The Study On Enzyme Characterization Of Glutamate Decarboxylase From Bacillus Megaterium

Glutamate decarboxylase which can convert sodium glutamate or glutamic acid into y-aminobutyric is the key enzyme for biological fermentation production of y-aminobutyric.y-aminobutyric with high value in the industrial biotechnology has been used in various foods and pharmaceutical products.However,many GAD enzymes derived from microorganisms are only active in acidic conditions and their activities will decrease significantly at neutral environment,which make them difficult for industrial applications.Therefore,it is very important to improve their industrial application by elevating the activity of glutamate decarboxylasdecarboxylase e in neutral pH conditions.The Bmgad was amplified from Bacillus megaterium CICC 10055 and expressed in E.coli BL21(DE3)by pET21b.After sequencing,the Bmgad was certified to be encoded by 1404 bp,and the recombinant BmGad was approximately 53 kDa.The BmGad was purified by homogeneity and the enzymatic properties was determined.The enzyme had a maximum specific activity at 50? and was stable at 30?.The optimum pH value for BmGad activity was 5,and it was stable at neutral pH.When recombinant BmGad was incubated with 2 mM of different metal ions,Zn2+,Fe2+,Fe3+,and Cu2+produced different degrees of reducing on the BmGad activity,but no metal ions could significantly promote the BmGad activity.The presence of PLP promoted the activity of BmGad in a dose dependent manner.The kinetic parameters of recombinant BmGad was measured at 50?in pH 5 buffer,the Km and Vmax vaule of the enzyme was 8.1±0.5 mM and 149.3± 4.7 U/mg,respectively.Compared with glutamate decarboxylases ever reported,the BmGad had higher activity at pH 5.0-6.5.In order to enhance the activity of BmGad at neutral pH,seven amino acids sites were chosen for site-directed mutagenesis.Compared with wild-type,mutants of BmGadGlu294Arg and BmGadHis467Ala had higher activity at pH 5.0-6.5,their Vm were respectively 210.8±6.9 U/mg and 179.8± 4.1 U/mg at 50?-in pH 5 buffer.The conversion capability of recombinant enzyme was certified by whole cell transformation,befor which the optimal express conditions of BmGad were engineered.The final express conditions of recombinant cell were cultured at 25? for 6 h after adding 0.4 mM IPTG when the cell were cultured for 2h.The y-aminobutyric was 347.9 g/L at 12 h when the 500 g/L L-glutamate was added and the mole conversion rate had reached 99.4%,through fed-batch cultivation in pH 7 buffer at 37?.