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- Title
Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.
- Authors
Sakaguchi, Kouta; Katoh, Toshihiko; Yamamoto, Kenji
- Abstract
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.
- Subjects
GLYCOCONJUGATES; GLYCANS; ENDOGLYCOSIDASES; GLYCOPROTEINS; RIBONUCLEASES; OXAZOLINE; HYDROLYSIS
- Publication
Biotechnology & Applied Biochemistry, 2016, Vol 63, Issue 6, p812
- ISSN
0885-4513
- Publication type
Article
- DOI
10.1002/bab.1433