Preparation of a new chromogenic substrate to assay for β-galactanases that hydrolyse type II arabino-3,6-galactans

• Published in: Carbohydrate research 2009-10, Vol.344 (15), p.1941-1946
• Main Authors: Ling, Naomi X.-Y., Pettolino, Filomena, Liao, Ming-Long, Bacic, Antony
• Format: Article
• Language: English
• Subjects: Arabinogalactan-protein; Chromogenic assay; Chromogenic Compounds - chemistry; Chromogenic Compounds - metabolism; De-arabinosylated gum arabic; Galactans - chemistry; Galactans - metabolism; Glycoside Hydrolases - metabolism; Hydrolysis; Molecular Structure; β- d-Galactanases
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/j.carres.2009.07.014

A colorimetric assay specific for β-(1→3)- and β-(1→6)- d-galactanases was developed based on the use of a chromogenic substrate. The substrate (RB5-dGA) was produced by covalently coupling Reactive Black 5 (RB5) dye to de-arabinosylated gum arabic (dGA), prepared by partial acid hydrolysis of gum arabic (GA) from Acacia senegal. A chromogenic assay using RB5-dGA, Reactive Black 5 (RB5) dye covalently coupled to de-arabinosylated gum arabic (dGA), was developed for rapid screening of β-galactanases. dGA was prepared by partial acid hydrolysis (0.25 M trifluoroacetic acid for 2 h at 90–95 °C) of gum Arabic (GA) from Acacia senegal. The dGA exhibited a median molecular mass of ∼10 kDa, corresponding to a degree of polymerisation (DP) ∼60. It was devoid of Ara residues, and contained mostly Gal p (68 mol %) together with Glc pA (30 mol %). The Gal p residues were (1,6)- (34 mol %), (1,3)- (3 mol %) and (1,3,6)- (26 mol %) linked, and the GlcA p residues were primarily terminal (28 mol %) together with a small amount of (1,4)-linked (2 mol %), as expected for a type II (3,6)-galactan. The new chromogenic assay is simple, cost effective, relatively sensitive, and is specific for either β-(1→3)- and/or β-(1→6)- d-galactanases. It will enable routine large-scale screening of β-galactanases from crude enzyme preparations and microorganism cultures, and is suitable for profiling activity during purification processes.