为提高Escherichia coli BL21(DE3)中漆酶基因lac1338的表达水平,采用响应面法对其发酵条件进行优化.首先用Plackett-Burman法筛选出3个影响较大的重要因素,分别为温度(Q1)、诱导时间(Q3)以及诱导前菌体浓度OD600 nm(Q4).继而结合响应面分析,建立以漆酶酶活为响应值的二次回归方程模型,即Y=75.33+3.30A+9.35B+5.35C-1.69AB+0.80AC+11.50BC-25.97A^2+1.83B^2-4.66C^2,从中获得最优的发酵条件:诱导温度为31℃,诱导时间为20 h,诱导前的菌体浓度OD600 nm值为1.8.采用该发酵条件,供试菌株的漆酶比酶活达到22.8 U/mg,较优化前漆酶的表达量(10.7 U/mg)提高了2.13倍,其试验值与预测值基本相符.说明预测模型可靠性高,可应用于漆酶发酵条件的优化.
In order to enhance the production of laccase in Escherichia coli BL21(DE3), response surface methodology was applied to optimize the fermentation conditions. Plackett-Burman design was used to investigate the main factors affecting laccase yield of E. coli BL21(DE3). The results showed that three factors played important roles in the fermentation conditions: the culture temperature(Q1), culture time(Q3) and the initial bacteria density of OD600 nm(Q4). The second-order regression equation model for the yield of laccase was established by response surface analysis as Y=75.33 + 3.30 A + 9.35 B + 5.35C- 1.69 AB + 0.80 AC + 11.50BC- 25.97A^2 + 1.83B^2- 4.66C^2. Based on this model, the optimal fermentation conditions were obtained as 20 h of culture time, 31 °C of culture temperature, and OD600 nm 1.8 of initial bacteria density. The ferment condition optimization increased the laccase production by nearly 2.13 times to 22.8 U/mg, with the experimental values in agreement with the predicted ones. These results suggested that the prediction model is reliable and suitable for the optimization of laccase fermentation conditions.