||Lin Q, Li D, Qin H
||Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2
||Electronic J Biotechnol
||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 GAD would 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 ±
. 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
. The immobilized CBM-GAD c ould
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.