Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides

Jolanda M van Munster, Peter Sanders, Geralt A ten Kate, Lubbert Dijkhuizen, Marc J E C van der Maarel

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The abundant polymer chitin can be degraded by chitinases (EC 3.2.1.14) and β-N-acetyl-hexosaminidases (EC 3.2.1.52) to oligosaccharides and N-acetyl-glucosamine (GlcNAc) monomers. Kinetic characterization of these enzymes requires product quantification by an assay method with a low detection limit, preferably compatible with the use of native, non-labeled substrates. Here we report a quantitative HPAEC-PAD method that allows fast separation of chitin oligosaccharides (COS) ranging from (GlcNac)1-6 at detection limits of 1-3 pmol and a linear range of 5-250 pmol. Quantification under intra- and interday precision conditions was performed with 2.1-5.4% relative standard deviation (RSD) and 1.2-10.3% RSD, respectively. This method was successfully used for the determination of the kinetic parameters of the Aspergillus niger chitinase CfcI with native COS. CfcI was recently shown to release GlcNAc from the reducing end of COS, a new activity for fungal chitinases. A Carbohydrate Binding Module of family 18 (CBM18) is inserted in the CfcI catalytic domain. Site directed mutagenesis was used to assess the functionality of this CfcI-CBM18: four of its key amino acids were replaced by glycine residues, yielding CfcISYNF. Comparison of the kinetic parameters of CfcI and CfcISYNF confirmed that this CBM18 is functionally involved in catalysis.

Original languageEnglish
Pages (from-to)73-8
Number of pages6
JournalCarbohydrate Research
Volume407
Early online date3 Feb 2015
DOIs
Publication statusPrint publication - 30 Apr 2015
Externally publishedYes

Bibliographical note

Copyright © 2015 Elsevier Ltd. All rights reserved.

Keywords

  • Acetylglucosamine/metabolism
  • Aspergillus niger/chemistry
  • Catalytic Domain
  • Chitin/chemistry
  • Chitinases/chemistry
  • Chromatography, Ion Exchange/methods
  • Fungal Proteins/metabolism
  • Kinetics
  • Mutagenesis, Site-Directed
  • Oligosaccharides/chemistry

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