Recently a bacterial cyclohexylamine oxidase (CHAO) has been reported as a possible new biocatalyst in the synthesis of chiral amine.However,the application of CHAO was limited because it showed little or no activity toward secondary and tertiary amines.To expand the application of the bacterial originated cyclohexylamine oxidase (CHAO) in the deracemization of secondary amines,site-saturation mutagenesis of wild-type CHAO was performed and the resulting libraries were screened with 2-methyl-1,2,3,4-tetrahydroquinoline as the substrate.Two positive mutants T198F and T198FL199F were obtained which displayed 97 and 76 folds higher catalytic efficiency than that of wt CHAO,respectively.The resting cells contained T198F and T198FL199F can efficiently catalyze the deracemization of 2-methyl-1,2,3,4-tetrahydroquinoline giving (R)-2-methyl-1,2,3,4-tetrahydroquinoline with high isolated yield (76% and 74%,respectively) and ee value (both were 96.5%).Enzyme-substrate docking studies showed that the distances between amine and C-4 of the flavin moiety for T198F and T198FL199F were shorter than that of wild-type CHAO,which may cause the higher catalytic efficiency of the mutant enzymes.