Cloning, Expression And Enzymatic Characterization Of Glutamate Decarboxylase

Glutamate decarboxylase (GAD) is a main enzyme for biotransformation of L-glutamic acid (L-glu) to gamma-aminobutyric acid (GABA). GABA, an important inhibitory neurotransmitter, has a variety of important physiological functions and can be widely used in healthy foods.Primers for PCR were designed according to the nucleotide sequence of GAD of Lactococcus lactis subsp. lactis IL1403. The target gene was amplified by PCR. Then the target gene were cloned into a vector pET-22b(+). The recombinant GAD gene (pET22b-ll-gad) was expressed in a host cell E. coli BL21(DE3) by inducing by IPTG, then purified by affinity chromatography. An approximately 54 kDa band was found on the SDS-PAGE. The results showed that the recombinant GAD was expressed correctly and efficiently.The purified recombinant GAD was studied in this thesis. The optimal reaction pH and temperature were 4.7 and 50 oC respectively; The enzyme was stable under 50 oC for 5 hours. The enzymes activity could be significantly inhibited by Cu2+. The Km, Vmax and kcat/Km of recombinant GAD were found to be 4.15 mM, 62.9 U/mg and 818 (min·mM)-1 respectively. The production of GABA by engineering bacteria with recombinant GAD was studied, including culture medium, inducing conditions and cell conversion. The optimum culture were found to be 5 g/L glucose, 20 g/L tryptone, 1 g/L K2HPO4·3H2O and 1g/L MgSO4·7H2O. The optimum inducing conditions were found to be cell OD600 1.0, temperature 25 oC, time 7 hours and lactose 0.5 g/L. After the fed-batch reaction, 0.31 g GABA was produced by the cell of 15 mL of fermentated liquor.The proportional relation between of H+ exhausted and GABA producted during the process of cell conversion was studied, the mole ratio was found to be 2:1, which provided a reasonable basis for rapid determination of GABA during the process of cell conversion.