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- -argininamide (BAA), specific for trypsin; N-benzyloxycarbonyl- -phenylalanyl -leucine (ZPL), specific for carboxypeptidases, and casein, essentially non-specific. 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.     

Hydrolases extracted from soil: Kinetic parameters of several enzymes catalysing the same reaction

The kinetic parameters (K_m and V_max) of ureases, phosphatases, casein and benzoylargininae-mide-hydrolysing proteases extracted from two different soils were determined. The Eadie-Scatchard plot, which is the most sensitive graphical technique for detecting deviations from Michaelis-Menten kinetics, was used. In the case of phosphatases extracted from two soils and of the benzoylarginineamide hydrolysing protease extracted from one soil the relationship between V/S and V produced two straight lines. The presence of at least two enzymes (or two forms of the same enzyme) catalysing the same reaction and characterized by markedly different K_m and V_max values, was indicated. A procedure to determine the true constants was reported. The conclusion was that the presence in soil of high and low-affinity enzymes permits an efficient reaction at either low or high substrate concentration.     

Fractionation of humus-urease complexes

Some techniques commonly used for enzyme purification were unsuitable to purify urease extracted by pyrophosphate from soil. An all-or-none behaviour seemed to be a prominent feature of such experiments but in some instances slight increases of specific activity were observed.The most effective purification of soil urease was achieved by exhaustively ultrafiltrating the soil extract against 0.1 m pyrophosphate at pH 7.1, separating the retained material into fractions of mol. wt. higher (A_I) and lower (A_II) than 10⁵, followed by gel chromatography.Increases in total activities were observed both after ultrafiltration and gel chromatography. Ultra-filtration increased the total activity of the extracted urease by about 8%. The specific activity of fraction A_I increased four fold, that of fraction A_II by more than three fold. Fractions obtained by gel chromatography accounted in toto for only 13.5% of the organic-C of the soil extract. Total urease activity increased by 45.6%. Specific activities increased to 6.9–18 times that of the soil extract.     

Bovine serum albumin and Triton X-100 greatly increase phosphomonoesterases and arylsulphatase extraction yield from soil

Extraction of arylsulphatase (aryS), acid (acP) and alkaline phosphomonoesterase (alkP) from six different soils using sodium pyrophosphate (0.14 M, pH 7.1) yielded brown extracts displaying enzymatic activity mostly below detection limit. Tris-HCl (50 mM, pH 7.5) gave an extraction yield, on average, lower than 0.5%, but addition of Triton X-100 or bovine serum albumine (BSA) to Tris buffer increased the extraction yield 2-8 times. When both Triton X-100 and BSA were added to the buffer, the extraction yield was more than additive and reached 2-13% for acP, 2-5% for alkP and 3-6% for aryS, depending on the soil. In addition, these extracts were colourless or at most light yellow, showing that besides the high yield enzymes were extracted along with negligible amounts of humic substances.     

Changes in hot water soil extracts brought about by nitrogen immobilization and mineralization processes during incubation of amended soils

Summary During incubation of an acid cambisol and an alkaline fluvisol, amended with glucose and nitrate, hot water soil extracts were analysed for N content, ultraviolet absorption, and fluorescence. Humic substances in the hot water extracts and in a neutral sodium pyrophosphate extract were fractionated on polyvinylpyrrolidone and measured spectroscopically. Changes in the hot water and pyrophosphate extract compositions were related to changes in microbial biomass, as estimated by substrate-induced respiration, and the hexosamine content of soil hydrolysates. During the incubation, the microbial population in each type of soil developed quite differently, according to the soil pH. Microbial growth and death in the alkaline soil sample induced a maximum of hot-water-extractable ultraviolet-absorptive non-fluorescent substances. The fluorescence of the hot water soil extract increased steadily with incubation time even after the microbial activity was reduced. A similar increase in fluorescence, in accord with the ultraviolet absorption, was found during incubation of the acid soil samples. After 95 days of incubation, the hot-water-extractable fluorescent and ultraviolet-absorptive substances were reduced. N immobilization induced an increase, and N mineralization a decrease, in dissolved organic N. The relative increase in humic substances in the hot water soil extract was much higher than in the pyrophosphate extract. Therefore, humic material, produced by microbial growth and death, is obviously extractable with hot water.     

Enzymatic hydrolysis of soil organic phosphorus by immobilized phosphatases

 In order to estimate the role of phosphatases in maintaining the potential bioavailable P pool in soils, water and 0.4 M NaOH soil extracts were incubated with immobilized acid phosphatase, alkaline phosphatase, phospholipase and nuclease, separately, and in combinations. Immobilized nuclease at an optimum pH of 7.0 hydrolyzed the most soluble unreactive P (SUP) both in water and 0.4 M NaOH extracts. The combination of immobilized alkaline phosphatase and nuclease increased the hydrolysis of SUP at pH 7.0 by up to 61% in 0.4 M NaOH extracts relative to that due to immobilized nuclease alone. The combination of immobilized acid phosphatase and nuclease, however, did not increase the hydrolysis of SUP in either extract relative to that due to immobilized nuclease alone. Immobilized alkaline phosphatase and phospholipase increased the hydrolysis of SUP at pH 7.0 by up to 62% in 0.4 M NaOH extracts relative to that due to immobilized phospholipase alone. Similarly, immobilized acid phosphatase and phospholipase increased the hydrolysis of SUP at pH 7.0 by up to 49% in 0.4 M NaOH extracts relative to that due to immobilized phospholipase alone. The similarities in the optimum pH of indigenous phosphatases in soils and the immobilized phosphatases used in this study, immobilized on positively charged supports, suggests that indigenous phosphatases could be immobilized on positively charged surfaces in soils. Received: 17 November 1998     

Response of phosphomonoesterases in soils to chloroform fumigation

The chloroform fumigation technique has been successfully employed to quantify intracellular and extracellular urease and arylsulfatase activities in soil. In this study, the same approach was evaluated for its ability to differentiate between various pools of phosphomonoesterase activities in soils and reference proteins purified from plant and microbial sources. The activities of acid and alkaline phosphatases were assayed in 10 surface soils and reference proteins at their optimal pH values before and after chloroform fumigation and in the presence and absence of toluene. Chloroform fumigation decreased the activities of acid and alkaline phosphatases in soils, on average, by 6 and 8%, respectively. Similarly, the activities of two purified reference enzyme proteins were decreased after fumigation, with acid and alkaline phosphatase activities exhibiting a reduction of 17 and 8%, respectively. Toluene treatment caused an increase in the activities of acid and alkaline phosphatases by 8 to 18% in nonfumigated soils, but showed no effect in the fumigated soils. Average enzyme protein concentrations, calculated for the 10 soils based on the activity values of the soils and the specific activity of the purified enzymes (i.e., activity values per mg protein), were 22.5 and 2.1 mg protein (kg soil)^—1 for acid and alkaline phosphatase, respectively. The decrease in enzyme activity by the fumigant was either by direct denaturing of the periplasmic and extracellular portion of the particular protein after lysis of the microbial cell membrane, by absorption and/or inhibition of the released phosphomonoesterases by organic and inorganic constituents or by degradation of the protein by soil proteases. The ratios of acid phosphatase protein concentrations relative to organic C in six soils were significantly, but negatively correlated with soil organic C, suggesting differences in organic C quality. Comparison of the activity values of soil phosphatases with those of the protein concentrations present in soils indicated that alkaline phosphatase has greater catalytic efficiency than does acid phosphatase.     

Phosphomonoesterase,phosphodiesterase, phosphotriesterase,and inorganic pyrophosphatase activities in forest soils in an alpine area: effect of pH on enzyme activity and extractability

We investigated different types of phosphatase activity (phosphomono-, phosphodi-, phosphotriesterase, inorganic pyrophosphatase) in five forest soils in Vorarlberg, Austria. Phosphatase activity was determined both in soils and in soil extracts prepared with different solutions (distilled water, 0.1M sodium pyrophosphate at pH 7, 0.1M sodium phosphate buffer/1M KCl at pH 6.5, and a modified universal buffer at pH 4, 6.5, 9, and 11). High phosphomonoesterase activity in these soils indicated a severe deficiency in available P. Acidic phosphomonoesterase prevailed over alkaline phosphomonoesterase activity. Phosphodiesterase was highest in the least acidic soil but no general trend towards an optimum pH was recognized. Phosphotriesterase activity was observed in only two of the five soils and favoured an alkaline optimum pH; this activity was not detected in strongly acid soils. Inorganic pyrophosphatase activity was high in soils with no phosphotriesterase. Phosphomonoesterase, phosphodiesterase and inorganic pyrophosphatase activities were much lower in soil extracts than in soils.     

Effect of Increasing Concentration of Inorganic Phosphate (Pi) and Pyrophosphate (PPi) on Growth and Phosphatases of Germinating Chickpea Seeds

Chickpea (Cicer arietinum L.) seedlings growing on different concentrations of inorganic phosphate and pyrophosphate in agar based MS-medium were studied for their growth and activities of phosphatases in cotyledon, shoots and roots. Growth of seedlings was affected with both Inorganic Phosphate (Pi) and Pyrophosphate (PPi). Germination was completely inhibited beyond 100 mM monopotassium phosphate (KH₂PO₄) and 20 mM sodium pyrophosphate. Specific activities of acid phosphatases of cotyledons, shoots and roots decreased under high Pi-supply however alkaline phosphatases were not affected. Addition of PPi increased specific activities of acid phosphatases of roots and shoots at 3 days after germination (DAG) stage, but decreased at later stages of seedling growth. There was an appearance of PPi-specific acid phosphatase in roots under PPi-supply.     

Regularities of extracting humic acids from soils using sodium pyrophosphate solutions

Regularities of extracting humic acids from different soil types (soddy-podzolic soil, gray forest soil, and all chernozem subtypes) with sodium pyrophosphate solutions at different pH values (from 5 to 13) have been studied. It is found that, regardless of soil type, the process occurs in two stages through the dissociation of carboxylic groups and phenolic hydroxyls, each of which can be described by a logistic function. Parameters of the logistic equations approximating the extraction of humic acids from soils at different pH values are independent of the content and composition of humus in soils. Changes in the optical density of humic acids extracted from soils using sodium pyrophosphate solutions with different pH values are described in the first approximation by the Gaussian function. The optically densest humic acids are extracted using sodium pyrophosphate solutions at pH 10. Therefore, it is proposed to use an extract with pH 10 for the characterization of organic matter with the maximum possible degree of humification in the given soil.     

Extraction,purification and properties of soil hydrolases

Invertase, cellulase, phosphatases, protease and β-glucosidase were extracted from permanent pasture soil with 0.2 M phosphate buffer (pH 8) in the presence of 0.2 M EDTA. This extract was further treated with ammonium and salmine sulphates. Attempts were made to fractionate these enzyme activities by gel and anion-exchange chromatography. Specific activities were estimated in all fractions and some characteristics of the purified enzymes (optimum pH, temperature and substrate concentration, and K_m and V_max) were investigated. The results indicated that extracted enzyme activities occurred partly in soil as a carbohydrate-enzyme complex and partly as a humo-carbohydrate complex.     

Fractionation of hydrolase-humus complexes by gel chromatography

Summary Purification of soil phosphatase-, urease-, casein- and benzoylarginamide-hydrolysing proteases was obtained by exhaustively ultrafiltering a soil extract using 0.1M pyrophosphate solution at pH 7.1, separating the retained material into fractions of molecular weight higher (A_I) and lower (A_II) than 10⁵ and eluting the fractions on gel chromatography.Three peaks of phosphatase and urease activity were obtained after gel chromatography of fraction A_I on Sephadex G200 using 0.1M pyrophosphate solution as eluant. Only one distinct activity peak was observed when casein- and benzoylarginamide-hydrolysing proteases were assayed in the eluted fractions. Elution diagrams obtained by gel chromatography of fraction A_II on Sephadex G100, using a water as eluant, were characterized by one peak each of phosphatase-, casein- and benzoylarginamide-hydrolysing activity and by two peaks of urease activity.Gel chromatography of both A_I and A_II, generally, but not always, increased specific activity on a C and N basis of derivative fractions. Both proteases showed the highest increase in specific activity due to a marked decrease in organic C and N and an increase in total activity.     

Humic materials from an organic soil: A comparison of extractants and of properties of extracts

Five extraction procedures and thirteen extracting reagents, which included dipolar aprotic solvents, organic chelating agents, pyridine, ethylenediamine, sodium hydroxide, ion-exchange resins and two salts (sodium pyrophosphate and ethylenediamine hydrochloride), were used to extract humic materials from an organic soil. Extractabilities increased in the general order: salts < organic chelating agents < dipolar aprotic solvents < pyridine < ethylenediamine = sodium hydroxide, and the amounts of the soil organic matter extracted by the reagents in the series ranged from 13 to 63%. Gel chromatography techniques indicated that extracts in dipolar aprotic solvents were predominantly of intermediate and low molecular-weight values, and it is suggested that the more highly oxidised soil humic materials were extracted in these. The more efficient solvents extracted materials with a range (high—low) of molecular-weight values. Data from elemental analysis and from E.S.R. measurements indicated that ethylenediamine altered the chemical nature and the composition of extracts. Dipolar aprotics, by the same criteria, were found not to alter the humic extracts, and can be regarded as mild reagents for the extraction of a less representative (of the total) fraction of soil organic matter. Sodium hydroxide in solution, despite its oxidation effects, was the best of the reagents tested for isolating extracts which were representative of a wide range of soil humic substances.     

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.     

Use of 0·1 m pyrophosphate to extract urease from a podzol

Sodium pyrophosphatc (0·1 m) at pH 7.1 and 37°C extracted a significant fraction of urease from a podzol. Maximum extraction values were obtained after 18 h. The yields of soil organic matter and urease activity during the extraction show a different pattern: the extraction of non-specific organic matter precedes and may facilitate the following extraction of an active urease organo-complex. The urease extracted by pyrophosphate is about 30 40 per cent of the total urease activity, as shown by plotting the urease activity against the population changes of ureolytic microorganisms, both in the original and extracted soil. The number of ureolytic microorganisms is unaffected by pyrophosphate, and the extracted urease is assumed to be extracellular.     

Characterization of free and adsorbed phosphatases in soils

Summary The kinetic and thermodynamic parameters of soil alkaline phosphatases, extracted humus-enzyme complexes, and water extracts were evaluated. The isolation procedure for humus-enzyme complexes involves exhaustive extraction of the soil with the chelating resin Chelex 100. The K _m values for alkaline phosphatases in the resin extracts of two soils were 0.881 and 2.236 mM, respectively, and were greater than those of the water extracts (0.320 and 0.527 mM) and the soil suspensions (0.445 and 0.652 mM). The v _max values varied considerably, indicating the fundamental role of the enzyme concentration in the fractions and soils. The temperature of inactivation in the water extracts was 30°C, being 10° and 20°C lower than the temperature needed to inactivate alkaline phosphatase in humus-enzyme complexes and in soils, respectively. The results indicated that only a small portion of the enzyme activity was in a soluble state and that this fraction was characterized by a low thermal stability. The sorption of alkaline phosphatases to humic substances gives them additional thermal stability and leads to a partial inhibition of the enzyme.Dedicated to the late Prof. Dr. W. Kühnelt     

DISTRIBUTION OF PYROPHOSPHATE-EXTRACTABLE IRON AND ORGANIC CARBON IN SOILS OF VARIOUS GROUPS

Potassium pyrophosphate (0.1m ) removes very little Fe from crystalline Fe oxides at pH 10, but peptizes finely divided hydrous amorphous oxides and organic matter in soils. Fe and C contents of extracts from each horizon of twenty-six British soil profiles show distinctive patterns, independent of the residual dithionite-soluble Fe. Thus extracts of humus Fe podzols have maximum Fe and C in the B horizon, peaty gley podzol has maximum Fe in the B horizon but maximum C in the surface. These groups are differentiated from non-podzols which have maximum pyrophosphate extractable Fe and C in the surface horizon, decreasing with depth. Intermediate patterns help to quantify differences in soils of classes having properties of more than one soil group.     

Effect of tillage and residue management on enzyme activities in soils: III. Phosphatases and arylsulfatase

This study was carried out to investigate the effect of tillage and residue management on activities of phosphatases (acid phosphatase, alkaline phosphatase, phosphodiesterase, and inorganic pyrophosphatase) and arylsulfatase. The land treatments included three tillage systems (no-till, chisel plow, and moldboard plow) in combination with corn residue placements in four replications. The activities of these enzymes in no-till/double mulch were significantly greater than those in the other treatments studied, including no-till/bare, no-till/normal, chisel/normal, chisel/mulch, moldboard/normal, and moldboard/mulch. The effect of mulching on activities of phosphatases was not as significant as on activities of arylsulfatase. The lowest enzyme activities were found in soil samples form no-till/bare and moldboard/normal treatments, with the exception of inorganic pyrophosphatase, which showed the lowest activity in no-till/bare only. Among the same residue placements, no-till and chisel plow showed comparable arylsulfatase activity, whereas the use of moldboard plow resulted in much lower arylsulfatase activity. The activities of phosphatases and arylsulfatase were significantly correlated with organic C in the 40 soil samples studies, with r values ranging from 0.71*** to 0.92***. The activities of alkaline phosphatase, phosphodiesterase, and arylsulfatase were significantly correlated with soil pH, with r values of 0.85***, 0.78***, and 0.77***, respectively, in the 28 surface soil samples studied, but acid phosphatase and inorganic pyrophosphatase activities were not significantly correlated with soil pH. The activities of phosphatases and arylsulfatase decreased markedly with increasing soil depth and this decrease was associated with a decrease in organic C content. The activities of these enzymes were also significantly intercorrelated, with r values ranging from 0.50*** to 0.92***. Received: 4 October 1995     

Isoelectric focusing of β-glucosidase humic-bound activity in semi-arid Mediterranean soils under management practices

Changes in β-glucosidase enzyme–humic complexes and conventional parameters (pH, total organic C, total N, water-soluble C, and bulk density) were studied in an almond-cropped soil prone to erosion under a rehabilitation practice. The experimental plan included three soil slopes (0%, 2%, and 6%) and two type of fertilization (organic and mineral), with sampling of rhizosphere and inter-row soils. The enzyme humic complexes were extracted by pyrophosphate, purified by ultrafiltration of the organic extracts on molecular mass exclusion membranes (mol wt > 10⁴) and fractionated by isoelectric focusing technique (IEF). The IEF on polyacrylamide rod gels with a restricted pH gradient ranging between 6.0 and 4.0 gave five humic bands on the basis of the little differences of their electric charges (pI). Under both organic and mineral fertilization, β-glucosidase activity bound to the fractionated humic substances, especially in the pH range 4.5–4.2 of the rhizosphere soil, was higher than that of the inter-row soil. This also occurred in 6% slope where the enzyme activity was lower than in soil with lower slopes. The higher number of the β-glucosidase active humic bands in rhizosphere than inter-row soil, particularly for the 0% slope, may be due to the presence of humic molecules capable of preserving the enzyme molecules in the active form, other than to the higher microbial activity synthesizing and releasing the tested enzymes.     

Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.