Determination of Glucosamine and N-Acetyl Glucosamine in Fungal Cell Walls

Abstract

A new method was developed to determine glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in materials containing chitin and chitosan, such as fungal cell walls. It is based on two steps of hydrolysis with (i) concentrated sulfuric acid at low temperature and (ii) dilute sulfuric acid at high temperature, followed by one-step degradation with nitrous acid. In this process, chitin and chitosan are converted into anhydromannose and acetic acid. Anhydromannose represents the sum of GlcN and GlcNAc, whereas acetic acid is a marker for GlcNAc only. The method showed recovery of 90.1% of chitin and 85.7-92.4% of chitosan from commercial preparations. Furthermore, alkali insoluble material (AIM) from biomass of three strains of zygomycetes, Rhizopus oryzae, Mucor indicus, and Rhizomucor pusillus, was analyzed by this method. The glucosamine contents of AIM from R. oryzae and M. indicus were almost constant (41.7 +/- 2.2% and 42.0 +/- 1.7%, respectively), while in R. pusillus, it decreased from 40.0 to 30.0% during cultivation from 1 to 6 days. The GlcNAc content of AIM from R. oryzae and R. pusillus increased from 24.9 to 31.0% and from 36.3 to 50.8%, respectively, in 6 days, while it remained almost constant during the cultivation of M. indicus (23.5 +/- 0.8%).

Production of Ethanol and Biomass from Thin Stillage Using Food-Grade Zygomycetes and Ascomycetes Filamentous Fungi

“…The AIM was separated via centrifugation (5000 × g, 5 min), washed until neutral pH and dried using a freeze-dryer (Labconco, Kansas City, MO, USA). The cell wall contents of glucosamine and N-acetylglucosamine were determined according to a previous method [22] with some modifications; specifically, the acid hydrolysate was diluted to a glucosamine range of 0.01-0.1 g· mL −1 and the pH was adjusted to 3 with 0.5 M NaAc before addition of 3-metyl-2-benzothiozolone-hydrazone-hydrochloride (MTBH) [23].…”

Characterization and Properties of Chitosan

“…HPLC of the reaction mixture of chitosan: (1) formic acid, (2) acetic acid, and (3) propionic acid (reproduced from Niola et al, 1993). (Niola et al,1993;Zamani et al, 2008). The degree of deacetylation (DD) was calculated according to formula 7:…”

“…• Elemental analysis (Davies & Hayes, 1988;dos Santos et al, 2009); • Titration (Arcidiacono & Kaplan, 1992;Balázs & Sipos, 2007;de Alvarenga et al, 2010;Hattori et al, 1999;Park et al, 1983;Raymond et al, 1993;Tôei & Kohara, 1976;Zhanga et al, 2011); • Hydrolytic methods (Davies & Hayes, 1988;Nanjo et al, 1991;Niola et al, 1993;Sato et al, 1998;Zamani et al, 2008); • HPLC -Ultraviolet (Aiba, 1986;Muzzarelli & Rocchetti, 1985); • Infrared (Baxter et al, 1992;Duarte et al, 2002;Kasaai, 2008;Miya et al, 1980;Moore & Roberts, 1980;Sannan et al, 1978); • 1 H nuclear magnetic resonance (Brugnerotto et al, 2001;de Alvarenga et al, 2010;Fernandez-Megia et al, 2005;Lavertu et al, 2003;Varum et al, 1991); • CP-MAS 13 C NMR (Heux et al, 2000;Manni et al, 2010;Raymond et al, 1993); • CP-MAS 15 N NMR (Kasaai, 2009;Yu et al, 1999); • Many other methods are described in the literature but with a somewhat smaller appeal. Some of these methods are: steric exclusion chromatography (Brugnerotto et al, 2001), nitrous acid deamination (Sashiwa et al, 1991), thermal analysis (Garcia Alonso et al, 1983), gas chromatography with columns packed with chitin and chitosan (Muzzarelli et al, 1980),...…”

Utilisation of Jatropha press cake as substrate in biomass and lipase production from Aspergillus niger 65I6 and Rhizomucor miehei CBS 360.62

“…All experiments were done in duplicate. The solids retained on the Miracloth filter were dried and the N--acetyl glucosamine content, as a measure of the amount of fungal biomass, was determined using the method described previously (Zamani et al, 2008).…”