Cloning, expression and characterization of a short-chain dehydrogenase from Pseudomonas fluorescens / 生物工程学报
Chinese Journal of Biotechnology
; (12): 1317-1325, 2011.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-304572
Biblioteca responsável:
WPRO
ABSTRACT
To explore the physiological role and biocatalytic properties of short-chain dehydrogenases from Pseudomonas fluorescens GIM1.49, we cloned the structural gene pfd and characterized its over-expressed product. The length of gene pfd was 684 bp encoding a short-chain dehydrogenase with 227 amino acid residues and calculated molecular mass of 24.2 kDa. The recombinant plasmid pET28b-pfd was constructed and functionally expressed in Escherichia coli BL21(DE3), resulting in the over-production of recombinant short-chain dehydrogenase PFD with a size of 28 kDa. The enzyme could oxidize alcohols including 4-chloro-3-hydroxbutanoate ester and reduce 4-chloro-acetoacetate ester using either NAD(H) or NADP(H) as coenzyme. The enzyme showed the highest activity against 4-chloro-3-hydroxbutanoate ester as substrate, with Km of 186.40 mmol/L and Vmax of 89.56 U/mg. When catalying the oxidative reaction, its optimal temperature was 12 degrees C and optimal pH was 10.5, in contrast to the values of 24 degrees C and pH 8.8 in the reductive reaction. The enzyme had high solvent tolerance and its activity was improved by the addition of Ca2+ (1 mmol/L) or EDTA (5 mmol/L). These results indicated that the enzyme from Pseudomonas fluorescens GIM1.49 was a novel short-chain dehydrogenase and might play a role in oxidative degradation of halogenated secondary alcohols.
Texto completo:
Disponível
Contexto em Saúde:
Doenças Negligenciadas
Problema de saúde:
Doenças Negligenciadas
/
Zoonoses
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Oxirredução
/
Proteínas de Bactérias
/
Proteínas Recombinantes
/
Pseudomonas fluorescens
/
Clonagem Molecular
/
Butiril-CoA Desidrogenase
/
Álcoois
/
Escherichia coli
/
Vetores Genéticos
/
Genética
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2011
Tipo de documento:
Artigo