title: A novel analytical method for d-glucosamine quantification and its application in the analysis of chitosan degradation by a minimal enzyme cocktail.
abstract: Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Sophanit
authors.initials: S
authors.lastname: Mekasha
authors.affiliation: Apronex s.r.o., Nad Safinou II/365, Vestec, 252 42 Jesenice u Prahy, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Toupalová
authors.affiliation: PT Biotech Surindo, Komp. Pelabuhan Perikanan Kejawanan Jl. Pelabuhan Perikanan No. 1, Cirebon, West Java 45113, Indonesia.
authors.forename: Eka
authors.initials: E
authors.lastname: Linggadjaja
authors.affiliation: PT Biotech Surindo, Komp. Pelabuhan Perikanan Kejawanan Jl. Pelabuhan Perikanan No. 1, Cirebon, West Java 45113, Indonesia.
authors.forename: Harish A
authors.initials: HA
authors.lastname: Tolani
authors.affiliation: Apronex s.r.o., Nad Safinou II/365, Vestec, 252 42 Jesenice u Prahy, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Anděra
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Magnus Ø
authors.initials: MØ
authors.lastname: Arntzen
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Gustav
authors.initials: G
authors.lastname: Vaaje-Kolstad
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway; Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs Lyngby, Denmark. Electronic address: jaag@kt.dtu.dk.
authors.forename: Jane W
authors.initials: JW
authors.lastname: Agger
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D048271:Chitosan / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005944:Glucosamine / Q000032:analysis* / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013291:Streptococcus / Q000201:enzymology; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D001617:beta-Glucosidase; D005944:Glucosamine
keywords: Chitosan; Chitosanase; Glucosamine; HPAEC-PAD; N-acetyl-d-glucosamine (PubChem CID:439174); d-glucosamine (PubChem CID:439213); exo-β-glucosaminidase