Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(-)-mandelic acid production.
Bioprocess Biosyst Eng
; 34(3): 315-22, 2011 Mar.
Article
en En
| MEDLINE
| ID: mdl-20960011
A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His6-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V(max) and K(m) parameters were determined to be 27.9 µmol min⻹ mg⻹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(-)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(-)-mandelic acid.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Estabilidad de Enzimas
/
Alcaligenes
/
Aminohidrolasas
/
Ácidos Mandélicos
Idioma:
En
Revista:
Bioprocess Biosyst Eng
Asunto de la revista:
BIOTECNOLOGIA
/
ENGENHARIA BIOMEDICA
Año:
2011
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Alemania