Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
; (12): 682-685, 2006.
Article
en Zh
| WPRIM
| ID: wpr-286227
Biblioteca responsable:
WPRO
ABSTRACT
The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Filogenia
/
Química
/
Clonación Molecular
/
Escherichia coli
/
Amidohidrolasas
/
Genética
/
Peso Molecular
/
Nocardia
Tipo de estudio:
Prognostic_studies
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Año:
2006
Tipo del documento:
Article