Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides.
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.
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: Jolanda.van_Munster@nottingham.ac.uk.
Jolanda M
JM
van Munster
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: petersanders@eurofins.com.
Peter
P
Sanders
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: g.a.ten.kate@rug.nl.
Geralt A
GA
ten Kate
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: L.Dijkhuizen@rug.nl.
Lubbert
L
Dijkhuizen
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands; Aquatic Biotechnology and Bioproduct Engineering Department, Institute for Technology and Management (ITM), University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Electronic address: M.J.E.C.van.der.Maarel@rug.nl.
Marc J E C
MJ
van der Maarel
D000117:Acetylglucosamine / Q000378:metabolism; D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry / Q000302:isolation & purification; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification
D005656:Fungal Proteins; D009844:Oligosaccharides; D002686:Chitin; D002688:Chitinases; D000117:Acetylglucosamine
Aspergillus niger; Carbohydrate binding module; Chitin oligosaccharide; Chitinase; HPAEC-PAD; Method validation