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

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 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.

Enhancement of gamma-aminobutyric acid in a fermented red seaweed beverage by starter culture Lactobacillus plantarum DW12

Lactobacillus plantarum DW12, a gamma-aminobutyric acid (GABA) producing strain, was used as a starter culture to produce a functional fermented red seaweed beverage (FSB). Optimal conditions for producing FSB were established using Central Composite Design by varying the amounts of monosodium glutamate (MSG), sucrose and the initial pH in MRS medium. After a verification test, 1 percent MSG, 6 percent sucrose and an initial pH of 6 were selected. Four treatments were tested: traditional formula (A), red seaweed-cane sugar-potable water = 3:1:10, w/w/v, initial pH 6; the traditional formula with a 5 percent starter culture consisting of 4.1 x 10(9) CFU of DW 12/ml (B); formula A modified by changing the amounts of cane sugar and MSG to 6 percent and 1 percent, respectively (C); formula C with a 5 percent starter culture added (D). Comparison among the 4 treatments showed that the treatment D produced the highest amount of GABA (4000 mg/L) during days 45-60 while the GABA content of A, B and C treatments was 340, 730 and 1690 mg/L, respectively. However, the results of the sensory test for the treatments C and D showed that the presence of MSG produced an unsatisfactory salty taste. All finished products from the 4 treatments met Thai standard guidelines for chemical and microbiological qualities after 120 days. The results indicated that enrichment of the GABA content in FSB is possible by adding MSG and the GABA producing strain DW12; however, the appropriate amount of MSG addition should be further studied.