|
type |
Journal Article |
authors |
Lin Q, Li D, Qin H |
title |
Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2 |
journal |
Electronic J Biotechnol |
Activity |
4.1.1.15 |
Family |
4.1.1.15.b |
sel |
selected |
ui |
nOtInNC-BI |
year |
(2017) |
volume |
27 |
pages |
8-13 |
| |
keywords |
doi 10.1016/j.ejbt.2017.03.002 |
abstract |
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 ±
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 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. |
last changed |
2018/05/14 11:45 |
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