Study On Glutamate Decarboxylase Of Bacillus Megaterium X-1 And Conversion Of γ-aminobutyric Acid

γ-aminobutyric acid(GABA)is a natural non-protein amino acid,which is widely distributed in animals,plants and microorganisms.GABA is an effective inhibititory neurotransmitter in the animals’central nervous system.It can act as the anticonvulsant and antiepileptic agents.It can also play a role in terminal sedation,blood pressure regulation,the vitality enhancement of brain,liverand kidney protection,nerve cell nourishing,and secretion promotion of growth hormone.Furthermore,GABA also plays a role in hormone regulation or the functions of the trophic factor in non-neural tissues,which means it has an important regulatory role in the animals’ normal physiological function.Therefore,it has a tremendous prospect and potential in the application of medicine and food industry Nowadays,GABA has been approved as a new resource of food additive,being added inbaking foods,dairy products,beverages and other products.Glutamic acid decarboxylase(GAD)is the key enzyme in the biosynthesis of GABA.It can catalyze L-glutamic acid and produce GABA through decarboxylation reaction.GAD can be found in both lowand high organisms.Production of GABA through microbial fermentation has several advantages.To be exact,microorganism can grow easily and fast without many constraints.Compared with plants enrichment and chemical synthesis to produce GABA,the cost of microbial fermentation is lower.As a result,microbial fermentation has a broad application prospects in terms of GABA production.At present,many microorganisms can produce GABA,including Lactic acid,Escherichia coli and monascus.However,GAD in these microorganisms are all endoenzymes,which are difficult for extraction and purification.Therefore,it cannot be easily applied in industry.The bacillus strainis a novel strain for medicinal compound production,which has been first reported to produce GABA in 1971.However,the fundamental knowledge about the production of GABA using bacillus strain is still limited.As a result,this paper was mainly focused on screening a bacillus strain producing GABA.The fermentation condition was optimized and the cloning and expression of GAD gene were also studied.All these results can provide guidance for producing GABA by mean of microbial fermentation in practical application.The results are shown as follows:1.Screening and identification of γ-aminobutyric acid producing strains.A strain producing GABA from Ecological Gardensoil Of Nantong,Jiangsu Province,was selected and named as X-1.This strain can produce 4.46 mmol/L GABA before optimization.Then it was preliminary identified as Bacillus megaterium by morphological,physiological and biochemical tests.After that,16S rDNA gene sequence and analysis were carried out.A similarity search was performed using Genbank databases and neighbor-joining phylogenetic tree was constructed.A typical characterization of the Bacillus megaterium 16S rDNA sequences(GenBank accession number NR074290)comparison study supported a strong relationship between strain X-1 and members of genus Bacillus.Highest homology(99%)with Bacillus megaterium was obtained..The result further indicated that strain X-1 belonged to Bacillus megaterium.2.Optimization of fermentation conditions of B.megaterium X-1 to product GABA.Fermentation conditions of B.megaterium X-1 were optimized.The results showed that the glucose,peptone,K2HPO4 and pyrodoxal-5’-phosphate(PLP)were the key facters.According to the orthogonal experiment,the optimal fermentation medium composition was obtained,namely glucose 30.0 g/L,peptone 30.0 g/L,K2HPO4 0.6 g/L,PLP 0.3 g/L,L-MSG 10.0 g/L,NaN03 3.0 g/L,MgS04 7H20 0.5 g/L,FeSO4 7H2O 0.01 g/L.Meanwhile,in the single factor experiment,it was found that inoculum,pH,fermental temperature and speeding were the key facters.After that,optimal fermentation conditions were obtained through the Taguchi experiment,namely inoculation 2%,pH 6.0,fermentation temperature 30℃,speeding 180 r/min.Based on repeated experiments,the final GABA concentration was 22.07±1.03 mmol/L,which was 3.94 times higher than that prior to optimization.3,Cloning and expression of B megaterium X-1 GAD.The gadB gene was amplified from B.megaterium X-1 genomic DNA by PCR.The PCR production was isolated and ligated into the pMD19-T vector and sequenced.The vector constructed above was pMD-gadB.The gadB gene was cloned into pET-30a and integrated in to E.coli BL21(DE3).Active recombinant GAD enzyme had been successfully expressed in Escherichia coli as protein expression with pET-30a by addition of IPTG.The activity of recombinant enzyme was 10.13 U/mL.The optimal expression conditions were shown as follows::inoculation 3%,IPTG 0.2 mM,temperature 55℃ and induction time 12 h.Under these conditions,the enzyme activity reached the highest,being 15.12 U/mL.In addition,the recombinant enzyme was isolated and purified by Ni affinity column.SDS-PAGE showed that the molecular weight of the enzyme was about 53 kDa.Through studying the properties of the recombinant enzyme,it was found that the optimum temperature and pH of the recombinant enzyme was 45℃ and 3.5,respectively.The enzyme was stable in the temperature range of 45-55℃ and pH 3.0-4.5.The Km of the enzyme was 13.10 mM and Vmax was 201.65 U/mg4.Optimization of recombinant Escherichia coli cell to preparate GABA.The optimal conditions of recombinant Escherichia coli to produce GABA was obtained by studying the effects of temperature,pH,surfactant and growth factor,metal ion and substrate concentration on GABA yield.When the temperature was 55℃,pH was 3.5,PLP was 0.02 mM and L-MSG was 125 mmol/L,the concentration of GABA produced by recombinant Escherichia coli reached 22.01±1.26 g/g·h.