NEW ASSAY FOR QUANTIFYING ENDO-BETA-D-MANNANASE ACTIVITY USING CONGO RED-DYE

A gel-diffusion assay for the quantification of endo-beta-D-mannanase (EC 3.2.1.78) activity has been developed. The assay is specific and detects activity as low as 0.07 pkatal yet is linear over five orders of magnitude to 14 nkat. For 70 and 7 pkatals of enzyme activity, the assay was equally effective at pH 3, 5 and 7, although detection was superior at pH 5 for 0.07 pkat enzyme activity. One per cent (w/v) Congo Red dye resulted in fast staining times (15 min) and good contrast. The edges of zones of clearing on gels comprised of 0.7% (w/v) Phytagel were most distinct at substrate concentrations of 0.1% w/v galactomannan in 0.1 M citrate/0.2 M phosphate buffer. The diameter of the clearing zones decreased linearly with increasing substrate concentration in the range of 0.1-1.0% w/v galactomannan. The assay was specific for this endo-enzyme, with no zone of clearing developing for a-galactosidase, and only a slight decrease in dye intensity with beta-mannosidase. The clearing zone diameter was greater in the gel-diffusion assay using Congo Red than using Remazol Brilliant Blue coloured Carob substrate at identical concentrations, enzyme activities and assay conditions. Two different consignments of commercially prepared Aspergillus niger enzyme were indistinguishable when activities were compared by the gel-diffusion assay using Congo Red. The detection sensitivity for the enzyme was similar to that of the viscometric assay (0.14 pkat). The assay was used to quantify the enzyme activity present in seeds, fruit, bulbs and fungi; and repeatability and sensitivity were excellent.