Trans-dihydrocarvone is widely used in flavoring, pharmaceuticals, agriculture, and other fields, and possesses high economic value. To enhance production of trans-dihydrocarvone through limonene biotransformation using Klebsiella sp. O852, we analyzed its extraction process and functionally characterized the key enzymes involved in trans-dihydrocarvone production by Klebsiella sp. O852. The optimal conditions for cell fragmentation were determined by single-factor experiments and response surface analysis. Effects of metal ions, organic solvents, and inhibitors on enzyme activity were analyzed, and optimal temperature and pH values for enzyme activity were determined. Optimal extraction conditions were as follows: ultrasonication time of 21 min, material-to-liquid ratio of 1:13, and amplitude of 36%. Ferrous, manganese (II), and zinc ions significantly increased enzyme activity, while copper (II) and ferric (III) iron exerted strong inhibitory effects. One percent (V/V) of dimethyl sulfoxide (DMSO) and isopropyl alcohol increased enzyme activity to 121.64% and 142.62%, respectively. When organic solvent concentrations were increased to 5% (V/V), they exerted enhanced inhibitory effects on enzyme activity. Chloroform exhibited the most significant inhibitory effect. Among the tested inhibitors, EDTA exhibited the most significant inhibitory effect on enzyme activity. The optimum temperature for enzyme activity was 20 ℃ and the optimum pH was 9.0. These results provide a theoretical basis and technical support for industrial application of this enzyme system.