[Heterologous expression and characterization of a thermostable acylCoA synthetase].
Wei Sheng Wu Xue Bao
; 56(9): 1477-85, 2016 Sep.
Article
em Zh
| MEDLINE
| ID: mdl-29738220
ABSTRACT
Objective:
An acyl-CoA synthetase (ACS) was characterized by in vitro biochemical assays to get a potential biocatalyst for the generation of polyketide precursors such as propionyl-CoA and butyryl-CoA.Methods:
An ACS was identified in Caldicellulosiruptor owensensis OL genome by BLAST program using malonyl-CoA synthetase as the query sequence. The enzyme was heterologously expressed in Escherichia coli cells and purified to homogeneity by affinity purification. In vitro biochemical assays were used to reveal the substrate profile, the optimum reaction condition, the stability and the kinetics of the enzyme while the site-directed mutagenesis was used to assay the function of active site residues.Results:
The enzyme was promiscuous enough to accept propionic acid, butyric acid, 2-methyl-propionic acid, pentanoic acid, 2-methyl-butyric acid, 3-methyl-butyric acid and cyclohexanecarboxylic acid as the substrates. It was most active at 30â and pH 7.0 and relatively stable because 45% activity remained after 8 hours incubation at 70â. Interestingly, the substrate specificity of the enzyme could be switched by engineering three residues of active site.Conclusion:
The ACS from C. owensensis OL was a potential biocatalyst for the generation of polyketide precursors.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Coenzima A Ligases
/
Firmicutes
Idioma:
Zh
Revista:
Wei Sheng Wu Xue Bao
Ano de publicação:
2016
Tipo de documento:
Article