Glucose oxidase(GOD)has many practical applications,but its poor thermostability limits its broader use.In this research,three primary mutants of wild-type GOD were designed using rational mutagenesis,and the GODm mut...Glucose oxidase(GOD)has many practical applications,but its poor thermostability limits its broader use.In this research,three primary mutants of wild-type GOD were designed using rational mutagenesis,and the GODm mutant was constructed by combinatorial design.The expression,purifcation,and enzymatic properties of the mutants were studied.The specifc enzyme activity of GODm was 2.10-fold higher than that of wild type,and the(k_(cat)/K_(m))value was increased by 1.45-fold.After treatment at 55℃for 3 h,GODm retained 37.5%of its enzymatic activity,and the half-life(t_(1/2))of GODm at 55℃and 65℃was 2.28-fold and 3.36-fold higher than that of wild type,respectively.By analyzing the three-dimensional structure of wild type and the GODm mutant,it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction,D315K optimized the surface electrostatic interaction,and A162T improved the efciency of the electron pathway.Thus,a novel mutant with improved thermostability and catalytic efciency was obtained in this research.展开更多
基金The authors are grateful for the fnancial support from the National First-class Discipline Program of Light Industry Technology and Engineering(Grant No.LITE2018-04)the Topnotch Academic Programs Project of Jiangsu Higher Education Institutions(TAPP),and the National Natural Science Foundation of China(NSFC)(32072162).
文摘Glucose oxidase(GOD)has many practical applications,but its poor thermostability limits its broader use.In this research,three primary mutants of wild-type GOD were designed using rational mutagenesis,and the GODm mutant was constructed by combinatorial design.The expression,purifcation,and enzymatic properties of the mutants were studied.The specifc enzyme activity of GODm was 2.10-fold higher than that of wild type,and the(k_(cat)/K_(m))value was increased by 1.45-fold.After treatment at 55℃for 3 h,GODm retained 37.5%of its enzymatic activity,and the half-life(t_(1/2))of GODm at 55℃and 65℃was 2.28-fold and 3.36-fold higher than that of wild type,respectively.By analyzing the three-dimensional structure of wild type and the GODm mutant,it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction,D315K optimized the surface electrostatic interaction,and A162T improved the efciency of the electron pathway.Thus,a novel mutant with improved thermostability and catalytic efciency was obtained in this research.
