Cloning, expression and characterization of a short-chain dehydrogenase from Pseudomonas fluorescens.
- Author:
Qun XUE
1
;
Xiangxian YING
;
Chi YANG
;
Zhao WANG
Author Information
1. College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China.
- Publication Type:Journal Article
- MeSH:
Alcohols;
metabolism;
Bacterial Proteins;
genetics;
metabolism;
Butyryl-CoA Dehydrogenase;
genetics;
metabolism;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Oxidation-Reduction;
Pseudomonas fluorescens;
enzymology;
genetics;
Recombinant Proteins;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2011;27(9):1317-1325
- CountryChina
- Language:Chinese
-
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.
