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

Fingerprint

Cellulase
Substrate Specificity
Bearings (structural)
Aspergillus oryzae
Trisaccharides
Cellulases
Mutation
Nucleophiles
Trichoderma
Sequence Alignment
Aspergillus
Substrates
Histidine
Alanine
Yeast
Saccharomyces cerevisiae

Keywords

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

Cite this

Blanchard, Sophie ; Cottaz, Sylvain ; Coutinho, Pedro M. ; Patkar, Shamkant ; Vind, Jesper ; Boer, Harry ; Koivula, Anu ; Driguez, Hugues ; Armand, Sylvie. / Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity. In: Journal of Molecular Catalysis B: Enzymatic. 2007 ; Vol. 44, No. 3-4. pp. 106-116.
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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.",
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author = "Sophie Blanchard and Sylvain Cottaz and Coutinho, {Pedro M.} and Shamkant Patkar and Jesper Vind and Harry Boer and Anu Koivula and Hugues Driguez and Sylvie Armand",
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Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity. / Blanchard, Sophie; Cottaz, Sylvain; Coutinho, Pedro M.; Patkar, Shamkant; Vind, Jesper; Boer, Harry; Koivula, Anu; Driguez, Hugues; Armand, Sylvie (Corresponding Author).

In: Journal of Molecular Catalysis B: Enzymatic, Vol. 44, No. 3-4, 2007, p. 106-116.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

AU - Blanchard, Sophie

AU - Cottaz, Sylvain

AU - Coutinho, Pedro M.

AU - Patkar, Shamkant

AU - Vind, Jesper

AU - Boer, Harry

AU - Koivula, Anu

AU - Driguez, Hugues

AU - Armand, Sylvie

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

KW - Glycosynthase

KW - Protein engineering

KW - Cellulase

KW - Oligosaccharide synthesis

KW - Enzymatic synthesis

U2 - 10.1016/j.molcatb.2006.08.009

DO - 10.1016/j.molcatb.2006.08.009

M3 - Article

VL - 44

SP - 106

EP - 116

JO - Journal of Molecular Catalysis B: Enzymatic

JF - Journal of Molecular Catalysis B: Enzymatic

SN - 1381-1177

IS - 3-4

ER -