Gamma aminobutyric acid (GABA) is a four-carbon non-proteinogenic amino acid synthesized in plants, fungi, vertebrates, and bacteria. GABA is an important inhibitory neurotransmitter in the mammalian central nervous system [1], [2], [3], [4]. It reduces neuronal excitability throughout the nervous system [5], [6]. GABA also enhances growth hormone levels, plasma concentrations, and protein synthesis in the brain [7], [8], [9], [10]. GABA deficiency may cause several neurological disorders, such as Parkinson’s and Alzheimer’s diseases [11], [12]. GABA functions as a compatible osmolyte and signaling molecule in plants [13], [14], [15]. In microbes, GABA production regulates the pH of cytosol under acidic conditions [16], [17]. For these physiological reasons, GABA is used in food supplements and the pharmaceutical industry to improve human health [18], [19], [20], [21], [22]. It is also a key molecule in the chemical industry, as a building block of 2-pyrrolidone and nylon 4 [23], [24], [25].

The bacterial biosynthesis of GABA occurs mainly via the pyridoxal-5’-phosphate (PLP)-dependent enzyme glutamate decarboxylase (GAD, EC 4.1.1.15), which converts L-glutamate to GABA [1]. These GAD-encoding genes are mainly distributed in lactic acid bacteria such as Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus reuteri [26], [27], [28]. Several studies have reported the production of GABA by fermentation with various lactic acid bacterial strains [26], [29], [30]. GABA can also be produced via biotransformation using whole-cell systems [31], [32], [33]. It has the advantages of a higher reaction speed and the formation of fewer byproducts than fermentation [34], [35], [36]. Therefore, whole-cell biotransformation using engineered Escherichia coli that overexpresses gad is a promising method [31], [32], [33]. Our previous study showed that 60 mg of GABA was produced by seven repeated whole-cell reactions of an E. coli BL21 (DE3) strain, LB GAD, which overexpresses gad originating from L. brevis KCTC 3498 [32].

In most cases, in whole-cell biotransformation using free whole cells, reuse is cumbersome and activity decreases rapidly [37], [38]. To increase the stability and reusability of whole-cell biotransformation, a method of immobilizing the whole cells has been used [39], [40]. Immobilization protects the cells from the harsh external environment of the biocatalyst, thereby increasing their stability [41], [42]. Entrapment is a widely used immobilization method because it is not complicated and it decomposes well [41], [43]. Alginate immobilization is one of the most commonly used methods and has good biocompatibility, low cost, easy availability, and easy preparation [39], [41]; however, to achieve high concentrations from reaction systems, strong immobilizing materials and improved production systems are needed [44].

In this study, we immobilized E. coli LB GAD, which produces GABA using monosodium glutamate (MSG) as a substrate and sodium alginate. We attempted to increase the reusability and stability of the whole cells and optimized various factors that affect immobilization. Furthermore, a continuous GABA production reaction system was established to increase convenience and productivity. This study is expected to contribute to the development of GABA production at an industrial level.