Enhancing the endo-activity of the thermophilic chitinase to yield chitooligosaccharides with high degrees of polymerization  

作　　者：Feifei Guan Xiaoqian Tian Ruohan Zhang Yan Zhang Ningfeng Wu Jilu Sun Honglian Zhang Tao Tu Huiying Luo Bin Yao Jian Tian Huoqing Huang 

机构地区：[1]Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China [2]Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China [3]College of Food Science and Technology,Hebei Agricultural University,Hebei Baoding 071000,China

出　　处：《Bioresources and Bioprocessing》2024年第1期407-419,共13页生物资源与生物加工(英文)

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];The Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304);the China Agriculture Research System of MOF and MARA(CARS-41).

摘　　要：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.

关 键 词：CHITIN CHITINASE Endo-activity MUTANTS Functional oligosaccharides 

分 类 号：TQ9[化学工程]