Preparation of a new chromogenic substrate to assay for beta-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 beta-galactanases. dGA was prepared by partial acid hydrolysis (0.25M trifluoroacetic acid for 2h at 90-95 degrees C) of gum Arabic (GA) from Acacia senegal. The dGA exhibited a median molecular mass of approximately 10kDa, corresponding to a degree of polymerisation (DP) approximately 60. It was devoid of Ara residues, and contained mostly Galp (68mol%) together with GlcpA (30mol%). The Galp residues were (1,6)- (34mol%), (1,3)- (3mol%) and (1,3,6)- (26mol%) linked, and the GlcAp residues were primarily terminal (28mol%) together with a small amount of (1,4)-linked (2mol%), as expected for a type II (3,6)-galactan. The new chromogenic assay is simple, cost effective, relatively sensitive, and is specific for either beta-(1-->3)- and/or beta-(1-->6)-d-galactanases. It will enable routine large-scale screening of beta-galactanases from crude enzyme preparations and microorganism cultures, and is suitable for profiling activity during purification processes.
Ling
Naomi X-Y
NX
University of Melbourne, Victoria, Australia.
Pettolino
Filomena
F
Liao
Ming-Long
ML
Bacic
Antony
A
D002863:Chromogenic Compounds / Q000737:chemistry* / Q000378:metabolism*; D005685:Galactans / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D015394:Molecular Structure
D002863:Chromogenic Compounds; D005685:Galactans; D006026:Glycoside Hydrolases; C089138:beta-galactanase