Journal
LWT-FOOD SCIENCE AND TECHNOLOGY
Volume 148, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.lwt.2021.111699
Keywords
Glutamate decarboxylase; Neutral pH; Overexpression; Gamma amino butyric acid
Categories
Funding
- Ferdowsi University of Mashhad [3/48396]
- Iran National Science Foundation [98000180]
Ask authors/readers for more resources
This study successfully cloned the GAD gene of Enterococcus faecium DO into PET21a (+) and overexpressed it in Escherichia coli, confirming its activity at neutral pH. The recombinant GAD exhibited high specific activity at 45 degrees C and pH = 6.6, making it a strong candidate for GABA production in the food and pharmaceutical industries.
In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since L-glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of gamma-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg(-1) at 45 degrees C and pH = 6.6. It was reduced rapidly when pH > 7.8. The Km, Vmax, and kcat values of recombinant GAD were 1.8 mM, 12.9 mu mol L-1 min-1, and 0.43 s(-1), respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available