Construction of a co-reaction system for ethanol-promoted gamma-aminobutyric acid synthesis by Pediococcus pentosaceus

The biotransformation of L-glutamic acid (L-Glu) to γ-aminobutyric acid (GABA) using glutamate decarboxylase (GAD) in microbial whole cells represents an ideal approach for biosynthesis of food-grade and pharmaceutical-grade GABA. To overcome the cell membrane barrier, enhance mass transfer efficiency in the whole-cell reaction system, and improve GABA biosynthesis efficiency, we established a novel ethanol-enhanced whole-cell biocatalytic co-reaction system through systematic investigations on the enzymatic characteristics of GAD in Pediococcus pentosaceus whole cells and the regulatory effects mediated by ethanol. The results showed that the optimal reaction pH and temperature for GAD in P. pentosaceus whole cells were 4.2 and 32 °C, respectively. A 7.5% (v/v) ethanol concentration significantly promoted the activity of whole-cell GAD, but reduced its stability. Through orthogonal test optimization, the optimal reaction conditions for ethanol-promoted GABA synthesis via P. pentosaceus whole-cell transformation were as follows: mixing 0.3 M monosodium glutamate (MSG) solution with 100 mg/ml cell suspension at a 1:1 volume ratio, adding 40 g/l of L-Glu/MSG (2:1) solid mixture, adjusting the final ethanol concentration to 3.75% (v/v), reacting at pH 4.2, 28 °C for 40 h. Under these conditions, the GABA yield reached 366.07 ± 5.57 mM, which was 21.44 ± 1.85% higher than that of the control group without ethanol. As an enhancer, ethanol demonstrates great application potential in GABA production via whole-cell transformation due to its high safety and ease of use.