillus which was a variety of Lactobacillus plantarum strain EW-p with similarities of 99%. The strain was named as (Lactobacillus plantarum) LP-GB 01. Applying UVltraviolet light to mutagenize LP-GB 01, a mutant strain LP-GB 01-21 was selectively bred by GABA resistance selecion. After 24 h transformation, the mutant strain gave a GABA output of 51.9 g/L,55.39% higher than that of the parent strain. The mutant strain is kept China Center for Type CultureCollection(CCTCC) as Lactobacillus plantarum CCTCC M 209102.Second, optimize culture conditions of LP-GB 01 in shake flask. The optimal medium composition in shake flask was determined as follows (g/L):glucose 10, peptone 15, yeast extract 10, Sodium succinate 4, MgSO4 0.75, K2HPO4 3, L-MSG 3. The culture should be performed at 30℃, pH 7.0, rotational speed 250 r/min and strain age is 36 h. Compared with the original culture conditions, under optimal culture conditions lactic acid bacteria biomass increased by 25.15%, GABA production rate reached 88.3%,135% higher than that of the original culture conditions.Third, conditions affecting transformation of GABA were studied. The technical conditions of biotransformation of GABA by LP-GB 01-21 were obtained, including appropriate temperature(35℃), pH of buffer(4.8), concentration of buffer(0.2 mol/L), rotational speed (150 r/min). Compared with the original conditions, lactic acid bacteria under optimal conditions has a increase in biomass and GABA content also has a substantial increase, reached53.1 g/L,140% higher than that of the original conditions.Fourth, the effects of dissolved oxygen conditions on GABA transformation by LP-GB 01-21 in 5 L fermentor were investigated. The results showed that high dissolved oxygen conditions are conducive to microbial growth and low dissolved oxygen conditions are conducive to generating. The fed-batch transformation of GABA was preliminarily studied also. After three times L-Glu addition, the yield of GABA reached 143.55 g/L at 80 h, Conversion rate was 97.56%. Also found that pH of transformation system had a greatr impact to transform into GABA, according to flask experiments to determine the optimum buffer pH 4.8 to 5.0, therefore, On the basis of earlier experiments,we have a test under controling the conditions of pH 5. experiment results show that the conversion rate of GABA is as high as 99.13% at 70 h, indicating that controling pH of transformation system can shorten reaction time and improve conversion efficiency. strain LP-GB 01-21 and GABA production process have been applied for national invention patent, which has been transferred to certain manufacturing enterprises in Shandong Province. Now, this project has been completedton-fermentation tank test, and expected to complete the set of industrial tequipment of microbial synthesis GABA.