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 d etects activity as low as 0.07 pkatal yet is linear over five orders o f magnitude to 14 nkat. For 70 and 7 pkatals of enzyme activity, the a ssay was equally effective at pH 3, 5 and 7, although detection was su perior 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. T he 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 galactoman nan in 0.1 M citrate/0.2 M phosphate buffer. The diameter of the clear ing zones decreased linearly with increasing substrate concentration i n 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-galactosid ase, and only a slight decrease in dye intensity with beta-mannosidase . The clearing zone diameter was greater in the gel-diffusion assay us ing Congo Red than using Remazol Brilliant Blue coloured Carob substra te at identical concentrations, enzyme activities and assay conditions . Two different consignments of commercially prepared Aspergillus nige r enzyme were indistinguishable when activities were compared by the g el-diffusion assay using Congo Red. The detection sensitivity for the enzyme was similar to that of the viscometric assay (0.14 pkat). The a ssay was used to quantify the enzyme activity present in seeds, fruit, bulbs and fungi; and repeatability and sensitivity were excellent.