Biochemical Characterization Of A Glycoside Hydrolase Family 19 Chitinase From Streptomyces Alfalae And Its Applications

Chitinase is a glycosyl hydrolase that can convert chitin to N-acetylglucosamine and N-acetyl chitooligosaccharides.It can be used for biotransformation of chitin and biocontrol of phytopathgenic fungi.In our previous work,we predicted only a Glycoside Hydrolase?GH?family 19 chitinase?design as SaChi19B?in the whole genome sequence of Streptomyces alfalae ACCC40021 a biofertilizer strain utilized in China widely,based on the CAZy?Carbohydrate active enzyme database?analysis.In this study,we cloned,expressed and characterized the chitinase SaChi19B,and further assessed the efficiency of bioconversion of chitin and antifungi.Then,we successfully improved the antifungi properties of SaChi19B significantly by using protein engineering technique.1.The chitinase SaChi19B was successfully cloned and expressed in E.coli BL21?DE3?.The recombinant enzyme displayed most active in an alkaline pH optimum of 8.0?phosphate buffer?and optimum temperature of 45?,and stable under different temperature and pH conditions.It showed a broad substrate spectrum with high activity towards colloidal chitin,ethylene glycol Chitin and?-chitin.The action mode of SaChi19B was endo-type.In addition,SaChi19B had different inhibitory effects on six plant pathogenic fungi,especially on Botrytis cinereal and Rhizoctonia solani.Furthermore,the binary chitolytic system consisted of SaChi19B and SaHEX converted shrimp shell chitin to GlcNAc efficiently with the yield of 95.19%and the product purity>98%in 8 hrs.This is the first report that GH19 chitinase can be used to hydrolyze the crystalline chitin.These findings indicated that SaChi19B has the great potential applied in the enzymatic conversion of chitin and biocontrol of phytopathgenic fungi.2.To develop a simple method to improve the enzymatic and antifungal activity of SaChi19B,CatD_Chi19B?catalytic domain?,rChi19B?N-terminal chitin-binding domain?and DChBD_Chi19B?double chitin-binding domain?were successfully constructed and expressed in E.coli BL21?DE3?.Compared to CatD_Chi19B and rChi19B,DChBD_Chi19B improved the binding ability and activity towards colloidal chitin,?-chitin and chitin from Aspergillus niger.Furthermore,antifungal activity was enhanced against plant pathogenic fungus Trichoderma longibranchiatum.Therefore,a simple,feasible and efficient method was developed to improve the enzymatic and antifungal activity of chitinase by fusion of chitin binding domain on both C-and N-terminus of the chitinase.3.In order to further improve the antifungal properties of chitinase Sa Chi19B,we successfully constructed the chimeric enzyme SaChi19B-Csn that fused chitosanase SaCsn on C-terminus of SaChi19B by seamless cloning.The enzyme was successful expressed in E.coli BL21?DE3?.Purified SaChi19B-Csn displayed significantly active in an alkaline pH optimum of 8.0?phosphate buffer?and optimum temperature of 30?,It showed a broad substrate spectrum,especially towards chitin and chitosan;Compared to the wild type chitinase SaChi19B,antifungal assay displayed that SaChi19B-Csn not only improved all tested plants inhibitory efficiency but also expand the antifungal spectrum.In summary,this study provides a new strategy to obtain efficient chitinase as a potential candidate for biocontrol agent of plant pathogenic fungi.