重组大肠杆菌产CotA漆酶的发酵条件优化

Optimization of Fermentation Conditions of CotA Laccase from Recombinant Escherichia coil

CotA漆酶在环境保护、食品工业和纸浆漂白等工业中具有重要的应用价值.将重组表达载体pET-22b/CotA转入大肠杆菌BL21(DE3),得到工程菌株,采用单因素实验和正交实验相结合的方法,研究诱导表达条件和发酵培养基对重组大肠杆菌产CotA漆酶量的影响.结果表明,初始pH 7.5的培养基中,添加0.6 mmol L-1 Cu2+,1 g L-1葡萄糖作碳源、15 g L-1蛋白胨和2 g L-1硫酸铵作氮源,以10%的接种量,37℃、200 r/min,直到菌液的D600 nm值为1.0,加入终浓度为1.0 mmol L-1的IPTG,25℃诱导12 h,漆酶的产量最高.优化前发酵液的粗提液的漆酶活性仅为1 190 U mL-1,优化后达到3 526 U mL-1,正交实验优化后漆酶活性提高了2.96倍.纯化的CotA漆酶最适反应温度为45℃,最适pH值为7.2.CotA漆酶对RBBR的脱色率在90%以上,此CotA漆酶在短时间内对染料能够有效地脱色,能够成为有潜力的工业用酶.

CotA laccase is important for its applications in industries such as environmental protection, food industry, and paper biobleaching. A recombined expression vector pET-22b（＋）/CotA was transferred into Escherichia coli BL21 （DE3）. The effects of induction expression conditions and the fermentation medium on the production of CotA laccase by recombinant E. coli were investigated using one factor method and orthogonal design. The strain was cultured first under the following conditions： 0.6 mmol L^-1 copper ions, 1 g L^-1glucose as carbon source, 15 g L^-1tryptone and 2 g L^-1 ammonium sulfate as nitrogen source, inoculum concentration 10%, temperature 37 ℃ and shaking speed 200 r/min. IPTG was added to the culture at 1.0 mmol L-t when the D600nm of culture reached 1.0. After 12 h cultivation at 25 ℃, the highest laccase yield was obtained. The laccase activity of the extraction of the fermentation broth after optimization （3 526 U mL^-1） was 2.96 times than that before optimization （only I 190 U mL^-1）. The optimum temperature and pH of the enzyme activity were 45 ℃ and 7.2, respectively. The decolorization rate of Remazol brilliant blue R （RBBR） by CotA laccases reached over 90%. The result indicated that the CotA laccase had the potential to be industrial enzyme. Fig 6, Tab 3, Ref 17