Abstract: To explore the effect of C-terminal region residues on the substrate specificity of a novel cyclic imide hydrolase (CIH), a recombinant cyclic imide hydrolase (CIH293) and its mutant enzymes deleted or substituted at C-terminus (CIH291, CIH290, KK292-293EE) were prepared by gene cloning. Their substrate specificity and kinetic parameters were analyzed by both the spectrophotometric assay and high-performance liquid chromatography. Results show that the substrate specificity of mutant enzymes was not obviously changed, but slightly low for the substrate affinity, compared with the wild-type enzyme, CIH293.