枯草芽孢杆菌漆酶BsCotA的定向进化及其在靛蓝脱色中的应用

Directed evolution of laccase BsCotA from Bacillus subtilis and its application in indigo decolorization

[目的]获得枯草芽孢杆菌(Bacillus subtilis)CotA漆酶活性及热稳定性提高的突变体并用于靛蓝脱色。[方法]利用易错PCR技术筛选获得2株酶活提高的漆酶突变体A1、A2,测定酶学性质,并进行靛蓝脱色研究。[结果]突变体A1、A2的最适pH值均为5.5,最适温度均为70℃;A1、A2在pH值2~7范围内,孵育30 min,剩余酶活均在40%以上,相比野生型(35%以下)有明显提高;A1、A2的比酶活为74.31 U/mg和80.96 U/mg,相比野生型(27.04 U/mg)分别提高了1.75倍、1.99倍;酶的三维结构分析显示,形成的4个突变位点中,1个突变位点通过氨基酸的替换,降低了主肽链的构象自由度,其余3个突变位点在漆酶的整体结构上引入了新的氢键;靛蓝染料脱色结果表明,3种漆酶对靛蓝的12 h脱色率均超过60%。[结论]筛选出来的漆酶突变体A1、A2,在pH值2~7范围内和65~80℃范围内的活性和稳定性比野生型显著提高,在靛蓝脱色等领域具有良好的应用基础。

[Objective]To obtain laccase mutants of BsCotA in Bacillus subtilis with improved activity and thermal stability,and use them in indigo decolorization.[Method]Two laccase mutants A1 and A2 with improved enzyme activity were screened by error-prone PCR,and their enzymatic properties were determined,and indigo decolorization was studied.[Result]The optimum pH value and temperature of A1 and A2 were 5.5 and 70℃respectively.The residual enzyme activity of A1 and A2 was above 40%when incubated for 30 min in the range of pH value 2 to 7,which was significantly higher than that of wild type(below 35%).The specific enzyme activities of A1 and A2 were 74.31 U/mg and 80.96 U/mg,which were 1.75 and 1.99 times higher than those of wild type(27.04 U/mg)respectively.Three dimensional structure analysis of laccase showed that one of the four mutated sites reduced the conformational freedom of the main peptide chain by substituting amino acids,and new hydrogen bonds were introduced into the overall structure of laccase in other three mutated sites.The decolorization results of indigo dye showed that the decolorization rates of the three laccases were all over 60%for 12 h.[Conclusion]The activity and stability of the screened laccase mutants A1 and A2 were significantly higher than those of the wild type in the range of pH value 2-7 and 65-80℃,which had good application foundation in the fields of indigo decolorization.