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

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 Galp (68 mol %) together with GlcpA (30 mol %). The Galp residues were (1,6)- (34 mol %), (1,3)- (3 mol %) and (1,3,6)- (26 mol %) linked, and the GlcAp 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.

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.Figure optionsDownload as PowerPoint slide