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Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production.
- Source :
-
Journal of Biotechnology . Jul2018, Vol. 278, p1-9. 9p. - Publication Year :
- 2018
-
Abstract
- 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 °C, 8.7 °C rise in melting temperature (Tm) and a 14.3 °C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T 15 50 ), 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01681656
- Volume :
- 278
- Database :
- Academic Search Index
- Journal :
- Journal of Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 129922378
- Full Text :
- https://doi.org/10.1016/j.jbiotec.2018.04.009