Specificity of enzymatic in vitro glycosylation by PNGase F: A comparison of enzymatic and non-enzymatic glycosylation

Hee Yong Jeong, Ji Youn Lee, Tai Hyun Park

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Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.

Original languageEnglish
Pages (from-to)587-591
Number of pages5
JournalEnzyme and Microbial Technology
Issue number6-7
StatePublished - 1 Dec 2004

Bibliographical note

Funding Information:
The authors wish to acknowledge the financial support of the Korea Science & Engineering Foundation through the Nano Bio-Electronic & System Center, Seoul National University, Seoul, Korea.


  • Acid hydrolysis
  • In vitro glycosylation
  • MALDI-TOF mass spectrometry
  • N,N′-Diacetylchitobiose
  • Peptide-N-glycosidase F (PNGase F)


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