고초균에서 유산균 균주의 글루탐산 탈탄산효소의 발현 및 분비: GABA 생성에 의한 생물학적 활성

Abstract

Gamma-aminobutyric acid (GABA) can be converted from monosodium glutamate (MSG) by glutamate decarboxylase (GAD). GABA is known to be a four-carbon non-protein amino acid existing in the human brain and eye. Recent studies have shown that GABA is effective in accelerating hypotension and anxiety relief, along with acting as a diuretic, antidiabetic, and antidepressant in humans and other mammals. Since the low activity of cell preparations has been a major limitation to commercial enzyme synthesis, it is worthwhile to look for methods to obtain a affordable recombinant enzyme with high GABA synthesizing activity. In order to maximize production of GABA using the culture broth without purification in Bacillus subtilis, the gene (gadB) first was obtained from chromosome of Streptococcus thermophilus (S. thermophilus) expressing the GAD with appropriate amount. The primer set with restriction enzyme sites and His-tag was designed by referring to the DNA sequence of glutamate decarboxylase enrolled in GenBank (DQ871217.2), amplified and cloned into pGemTeasy vector. The right clone was screened and characterized by DNA sequencung analysis. As a result, the DNA sequence was different from that of GenBank in codons encoding 4 amino acids (Tyr 189 , Gly 282 , Ser 301 , and Met 425 ) by point mutations, which were changed to Cys 189 , Asp 282 , Gly 301 , and Thr 425 , respectively. To secret the GAD in culture medium, the recombinant vector (pRBASGadB, 7.83 kb) containing the gadB gene was constructed using pRBAS secretion vector with alkaline protease promoter (aprE-P, 0.45 kb) and signal sequence (aprS, 87 bp) and then transferred into Bacillus subtilis LKS87. The secretion levels of GAD were analyzed by SDS-PAGE and western-blot and the level in B. subtilis LKS87 harboring the pRBAS-GadB vector was increased to approximately 10-12% of total secreted proteins. The conversion of MSG (monosodiumglutamate) to GABA was measured by paper chromatography using the culture broth and PBS-eluted cell pellet. As a result, the culture broth shows 16% GABA conversion rate from MSG, whereas the pellet shows higher conversion (~43%), indicating that the expressed GAD was not sufficiently secreting into the culture broth caused by the inefficient processing of aprS signal sequence or presence of self signal sequence of the GAD gene. By measuring the ABTS+ scavenging ability, it was confirmed that the GABA converted by GAD showed higher antioxidant activity (55%) than that (50%) of ascorbic acid used as positive control. The survival recovery rate of STZ-treated RINm5F cells was increased to 28.2% by the GABA treatment and also, production of nitric oxide (NO) was drastically reduced with 78.7% in STZ-treated RINm5F cells, suggesting that the GABA possess antidiabetic and antioxidant activities. Finally, although GABA is showing various biological activities, it might be needed to optimize secretion efficiency for industrial applications.