Preparation of a new chromogenic substrate to assay for b-galactanases that hydrolyse type II arabino-3,6-galactans Naomi X.-Y. Ling, Filomena Pettolino, Ming-Long Liao, Antony Bacic * CRC for Bioproducts, School of Botany, University of Melbourne, Victoria 3010, Australia article info Article history: Received 9 June 2009 Received in revised form 24 July 2009 Accepted 27 July 2009 Available online 12 August 2009 Keywords: b-D-Galactanases Arabinogalactan-protein Chromogenic assay De-arabinosylated gum arabic abstract 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 b-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 b-(1?3)- and/or b-(1?6)-D-galactanases. It will enable rou- tine large-scale screening of b-galactanases from crude enzyme preparations and microorganism cul- tures, and is suitable for profiling activity during purification processes. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction b-Galactanases that cleave (1,3)- and/or (1,6)-D-galactosyl link- ages (referred to simply as b-galactanases) are useful in the analy- sis of the fine structure of type II arabino-(3,6)-galactans (AG), such as those in arabinogalactan-proteins (AGPs). 1–5 Defining the pre- cise structure of type II AGs will assist in furthering our under- standing of their biosynthesis, as well as their biological functions and industrial applications. As none of the known b- (1?3)-galactanases 1,3,6–9 or b-(1?6)-galactanases (no EC num- ber) 2,4,5,10,11 are commercially available, there is a need to screen a range of microorganisms to isolate b-galactanases that can then be cloned and expressed for commercial production. 12 However, a suitable substrate is required in order to do this in a high throughput manner. During the purification and characterisation of enzymes specific for the hydrolysis of type II AGs, both glycosidase and glycanase activities must be monitored. 1–3 Glycanase activities can be deter- mined by using a range of methods. The most common is the vis- cometric approach that measures glycanase activity based on the decrease in solution viscosity due to a molecular size reduction of the substrate. This assay is particularly useful for measuring the activities of endo-glycanases in the presence of exo-glycanases, because comparatively minor viscosity changes will be observed for exo-glycanases over short incubation periods. 13 However, this assay is not convenient for routine large scale screening or where materials (enzyme and substrate) are available in very limited amounts. Furthermore, the sensitivity of the assay depends on the initial viscosity of the substrate that must be sufficiently high to enable detection of viscosity changes associated with the molec- ular size reduction. Unlike many polysaccharides, type II AGs, such as gum arabic, exhibit very low viscosity even at a high concentra- tion; for example, a 50% (w/v) solution of GA has a viscosity similar to that of xanthan gum at 1.5% (w/v). 14 Other than the viscometric assay, there are basically three types of biochemical assays used to measure glycanase activities: (1) measuring the reducing sugar released (e.g., Nelson-Somogyi 15,16 and 2,2 0 -bicinchoninate 17 ), (2) a coupled assay 1,3,18 to detect spe- cific monosaccharides released (e.g., galactose dehydrogenase and glucose oxidase) and (3) the use of specific chromogenic re- agents. 19,20 The first two do not allow one to distinguish between glycanase and glycosidase activities and therefore the preferred option is to use covalently coupled chromogenic substrates. Chromogenic substrate based methods have been reported for the rapid, routine assay of glycanases, such as b-D-mannanase (EC 3.2.1.78), 19 a-amylase (EC 3.2.1.1) 20 and b-(1?4)-D-glucanase (EC 3.2.1.4). 20 These chromogenic assays are sensitive and highly specific for the target enzymes. The substrates have reasonable sol- ubility and their preparation appears to be relatively cost effective. Chromogenic assay approaches have been adapted to a wide range of glycanases, but none are currently available for b-galactanases. Here, an appropriate chromogenic substrate and an assay system for large-scale screening of b-galactanases were established. 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.07.014 * Corresponding author. Tel.: +61 03 83449847; fax: +61 03 93471071. E-mail address: abacic@unimelb.edu.au (A. Bacic). Carbohydrate Research 344 (2009) 1941–1946 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres