Molecular cloning, expression and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Abstract

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, cbm3 (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.