Premium
Transglycosidase‐like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor
Author(s) -
Sakaguchi Kouta,
Katoh Toshihiko,
Yamamoto Kenji
Publication year - 2015
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1433
Subject(s) - endoglycosidase , glycan , chemistry , biochemistry , glycoconjugate , mutant , rnase p , glycoside hydrolase , enzyme , natural product , glycobiology , glycoprotein , gene , rna
Glycan conversion of glycoprotein via the transglycosylation activity of endo‐β‐ N ‐acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio‐medicines with a homogeneous glycoform. Although Endo‐M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo‐M mutant enzymes including N175Q with glycosynthase‐like activity and/or transglycosidase‐like activity. We found that the Endo‐M N175H mutant showed glycosynthase‐like activity comparable to N175Q as well as transglycosidase‐like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo‐glycan onto an N ‐acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo‐M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.