Expression And Characterization Of Endo-?-N-Acetylglucosaminidase From Streptomyces Alfalae

Endo-?-N-acetylglucosaminidase is a member of the glycosidase family and its action mode is to hydrolyze the?-1,4 glycosidic bond between two N-acetylglucosamines.Endo-?-N-acetylglucosaminidases assigned to the GH18 family and the GH85 family respectively.They are used in the study of glycomics and glycobiology in attribute to the efficiency of deglycosylation.Based on the whole genomic sequencing of Streptomyces alfalae ACCC40021,a new endo-?-N-acetylglucosaminidase EndoSA was predicted by bioinformatic analysis.The CAZy(Carbonate Activity Enzyme database)analysis revealed that the enzyme belongs to the GH18 family,and the similarities sharing with the EndoFV and EndoT sequences of the GH18 family were 38.19%and 38.21%,The phylogenetic tree was constructed by homologous comparison with other endo-?-N-acetylglucosaminidase and other chitinase genes reported in the NCBI database.According to the results,This enzyme has the closest relationship with the chitinase type III from Coccidiodes posadasi.The fragment encoding target gene sequence was amplified by polymerase chain reaction,and the recombinant plasmid was constructed and transformed into E.coli BL21competent cell.The recombinant enzyme was successfully expressed,and purified by nickel column to apparent homogeneity by SDS-PAGE analysis.In this study,RNaseB was employed as substrate to determine the enzymatic properties.The results showed that the enzyme has high specific activity of 1.0×10~6 U/mg.The recombinant enzyme exhibited the optimum reaction temperature at 35?,and had good thermal stability from 30?to 45?.The optimum pH of the enzyme is 6,and the enzyme displayed good stability between pH 4-8.The?-1,4-Glucan glucohydrolase,pectinase and acid protease expressed by yeast were used as substrates to explore the deglycosylation of this enzyme.The results show that the enzyme deglycosylated all tested substrate completely under the optimal condition.Our result is to provide a new tool enzyme for glycoprotein glycosylation site research.This?-N-acetylglucosaminidase was also used to treat the by-product whey processed by dairy products such as cheese,and0.95 mg of the oligosaccharide per milliliter of whey was obtained per minute using 1 mg enzyme for cleavage.