Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Sophie Blanchard, Sylvain Cottaz, Pedro M. Coutinho, Shamkant Patkar, Jesper Vind, Harry Boer, Anu Koivula, Hugues Driguez, Sylvie Armand (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity.
Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209.
Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite.
The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.

Original languageEnglish
Pages (from-to)106-116
JournalJournal of Molecular Catalysis B: Enzymatic
Volume44
Issue number3-4
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

Keywords

  • Glycosynthase
  • Protein engineering
  • Cellulase
  • Oligosaccharide synthesis
  • Enzymatic synthesis

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