Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic,
and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and
economical method of preparing and immobilizing GADwould be helpful for GABA production. In this study, the
GAD from
Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was
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
PrpoS,
cbm3 (family 3 carbohydrate-binding module from
Clostridium thermocellum
) coding
sequence, the
gadB gene from L.
fermentum YS2 coding for GAD, and the T7 terminator. After a one-step
purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis
revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ±
0.7 U·mg
-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA
synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g
-1. The immobilized CBM-GAD could
repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the
conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the
productivity reached 3.09 g/L·h.
Conclusions: RAC could be used as an 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.
