Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

PLOS ONE, Dec 2019

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.

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Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

July Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid Yan Huang 0 1 Lingqia Su 0 1 Jing Wu 0 1 0 State Key Laboratory of Food Science and Technology, Jiangnan University , Wuxi , China , 2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University , Wuxi , China 1 Editor: Vivek K. Bajpai, Yeungnam University , REPUBLIC OF KOREA 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. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work received financial support from the National Science Fund for Distinguished Young Scholars (No.31425020), received by Jing Wu and the 111 Project (No. 111-2-06), received by Jing Wu. Competing Interests: The authors have declared that no competing interests exist. Introduction γ-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 [ 2–5 ]. At present, GABA is widely used to reduce the concentration of the blood ammonia [1] and to treat hepatic coma [ 6 ]. GABA has also attracted interest for its potential use as a biopolymer precursor. For example, 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. The currently method of GABA production include enrichment from food, chemical synthesis and biosynthesis. The amount of GABA obtained by enrichment from food is quite low; it is used as a nutritional supplement in products such as GABA-green tea [ 8 ] and GABAbrown rice [ 9 ]. The chemical synthesis of GABA consumes large amounts of energy and is not environmental friendly [ 10 ]. The isolation of GABA-producing lactic acid bacteria from traditional fermented foods has attracted much interest recently. However, the highest level of GABA produced through fermentation has reached only 35.6 g L-1 [ 11–13 ]. Furthermore, the recovery of GABA from the complex fermentation broths required by these organisms is generally difficult and expensive to perform. These drawbacks have limited the widespread use of GABA. In contrast, enzymatic synthesis, which uses the pyridoxal 5’-phosphate (PLP)-dependent enzyme glutamate decarboxylase (GAD) to catalyze the irreversible decarboxylation of L-glutamate to GABA, is performed under mild conditions with low cost and low energy consumption. Therefore, enzymatic synthesis is highly desirable as a method of industrial GABA production [ 14 ]. Due to the industrial importance of GABA, glutamate decarboxylases have recently been overexpressed in various hosts to improve GABA production. Among the GAD enzymes reported to date, the GAD from E. coli K12 exhibited the highest yield of GABA, reaching 280 g L-1 [ 15–20 ]. Therefore, E. coli GAD overexpressed in E. coli BL21 (DE3) was investigated further in the present study. GAD requires PLP for its glutamate decarboxylase activity [ 21 ]. Although PLP can be generated by the phosphorylation of pyridoxal aldehyde in the bacterium, this route cannot meet the demands of recombinant GAD production. Therefore, most researchers add a certain amount of PLP to the enzymatic conversion mixture to assist in the production of GABA [ 20 ]. At present, only Plokhov et.al have reported a preparation method in which 0.02 mM PLP is added to the fermentation medium to enhance the yield of GAD. The production of GAD increased 2–2.5-fold in the presence of 0.02 mM PLP [ 19 ]. However, the high price, poor availability, and poor stability of PLP have limited its industrial application. A (...truncated)


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Yan Huang, Lingqia Su, Jing Wu. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid, PLOS ONE, 2016, Volume 11, Issue 7, DOI: 10.1371/journal.pone.0157466