LAUSR

Background: GABA (γ-aminobutyric acid) is a four-carbon non-protein amino acid and has hypotensive, diuretic, and tranquilizing effects. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple, economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-induced promoter, purified and immobilized in a fusion form and its reusability was investigated. Results : The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB- gadB , which contained promoter P rpoS , cbm 3 (family 3 carbohydrate-binding module from Clostridium thermocellum ) coding sequence, gene gadB coding for glutamate decarboxylase from L. fermentum YS2 and T7 terminator. After one-step purification of CBM-GAD using RAC (regenerated amorphous cellulose) as adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa, and the specific activity of purified fusion protein CBM-GAD reached 83.6 ± 0.7 U mg -1 . After adsorption onto RAC, the immobilized GAD with CBM3 tag was used repeatedly for GABA synthesis. The protein binding capacity of RAC was calculated to be 174 ± 8 mg g -1 . The immobilized GAD with CBM3 tag could catalyze GABA synthesis in repeated reactions, and 8% of initial activities was retained after 10 uses. The test of conversion of MSG to GABA by the immobilized enzyme demonstrated that the yield reached 5.15 g/L and the productivity achieved 3.09 g/L·h. Conclusions : RAC could be used as adsorbent in one-step purification and immobilization of CBM-GAD and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif;}