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Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

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Abstract

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca2+ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn2+ can also improve the activity of recombinant enzyme, but the co-effect of Ca2+ and Mn2+ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca2+ (174%), Mn2+ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.

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Acknowledgments

This work was supported by the special funding from NJNU for talent faculty. We thank zhili Liu, associate professor, for performing the HPLC analysis.

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Authors and Affiliations

  1. Jiangsu Province Key Laboratory for Molecular and Medicine Biotechnology, College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing, 210046, Jiangsu, People’s Republic of China

    Qi Wang, Yinqiang Xin, Feng Zhang, Zhiyong Feng, Jin Fu & Zhimin Yin

  2. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, People’s Republic of China

    Lan Luo

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  1. Qi Wang
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  3. Feng Zhang
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Correspondence to Lan Luo or Zhimin Yin.

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Wang, Q., Xin, Y., Zhang, F. et al. Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli . World J Microbiol Biotechnol 27, 693–700 (2011). https://doi.org/10.1007/s11274-010-0508-2

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