The Expression Of Glutamic Acid Decarboxylase In E.coli BL21(DE3)and The Production Of ?-aminobutyric Acid By Immobilized Enzyme

?-aminobutyric acid(GABA)is a natural non-protein amino acid,which can be produced from glutamic acid catalyzing by the enzyme glutamic acid decarboxylase(GAD).GABA plays important physiological functions in reducing blood pressure,curing epilepsy,strengthening the liver and kidney,controlling asthma,regulating hormone secretion,tranquilizing effect,and enhancing memory.Meanwhile,GABA is one of the recommended ideal ingredients in the green and organic food,and is the ideal food and medicine for the prevention of senile disease.Microorganism was utilized to produce ?-aminobutyric acid in this study,which was characterized by safety and low cost.First,the engineering strain E.coli was constructed based on the genetic engineering technology,which can produce the protein glutamic acid decarboxylase(GAD)efficiently.Second,the enzyme GAD was immobilized,and the immobilized GAD was used to ferment and produce GABA.The fermentation using the immobilized GAD can not only avoid the potential safety risk brought by the production utilizing the engineering strain,but also reduce the production cost through the repeated use of the immobilized enzyme.The gene gad was cloned from the strain Lactobacillus plantarum 22144 and E.coli AS1.505,respectively.Two engineered strains E.coli BL21(DE3)(pET-28a-22144-gad)and E.coli BL21(DE3)(pET-28a-AS1.505-gad)were constructed,and both of them can produce GAD protein,with the molecular weight of 53kDa,induced by IPTG.The,gad gene of L.plantarum 22144 and E.coli AS 1.505 shared an identity of 58%and the amino acid sequence similarity was lower at 45%.Compared with the strain E.coli BL21(DE3)(pET-28a-AS1.505-gad),the strain E.coli BL21(DE3)(pET-28a-22144-gad)had higher enzyme activity and was selected as the object for further study.To promote the soluble expression of the recombinant protein,secretory expression using the signal peptide,fused expression using the fusion tag,and co-expression with the molecular chaperone were used.Six recombinant strains were constructed,and they were E.coli BL21(DE3)(pET-22b-22144-gad);E.coli BL21(DE3)(pET-22b-22144-gad,?pelB);E.coli BL21(DE3)(pMAL-c2X-22144-gad);E.coli BL21(DE3)(pMAL-c2X-22144-gad(TGA));E.coliBL21(DE3)(pET-28a-SUMO-22144-gad)and E.coli BL21(DE3)(pACYCDuet-l-GroEL-ES,pET-28a-22144-gad),respectively.The soluble expression and the enzyme activity of the protein GAD were compared among these recombinant strains,hence the strain E.coli BL21(DE3)(pACYCDuet-1-GroEL·ES,pET-28a-22144-gad)was determined to produce the recombinant protein GAD with a conversion rate up to 95%.To further enhance the soluble expression of the exogenous protein GAD in the recombinant strain E.coli BL21(DE3)(pACYCDuet-1-GroEL·ES,pET-28a-22144-gad),the induction condition temperature and IPTG concentration were optimized.In order to promote the percentage of cell disruption and reduce its effect on the enzyme activity,the buffers used in the ultrasonication of cells were optimized.The optimized condition for the induction of protein expression was O.1mM IPTG,25?,140 rpm for 10h,and the buffer used was 0.5ml TGE for 5 OD bacteria.The crude GAD enzyme extracted from the broken cells was analyzed,and the optimal pH for GAD was 4.6,the optimal temperature was 37? and the enzyme activity was up to 1.86 U/mL.To immobilize the enzyme,the anion exchange resin,cationic exchange resin,epoxy resin and amino resin were utilized,and the results indicated that the amino resin LX-1000EA in pH 4.6 buffer was the best for the immobilization of the enzyme at about 50%of the enzyme recovery.Single factor experiment and Box-Benhnke experiment design were used to find the optimal conditions for the immobilization of glutamic acid decarboxylase,the immobilization time was 7.5 h,the temperature was 42°C,the ratio of the carrier and the enzyme was 1:7.5,and the enzyme activity was 4.22±0.1 U/g.The optimal pH of the immobilized GAD(IGAD)enzyme was 4.8 higher than the free enzyme,and the enzyme IGAD was more stable in pH 3-5.The optimal temperature of IGAD was the same with free enzyme which is about 37 ?,and the immobilized enzyme activity was up to about 4.4 U/g.But the thermostability was improved and the IGAD can retain 80%enzyme activity preserved in 45 ? for 8 h.The higher the temperature,the lower the enzyme activity,hence the enzyme IGAD should be preserved under the condition of partial acid at low temperature(4?).Finally,the IGAD was used to produce GABA in the 3L fermentation tank by adding the MSG in batch,the GABA yield was 117 g/L and the conversion rate was 97%after 72 h transformation.After the process of separation and purification,it can meet the requirements of industrial production.