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Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli , and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase. [ABSTRACT FROM AUTHOR]