Background: Endo-D is an endoglycosidase that can deglycosylate N-glycan core from IgG antibodies. Results: Endo-D showed distinct activities toward core-fucosylated and nonfucosylated substrates, and novel mutants were generated that demonstrated remarkable transglycosylation activity. Conclusion:The new discovery expands the repertoire of endoenzymes for glycoprotein research. Significance: This study reveals interesting substrate specificity of Endo-D and provides new enzymatic tools for glycosylation engineering of glycoproteins such as IgG-Fc.

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α-L-Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α-L-fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli. Afc1 had the α-L-fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined. The putative acid/base residue of Afc1, Ser205, was replaced by a glutamic acid which is conserved in GH29-B α-L-fucosidases. However, the mutant Afc1-S205E still failed to show enzyme activity. Afc2 and Afc3 were determined to be 1,3-1,4-α-L-fucosidase of GH29-B subfamily and 1,2-α-L-fucosidase of GH95 family, respectively, and both of them could release fucose from porcine gastric mucin (PGM). When C. perfringens ATCC 13124 grew with the presence of PGM, the transcription of afc1 decreased slightly, while those of afc2 and afc3 increased to 2.2-fold and 1.4-fold, respectively, and the enzyme activities of Afc2 and Afc3 in the culture increased to 2.2-fold and 2.6-fold, respectively. These results suggest that Afc2 and Afc3 are involved in the degradation of intestinal fucosyl glycans by C. perfringens ATCC 13124.

Throughout the article, some of the Asn-322 mutants were mistakenly typed as Asn-332 mutants. All instances of "N332A" or "N332Q" should be corrected as "N322A" or "N322Q" mutant.

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