Protease extraction from soil by sodium pyrophosphate and chemical characterization of the extracts

Two arable soils and one pasture soil had previously been air-dried for 6 d and stored at room temperature. The enzyme activities remaining after this treatment were constant. The soils were then extracted with 140 mM sodium pyrophosphate at pH 7.1. Amino acid N and total organic C content of soils and soil extracts, together with humic and fulvic acids content of soil extracts were determined. Total organic C was determined in soil residues obtained after extraction. Chemical characterization of the organic matter of soils, soil extracts and soil residues was carried out by pyrolysis-gas chromatography (Py-GC). Protease activity was determined in soil extracts and soil residues by using three different substrates: N-benzoyl-L-argininamide (BAA), specific for trypsin; N-benzyloxycarbonyl-L-phenylalanyl L-leucine (ZPL), specific for carboxypeptidases, and casein, essentially nonspecific. Comparative studies between specific activities referred to organic C in soils, soil extracts and soil residues and their corresponding pyrogram composition, and also between total extracted or residual activity and the humine or unhumified organic matter content of the corresponding soil, allowed us to establish hypotheses about the type of organic matter the enzymes are associated with. From 12% to 21% of the soil organic C (33% to 39% of which were humic acids) and from 3% and 18% of amino acid N were extracted from soil using pyrophosphate. Py-GC analyses showed that pyrophosphate was effective in extracting condensed humic substances and glycoproteins and that the organic matter present in soil extracts was especially rich in intact or partially-decomposed fresh residues of carbohydrate origin and also in certain humus-associated proteins. Extracted BAA-hydrolysing activity accounted for 11% to 36% of the soil activity, depending on soil type. Extracted ZPL- and casein-hydrolysing activities were, with one exception, remarkably high, accounting for about 100% or even more of the soil activity, depending on soil type. According to the results BAA-hydrolysing proteases are probably mostly associated with highly condensed humus, ZPL-hydrolysing proteases with less condensed humic substances and casein-hydrolysing proteases with fresh organic matter.