Abstract: 【Objective】The objective of this study is to optimize fermentation parameters for the laccase production by Setosphaeria turcica, investigate its enzymatic properties, and to develop the applications of the laccase.【Method】According to the results of single factor experiments to confirm the categories and concentration scales of the carbon source, nitrogen source and Cu²⁺ as single parameters, the response surface experiments of fermentation using S. turcica were preformed which the enzymatic activity was chosen as the response value. The experiments were designed by the central composite design (CCD), and analyzed by Multivariate Quadratic Regression Analysis of Design Expert for three factors and three levels, then the optimal fermentation medium for laccase production by S. turcica was determined. And the properties of laccase isolated from the fermentation culture were characterized using ABTS as substrate, including the optimum temperature and pH, the stability at different pH and temperatures, and the kinetics constants of Km and Vm.【Result】The multivariate quadratic regression model which the enzymatic activity was chosen as response value was significantly different (P=0.0001), which meant the established regressing equation for activity of laccase had an excellent goodness of fit and the laccase fermented by S. turcica could be analyzed by this model. According to the model, the factors influencing the activity of laccase were in the order as follows: Cu²⁺＞glucose＞urea and the interaction of glucose and urea was very significant. The optimum conditions, which were predicted by this model, were glucose 50.05 g?L^-1, KH₂PO₄ 1 g?L^-1, urea 1.46 g?L^-1, MgSO₄ 0.5 g?L^-1, peptone 2 g?L^-1, maize steep liquor 0.5 g?L^-1, CuSO₄ 0.07 g?L^-1 and Tween80 3 mL?L^-1, at 28℃, and 150 r/min. The maximal enzyme activity was (40.00±1.20) U?mL^-1 after 7 d of cultivation. The laccase of S. turcica had a molecular weight of approximately 80 kD analyzed by SDS-PAGE after preliminarily purified. It was demonstrated that the optimum pH and temperature were pH 4.2 and 50℃, respectively, and the laccase of S. turcica had higher activity at higher temperature and weak acidic condition, using ABTS as substrate. It also exhibited high thermal stability and the stability of pH, retaining its activity basically unchanged after incubation at pH 4.2 for 14 h and above 60% at 50℃ for 14 h. The Km was 0.036 mmol?L^-1 and Vm was 28.63 mmol?L^-1·min^-1, at room temperature and pH 4.2. 【Conclusion】The enzymatic properties of the laccase of S. turcica were investigated, and the fermentation medium by response surface methodology was optimized. The laccase of S. turcica has a potential value for further research due to the advantages of high activity, short fermentation period and high stability.

Key words: Setosphaeria turcica, laccase, response surface methodology, enzymatic characters