Micro-plate colourimetric assay for Endo -acting cellulase, xylanase, chitinase, 1,3-β-glucanase and amylase extracted from forest soil horizons

Abstract

A new approach, based on the application of soluble, dye-labelled and acid-precipitable polysaccharide derivatives, is introduced for the sensitive assay of polysaccharide endo-hydrolases extracted from soil.An extraction procedure involving sodium acetate-acetic acid buffer (pH 5, 0.5M; 5ml g−1 soil) and an assay system adapted to microtitre plates were developed for routine determinations of endo -acting cellulase, xylanase, chitinase, 1,3-β-glucanase and amylase activities. An Acid Brown Earth under a mature beech forest (Fagus sylvatica L., humusform: typical Moder) was studied with respect to distinct, clearly-developed soil horizons (L, F, H, Ahh, Aeh).Enzymes purified by (NH4)2SO4-precipitation and dialysis were assayed for pH and temperature activity profiles. pH optima were determined in the range of 4.5–5.5, temperature optima were in the range of 40–55°C, revealing stable and fairly similar characteristics of these enzymes in the horizons under study.In routine investigations, highest enzyme activities were determined in the L and F horizons. Significantly decreasing activities with increasing soil depth and decreasing organic matter content were determined in the H, Ahh and Aeh horizons, respectively.Cellulase, xylanase and chitinase activities were highly correlated with total-C content of the soil horizons under study 0.871 ⩽r2⩽ 0.954).