Journal
JOURNAL OF BIOTECHNOLOGY
Volume 278, Issue -, Pages 1-9Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2018.04.009
Keywords
Glutamate decarboxylase; gamma-Aminobutyrate; Site-directed saturation mutagenesis; Thermostability; Hydrogen bonds
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Funding
- National High Technology Research & Development Program of China (863 Program) [SS2014AA021202]
- Shuguang Program project - Shanghai Education Development Foundation
- Shanghai Municipal Education Commission [15SG28]
- Fundamental Research Funds for the Central Universities [222201717026]
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Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert L-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 degrees C, 8.7 degrees C rise in melting temperature (Tm) and a 14.3 degrees C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T-50(15)), compared to wild-type enzyme. Protein 3D structure analysis showed that the induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in E. coli GadB homo-hexamer may be responsible for the improved thermostability. Increased thermostability contributed to increased GABA conversion ability. After 12 h conversion of 3 mol/L L-glutamate, GABA produced and mole conversion rate catalyzed by M6 whole cells was 297 g/L and 95%, respectively, while those by wild-type GAD was 273.5 g/L and 86.2%, respectively.
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