High-throughput screening of glycosynthases using azido sugars for oligosaccharides synthesis

Mohit Kumar, Chandra Kanth Bandi, Shishir P.S. Chundawat

Research output: Chapter in Book/Report/Conference proceedingChapter


Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar. We created a diverse library of fucosynthase mutants using semi-random and random error prone mutagenesis and then identified improved fucosynthase mutants with desired activity using two distinct screening methods developed by our group to detect glycosynthase activity (i.e., by detecting azide formed upon completion of fucosynthase reaction); (a) pCyn-GFP regulon method, and (b) Click chemistry method. Finally, we provide some proof-of-concept results illustrating the utility of both these screening methods to rapidly detect products of glycosynthase reactions involving azido sugars as donor groups.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
StateAccepted/In press - 2023

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology


  • Carbohydrate-active enzymes
  • Carbohydrates
  • Directed evolution
  • Enzymology
  • Glycans
  • Glycoconjugates
  • Glycosyl hydrolase
  • Glycosynthase
  • High-throughput screening
  • Human Milk oligosaccharides
  • Rational protein engineering


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