Construction Of The Gamma-aminobutyric Acid Producing Strain By Whole-cell Catalyzing And Its Fermentation

Γ-amino butyric acid(GABA)is an endogenous non-protein amino acids in vivo,which has important physiological functions and is widely used in medicine,health products,food and feed industries.This paper constructed a recombinant strain that efficiently catalyzing glutamic acid to y-amino butyric acid,optimized its whole-cell catalyzing system and discussed the low temperature crystallization process of the conversion fluid.Firstly,we build a high-glutamic acid decarboxylation dynamic recombinant strain.Constructing engineered strain of E.coli by cloning glutamic acid decarboxylase gene of different lactic acid and using different copy number expression plasmid.According to the whole-cell catalytic efficiency,we chose E.coli BW25113/PDB1S-LigadB whose gadB come from Lactococcus lactis and expression vector is moderate copy PDB1S.The GABA yield reached 77.32 g/L when this strain reacted in 1 M Glu solution for 5 hours under the temperature of 37 ℃.Secondly,we established an efficient whole-cell catalyzing system.Through single factor experiment we optimized the culture conditions and conversion system and determined that catalytic efficiency is highest when using 10 OD cells which cultivated for 16 hours under temperature of 30 ℃ reacted in 2 M GLU solution.GABA production reached 203.47 g/L after reacting for 6 hours;the conversion efficiency was 4.23 g/L/h/OD.Cell reuse test showed that the recombinant strain can be reused for at least three times.GABA yield of each batch reached 519.27 g/L,and cell catalytic efficiency was 8.87 g/OD.In addition,the medium component optimization experiments showed that adding some VB6,GABA conversion efficiency increased to 62.59 g/L/h.Finally,we discussed the low temperature crystallization process of conversion liquid.The decolorization process optimization results were analyzed by partial least squares quadratic polynomial regression analysis.By adding 2.05%of activated carbon deal with 29.4 min at 40 ℃ is the optimal conditions for decolorization.The decolorization rate was 95.71%,and the GABA retention rates were 90.09%.GABA lost about 3%after filtering with membrane(0.45 p,m).Through concentrating and crystallizing conversion liquid at low temperature(4 ℃)for three times,the eventual crystals yield was 87.88%,the crystal purity was 98.44%and the total GABA yield was 86.1%.