Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides
| Autor: | Jolanda M. van Munster, Peter Sanders, Geralt A. ten Kate, Lubbert Dijkhuizen, Marc J.E.C. van der Maarel |
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| Přispěvatelé: | Host-Microbe Interactions, Bioproduct Engineering |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Method validation
PULSED AMPEROMETRIC DETECTION Oligosaccharides Chitin Biochemistry ELECTROPHORESIS Chitin oligosaccharide Analytical Chemistry Acetylglucosamine Cell wall Fungal Proteins HEVEIN DOMAINS Carbohydrate binding module ANION-EXCHANGE CHROMATOGRAPHY Catalytic Domain BINDING HPAEC-PAD AFFINITY biology CHITOOLIGOSACCHARIDES Chemistry CARBOHYDRATE ANALYSIS Organic Chemistry Aspergillus niger Chitinases Chitinase RECOGNITION General Medicine biology.organism_classification Chromatography Ion Exchange TRICHODERMA-HARZIANUM Kinetics Mutagenesis Site-Directed Carbohydrate active enzymes |
| Zdroj: | Carbohydrate Research, 407, 73-78. ELSEVIER SCI LTD |
| ISSN: | 0008-6215 |
| Popis: | The abundant polymer chitin can be degraded by chitinases (EC 3.2.1.14) and beta-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 CfcI(SYNF). Comparison of the kinetic parameters of CfcI and CfcISYNF confirmed that this CBM18 is functionally involved in catalysis. (C) 2015 Elsevier Ltd. All rights reserved. |
| Databáze: | OpenAIRE |
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