嵌合突变嗜酸普鲁兰芽孢杆菌普鲁兰酶结构域B对酶学性质及功能的影响

摘要

为考察嗜酸普鲁兰芽孢杆菌(Bacillus acidopullulyticus)普鲁兰酶结构域B对热稳定性及其他酶学特性和功能的影响,采用嵌合蛋白技术将B.acidopullulyticus普鲁兰酶的结构域B置换为Geobacillus thermoleovorans普鲁兰酶结构域B.嵌合突变体在60℃下的半衰期由34.9 min提高至168.4 min,解折叠一半时的温度由65℃提高至72℃,嵌合突变体较野生型具有更加优良的动力学稳定性和热动力学稳定性.结构域B置换后,最适pH碱向偏移至pH6.5,最适温度提高至70℃.比酶活及底物特异性实验结果显示,嵌合突变削弱了酶分子与普鲁兰多糖的结合,转而有利于与糊精及可溶性淀粉的结合.淀粉糖化结果显示,嵌合突变不影响突变体的实际应用性能.上述结果表明,B.acidopullulyticus普鲁兰酶结构域B对酶蛋白酶学性质影响显著,可通过同源置换构建适合于不同淀粉糖化工艺的突变体.%Chimeric protein technology was used to replace domain B of Bacillus acidopullulyticus pullulanase with domain B from Geobacillus thermoleovorans pullulanase to investigate the effects of chimeric mutation on thermostability,enzymatic characteristics,and function.The results showed that the half-life of chimeric mutant at 60 ℃ was increased from 34.9 min to 168.4min,and its melting temperature increased from 65 ℃ to 72 ℃.Furthermore,chimeric mutant had more excellent kinetic stability and thermodynamic stability compared to wild-type enzyme.The optimal pH and optimal temperature were 6.5 and 70 ℃,respectively after domain B replacement.Specific activity and substrate specificity results showed that the chimeric mutation weakened the combination of enzyme with pullulan,increased the combination of enzyme with dextrin and soluble starch.Starch saccharification results showed that the chimeric mutation did not affect the practical application.These results demonstrated that B.acidopullulyticus pullulanase domain B had significant impact on enzyme properties.The mutants suitable for different starch saccharification processes can be constructed by homologous replacement.