Studied On γ-Amino-Butyric Acid (GABA)Synthesis By Microbial Fermentation

Gamma-aminobutyric acid (GABA) is widely existed in animal and plant, because it has a variety of physiological functions, and therefore is widely used as an additive in food,feed and other areas. Screened a high-yielding Lactobacillus strain to producing GABA directly is a hotspot research for many researchers in recent years,a high-yielding strain was isolated from the wastewater of tofu sample on this study., and its identification, domesticated, optimize training and further study of the glutamic acid decarboxylase (GAD) gene, the specific test results are as follows:(1) After screening and rescreening, a high-yielding strain producing GABA was isolated from the wastewater of tofu sample. After morphology analysis, physiological and biochemical properties and with all16SrDNA sequences in GenBank indicated that the strain belong to Lactobacillus genus.Then, the result of phylogenic tree of the strain which was generated by MEGA6.0software indicated that the strain which was a variety of Lactobacillus Plantarum with similarities of99%. Then it was numbered Lp-Dfa301.Finalized in the TYG fermentation broth GABA production could reach5.833g/L.(2) With the best mutagenesis conditions by test to treat the original strain:UV irradiation90s; the content of DES was25%and the dealing time was20min,After several times of mutagenesis, a mutant strain named Lp-Dfa301-49was bred by GABA resistance selection. After passage the genetic was stability, the average yield of GABA was6.899g/L, which had been improved18.28%than the wild-type strain.(3) Based on the single factor and orthogonal experiment to optimize the culture medium with Lp-Dfa301-49as the test strains, the optimized fermentation conditions were as follows:substrate concentration of20.00g/L;glucose dosage11.00g/L, sodium succinate dosage5.00g/L, yeast extract dosage18.00g/L, tryptone dosage14.00g/L;10.OOg/L of P3+,Mn2+,K+,Mg2+,Ca2+add volume were0.30,0.35,0.40,0.60,0.80mL/20mL(broth); then optimize the culture conditions on the basis of single factor test by response surface methodology, the best culture conditions were:initial pH5.90, inoculum concentration2.70%, culture time71.00h. In the optimized medium, the yield of GABA can be reached12.863g/L and increased by86.45%than that of the pre-optimization conditions. (4) The paper also explored and optimized the fermentation conditions of GABA by single factor and response surface experiments with the raw materials of agricultural, and got a set of optimum combinations of conditions as follows:Corn saccharification liquid addition of5.75mL/100mL (fermentation broth), corn flour dosage19.90g/L, soybean powder dosage35.20g/L, the yield of the fermentation broth GABA was3.371g/L. This test laid the foundation for developed of GABA-rich fermented foods, beneficial the resources recovery and reuse.(5) The last, glutamic acid decarboxylase (GAD) gene of Lp-Dfa301-49were test and verify by the technology-specific of PCR amplification and Bioinformatics analysis, the Exploring-Local Vector NTI Database software had been used for stitching, homology analysis and translation. The physical and chemical properties,transmembrance region,signal peptide,cellular localization,protein structure of ipgadB gene were analyzed and predicted by using bioinformatics methods. Results showed that:the IpgadB gene protein is hydrophilic and stable protein; does not exist signal peptide and transmembrane region or belong to secreted proteins; there are two N-sugar glycosylation sites; belong to the AAT protein superfamily (fold type I) pyridoxal phosphate (PLP)-dependent enzyme: protein tertiary structure analysis shows that in order to3fz6.1.A,1pmm.1.A, lxey.1.A,2dgk.1.A.3hbx.1.A as a template for modeling, coverage can be more than92%, similar to the consistency of more than42.00%.