Abstract: Chemical modification is a useful method to change the properties of enzymes. Laccase is a phenol oxidase belonging to a multicopper protein, which catalyzes the oxidation of many phenolics. DeniLite IIS, a commercial laccase preparation from the Novozymes China Company, was purified by ammonium sulfate fractional precipitation. Succinic anhydride (SA) was used as a modifier for the chemical modification of the purified laccase. The effects of modification were characterized using the 6-trinitrobenzene sulfonic acid method, ultraviolet spectroscopy, and fluorescence spectroscopy. The pH stability, thermal stability, and the phenolics removal efficiency for the native and modified laccases were compared. The results showed that the average amino modification yield of the modified laccase was 85% and the modified laccase had a blue shifted ultraviolet peak and fluorescence emission peak as well as a decrease in the ultraviolet absorbance and an increase in the fluorescence intensity. Although chemical modification with SA did not change the optimum temperature for the catalysis of the laccase, it caused the optimum pH of the catalysts to shift from 4.5 to 5.5 and the enzymatic activity increased by 60%. Compared with the native laccase, the modified laccase exhibited remarkably higher pH stability and thermal stability and its catalysis efficiency (k_cat) and substrate affinity (k_cat/K_m) increased by 53% and 122%, respectively. The phenolics removal efficiency (o-, m-, p-dihydroxybenzene) of the modified laccase increased by 48%, 57%, and 18%, respectively. These results indicate that the modified laccase with higher stability and higher efficiency is suitable for application in industrial production and for the treatment of phenolics-polluted water.

Key words: succinic anhydride, laccase, chemical modification, stability, kinetics, phenolics removal efficiency