Engineering chitinases for the synthesis of chitin oligosaccharides: Catalytic amino acid mutations convert the GH-18 family glycoside hydrolases into transglycosylases

E. A. Martinez, Harry Boer, Anu Koivula, E. Samain, H. Driguez, S. Armand, S. Cottaz (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

28 Citations (Scopus)

Abstract

Two family GH-18 chitinases mutated on the key catalytic amino acids were evaluated as “glycosynthases” for the coupling of oxazoline activated donor to chitin oligosaccharide (COs) acceptors obtained from microbial fermentation. Bacillus circulans WL-12 chitinase A1 (Bc ChiA1) and Trichoderma harzanium chitinase 42 (Th Chit42) were individually mutated on the three conserved carboxylic acids, all suggested to have a role in catalysis: the general acid/base glutamate and the two aspartates, defined as the putative stabilizer and its assistant. The mutants D200A and D202A of Bc ChiA1, together with D170N and to a lesser extent D170A of Th Chit42 proved to be active for chitinbiose oxazoline polymerization, and also for coupling reaction between Gal(β1 → 4) chitinbiose oxazoline and chitinpentaose at neutral pH. These mutants have additionally retained the ability to catalyze transglycosylation reaction on natural COs, whereas their hydrolytic activity is abolished. Such mutants can be considered as chitin transglycosylases, verifying also the fact that the two conserved aspartic acids, the stabilizer and its assistant, are not a prerequisite for the formation of oxazolinium intermediate by acetamido anchimeric assistance, i.e. for the substrate-assisted catalysis mechanism of family GH-18 chitinases.
Original languageEnglish
Pages (from-to)89-96
Number of pages8
JournalJournal of Molecular Catalysis B: Enzymatic
Volume74
Issue number1-2
DOIs
Publication statusPublished - 2012
MoE publication typeA1 Journal article-refereed

Keywords

  • Oligosaccharide
  • Enzymatic synthesis
  • Chitinase
  • Protein engineering
  • Glycosynthase

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