Abstract
Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.
Highlights
Introduction γAminobutyric acid (GABA), a four carbon, non-essential amino acid that is widely distributed in nature, plays a major role as an inhibitory neurotransmitter in the mammalian central nervous system [1]
The resulting recombinant plasmid, gadB/pET-24a, was used to transform E. coli BL21 (DE3) for Glutamate decarboxylase (GAD) expression. When this recombinant E. coli was cultivated in standard TB medium and GAD expression was induced with IPTG, the GAD activity in the cytoplasm increased gradually, reaching its highest value (87.5 U mL-1) 18 h after induction (Fig 1A)
SDS-PAGE analysis the cytoplasmic fraction showed a major band at approximately 54 kDa (Fig 1B), which is consistent with the molecular weight of GAD previously reported [20, 26] This enzyme, which was used as the control for further experiments, will be referred to as GAD-C
Summary
Aminobutyric acid (GABA), a four carbon, non-essential amino acid that is widely distributed in nature, plays a major role as an inhibitory neurotransmitter in the mammalian central nervous system [1]. GABA has several physiological activities, including diuretic and tranquilizer effects, and has been used in the treatment of epilepsy and the prevention of obesity [2,3,4,5]. GABA is widely used to reduce the concentration of the blood ammonia [1] and to treat hepatic coma [6]. GABA has attracted interest for its potential use as a biopolymer precursor. GABA can be converted to 2-pyrrolidone, a monomer of nylon 4 [7]. Due to its multiple functions, GABA is widely used in medicine, functional foods and the chemical industry
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