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Glycobiology Advance Access originally published online on July 17, 2008
Glycobiology 2008 18(10):799-805; doi:10.1093/glycob/cwn069
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Novel endo-{alpha}-N-acetylgalactosaminidases with broader substrate specificity

Dimitris Koutsioulis, David Landry and Ellen P Guthrie1

New England Biolabs, Inc., Ipswich, MA 01938-2723, USA

1 To whom correspondence should be addressed: Tel: +1-978-380-7231; Fax: +1-978-921-1350; e-mail: guthrie{at}neb.com

Received on May 16, 2008; revised on July 11, 2008; accepted on July 13, 2008

In an effort to identify novel endo-{alpha}-N-acetylgalact- osaminidases (endo-{alpha}-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-{alpha}-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Galβ1,3GalNAc{alpha}1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest kcat for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAcβ1,3GalNAc{alpha}1pNP. This is the first report of endo-{alpha}-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Galβ1,3GalNAc{alpha}1pNP or GlcNAcβ1,3GalNAc{alpha}1pNP was incubated with various 1-alkanols in the presence of the endo-{alpha}-GalNAcases tested in this work.

Key words: deglycosylation / endo-{alpha}-N-acetylgalactosaminidases / O-glycosylation


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