Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scar...Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.展开更多
基金supported by the National Key R&D Program of China[Grant No.2021YFC2103002]the National Natural Science Foundation of China[Grant No.32202720]+1 种基金The Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304)the China Agriculture Research System of MOF and MARA(CARS-41).
文摘Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.
基金supported by the National Key R&D Program of China(2022YFC2105501)the National Natural Science Foundation of China(32202720)+2 种基金the Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304 and CAAS-ASTIPG2022-IFST-07)the Central Public-interest Scientific Institution Basal Research Fund(1610392020001)the Agricultural Science and Technology Innovation Program(ASTIP)。
