Chapter 16
Enzyme Extraction from Soil Flavio Fornasier,* Yves Dudal, and Herve Quiquampoix
16–1 INTRODUCTION
Enzymes, like proteins in general, have a strong affinity with soil surfaces (Quiquampoix, 2008). The flexibility of their polypeptide chains and the various physicochemical properties of the lateral chains of the individual amino acids give rise to a range of possible interactions with solid surfaces: electrostatic forces, Lifshitz–van der Waals forces, hydrophobic interactions, and increases of conformational entropy (Quiquampoix et al., 2002). The adsorption of enzymes on soil surfaces is thus a complex phenomenon that involves several subprocesses (release of the exchangeable cations of clays, dehydration of surfaces, incorporation of cations in the adsorbed layer, rearrangement of protein structure). The combination of all these different subprocesses is often responsible for a quasi-irreversibility of the enzyme adsorption by dilution of soil components in water. Only more drastic treatments can desorb enzymes from soil particles. The challenge is to preserve the fragile structure of enzymes, and their catalytic activity, and to minimize the co-desorption of other compartments of soil organic matter. The stability of enzymes within the soil matrix, in addition to being controlled by surface reactions with minerals, is also controlled by various reactions with organic components of soils. Proteins can bind to humic substances through hydrogen, ionic, or covalent bonds. However, Simonart et al. (1967) showed that very little of the activity of the enzymes studied could be attributed to hydrogen bonding. Ionic binding does occur, but based on research that use ionic displacing extractants, the amount of activity is relatively low (Butler and Ladd, 1969; Hayano, 1977; Hayano and Katami, 1977; Ceccanti et al., 1978) Reactions of enzymes with both inorganic and organic substances were summarized by Weetall (1975). It was suggested that these mechanisms included adsorption, microencapsulation, cross-linking, copolymerization, entrapment, ion exchange, a combination of adsorption and cross-linking, and covalent bonding.
Flavio Fornasier, C.R.A.- R.P.S. Consiglio per la Ricerca e la Sperimentazione in Agricoltura - Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, 23, Via Trieste, 34170 Gorizia, Italy (flavio.
[email protected]), *corresponding author; Yves Dudal, ENVOLURE SAS, Campus La Gaillarde, Bât 12, 2 place Pierre Viala, 34060 Montpellier cedex 2, France (
[email protected]); Hervé Quiquampoix, INRA, UMR Eco&Sols, 2 place Pierre Viala, 34060 Montpellier, cedex 2, France (
[email protected]). doi:10.2136/sssabookser9.c16 Copyright © 2011 Soil Science Society of America, 5585 Guilford Road, Madison, WI 53711-5801, USA. Methods of Soil Enzymology. Richard P. Dick, editor. SSSA Book Series, no. 9.
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It seems likely there is a physical protection of stabilized enzymes that occurs and keeps them safe from microbial attack and free proteases. Today, the importance and interest in extracting enzymes and proteins from soil is even higher than in the past, due to the development of soil proteomics (Nannipieri, 2006; Ogunseitan, 2006). Unfortunately, extraction of soil enzymes has always been a highly difficult task because, due to the various interactions previously described, a low yield is usually obtained and enzymes are extracted together with other soil components, mainly humic substances. As Tabatabai and Fu (1992) pointed out some 20 years ago, no enzyme extracted from soil has been purified to the same extent as those extracted from biological tissues. The same is still true today and strongly inhibits soil enzymology and soil proteomic research. 16–1.1 Brief History of Soil Enzymes Extraction As reported by Skujiņš (1967), the first three reports on extraction of a soil enzyme activity were done by Fermi in 1910, who extracted a proteolytically active fraction with phenol; by Subrahmanyan in 1927, who precipitated the glycine deamination active principle; and by Ukhtomskaya in 1952, who desorbed several enzymes from soil using phosphate solutions. Antoniani et al. (1954) detected a cathepsin-like activity in a precipitate obtained from a soil extract using ammonium sulfate and sodium tungstate. Martin-Smith (1963) isolated two enzymes that decomposed uric acid from a soil; that same year, Briggs and Segal (1963) obtained the first solid preparation (12 mg of total protein from 25 kg of soil) containing an enzyme (urease). Bartha and Bordeleau (1969) extracted a peroxidase; Getzin and Rosefield (1971) extracted an enzyme (carboxylesterase) that decomposed malathion; and Chalvignac and Mayaudon (1971) obtained an extract that was enzymatically active (oxygenase effect) toward l-tryptophan from a forest soil. Burns et al. (1972) extracted urease; Pancholy and Lynd (1972) extracted lipase; and Ladd (1972) extracted proteases from soil. Starting in 1973, an increasing number of papers regarding extraction of enzymes from soil have been published (see Tabatabai and Fu [1992] for a detailed list of enzymes extracted from soil up to 1992). Various research efforts have investigated different parameters involved in the extraction process: pH, buffer composition, chelating resins, agitation, chaotropic agents, and others (Tabatabai and Fu, 1992; Nannipieri et al., 1996). Except for some cases when organic soils have been used (e.g., Nannipieri et al., 1980; Vepsalainen, 2001), the yield is usually very low as only a limited percent of the total soil enzymatic activity is extracted even with procedures lasting several hours. It is probably for this reason that extraction yield (i.e., the ratio between extracted activity and activity measured in whole soil) often was not reported. 16–1.2 Choosing the Extraction Procedure An important decision to make before starting to extract enzymes from soils is which method to use. This should be guided by the research objective and by consideration of potential chemical interferences that might occur for various extractants relative to data interpretation. Important factors to consider for using a particular method are: enzyme fraction that is to be studied (free, nonadsorbed enzymes or stabilized enzymes), choice of enzymes, location (e.g., distribution across aggregate sizes), and interference from clay and/or humic substances.
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No chemical extractant can exactly remove a specific enzyme fraction. However, depending on the chemistry and degree to which a given extractant solubilizes soil organic matter, a general inference can be made on the fraction of enzyme(s) that are being assayed in soil extract. The amount of soil organic matter released during the extraction is important. First, stronger extractants likely release enzymes that may be physically or chemically protected, or humus–enzyme complexes that remain catalytic. Second, the release of humic substances may react with free enzymes. The methods outlined in this chapter utilize either mild extractants (e.g., water) that only remove enzymes in soil solution or that are unbound on soil colloids, or stronger extractants (e.g., pyrophosphate) that co-extract significant amounts of humic substance. The degree of humic substance solubilization can be determined qualitatively by the visual color intensity of extracts or quantitatively by measurement of the absorbance at 450 nm (Ladd, 1972). Another factor to consider is whether an extractant will lyse cells. This may or may not be desirable depending on the goal. If one is interested in extracellular activity, cell lysis should be avoided. Alkaline extracts would be expected to lyse cells, releasing enzymes that are likely involved in only internal cellular reactions, not in extracellular processes that drive biogeochemical reactions. This might lead to overestimation of the enzyme activity associated with biogeochemical cycles that are important in soil functions. 16–1.3 Extractants Enzymes are bound to soil with different strengths, as previously described, and consequently “stronger” extractants should yield a higher amount of enzyme. In fact, thus far several extractants have been used. In general, buffers such as acetate, phosphate, pyrophosphate, tris(hydroxymethyl)aminomethane, and borate have been the most used (Tabatabai and Fu, 1992). Compounds that can specifically break ionic or hydrogen bonds, or metal bridges (chelating agents) have been used, but generally they did not improve the extraction yield (Nannipieri et al., 1996). In any case, significant amounts of enzyme can be brought out in solution by using only alkaline solution, which also yields high amounts of humic substances in solution (Nannipieri et al., 1996). Unfortunately, solutions with a high pH (i.e., higher than 8) also can denature enzymes. Complexes between enzymes and humic substances and/or minerals are thought to be dominant in soil. It should, however, be taken into account that the extraction yield does not depend strictly on the type of extractant, but also on its concentration (Busto and Perez-Mateos, 1995). Consequently, the extraction yield is not predictable. 16–1.4 Extraction Procedures There is no single, well-defined enzyme extraction procedure. Each author has tried to optimize the extraction for the specific soil being extracted and investigations comparing several extractants in different soils are rare. Ladd (1972) found little difference among the following buffers for the extraction of proteolytic enzymes in one soil: Tris, Tris-borate, sodium phosphate, Tris-citrate, Tris-EDTA, and distilled water. The author concluded that much of the protease activity was not tightly bound to that soil. Fornasier and Margon (2007) compared extraction
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yield of pyrophosphate, (one of the most commonly used and efficient extractants) with that obtained using a tris(hydroxymethyl)aminomethane solution in which either the detergent Triton X-100 or bovine serum albumin (BSA), or both, were added. The simultaneous use of Triton and BSA led to an extraction yield about 10 times higher than with pyrophosphate for three enzymes in six soils. A major issue that should be carefully considered when extracting enzymes from soil is the speciation of the enzyme in solution. Although humus–enzyme complexes originating from the soil are generally thought to be the dominant fraction, presence of free enzymes and artifacts, including complexes between extracted humic substances and enzymes formed during extraction, cannot be excluded a priori (Nannipieri et al., 1996) and cannot be distinguished from the complexes extracted from soil. Another important factor regarding the extraction procedure is the time dedicated to the extraction: it can be set from a half hour (Fornasier and Margon, 2007) to 24 h (Nannipieri et al., 1974; Nannipieri et al., 1980). For short-time extractions (few hours), microbial proliferation is thought to be nonsignificant, but for longer extraction periods, a biostatic agent generally is required. However, such a plasmolytic agent should be chosen with great care. Chloroform is an efficient biostatic agent, but it also breaks microbial cells (Jenkinson and Ladd, 1981) leading to the simultaneous extraction of intra- and extracellular enzymes. Toluene is a biostatic agent commonly used in enzyme assays, especially for hydrolases (Alef and Nannipieri, 1995; Frankenberger and Johanson, 1986; Tabatabai, 1994). Toluene permeabilizes the cell membrane specifically to small molecules, such as enzyme substrates (Skujiņš, 1967), and therefore can lead to an increase in the measured enzymatic activity, such as for arylsulfatase. However, toluene is suspected to act as an effective plasmolytic agent for certain groups of microorganisms; this could cause release of intracellular enzymes (Skujiņš, 1967) leading to simultaneous extraction of both intracellular and extracellular enzymes. The same co-extraction occurs when extraction of soil enzymes is performed on air-dried soil. 16–1.5 Selectivity of Extraction Among the 10 locations of enzymes in soil listed by Burns (1982), only those in free solution can be extracted selectively and quantitatively. Unfortunately, free enzymes in solutions constitute a very small fraction of the total enzyme pool in soil. Although selective extraction of extracellular enzymes can be performed using buffers, selective extraction of intracellular enzymes cannot be achieved. This is because enzymes released from cells are readily adsorbed by soil components (Quiquampoix, 2000) and subsequently can be extracted only together with extracellular enzymes. When using any cell lytic action, caution should be used as plasmolytic agents can interfere with enzymatic activity (Acosta-Martínez and Tabatabai, 2002; Frankenberger and Johanson, 1986). Margon and Fornasier (2008) increased extraction of arylsulfatase (an enzyme located both intra- and extracellularly) by lysing the cells before extraction using dichloromethane with a fast fumigation procedure (5 minutes). This procedure has the advantage of completely and easily removing the fumigant before the extraction and therefore does not interfere with enzymatic activity. In addition, microbial proliferation (if any) during extraction should be reduced to a minimum.
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In this chapter, five extraction procedures are presented. Extractions with water, neutral phosphate, and neutral pyrophosphate buffers exhibit increasing extraction strength, respectively. Extraction in acetic acid also is presented, as it could be useful in organic soils having an acidic pH. Indeed, neutral or alkaline extractants would extract high amounts of humic substances, causing strong interferences in the activity measurement. Finally, a new method, based on the desorption of enzymes using Triton X-100 and bovine serum albumin is presented (Fornasier and Margon, 2007). Determination of enzymatic activity in the extracts is not reported because the buffers and substrates have been the same when using soil instead of extract. Therefore, the methods described in the other chapters of this book can be utilized providing the incubation time is long enough to detect an appreciable concentration of the product of the enzymatic reaction. Times usually range from 4 to 16 h.
16–2 EXTRACTION WITH WATER
16–2.1 Introduction Ladd (1972) showed that on average extraction of protease with water resulted in the lowest amount of activity in comparison with extraction activity of Tris-borate, Tris, sodium phosphate, Tris-citrate, or Tris-EDTA. At the same time, the water extractant had the lowest absorbance at 450 nm, indicating it had the lowest level of humic substance extraction. Clearly, water extractants would act on extracellular enzymes that are likely free, in soil solution, or very loosely held on organic matter or mineral surfaces by ionic binding. This approach would be useful in characterizing what are likely recently released enzymes that have been excreted by viable microorganisms, or less likely from decomposing organic matter. 16–2.2 Principle This method uses water as an extractant and would not remove enzymes bound to inorganic and organic surfaces. It would extract enzymes that are in soil solution or, if on a soil colloid, would be held by extremely weak binding mechanisms. 16–2.3 Assay Method (Ladd, 1972) 16–2.3.1 Apparatus • Plastic bottles, 50 mL • Centrifuge tubes, 50 mL • End-over-end or reciprocating shaker • Centrifuge, 4000 g • Glass-fiber filters, 0.7 μm 16–2.3.2 Reagents 1. Distilled water 16–2.3.3 Procedure 1. Weigh 5 g (oven-dry basis) of soil into the plastic bottle, add 25 mL of distilled water, close the bottle, and place the bottle in the shaker. Adjust the shaker speed so that the soil suspension is well mixed and shake for 1 h.
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2. Transfer the suspension into centrifuge tubes and centrifuge at 4000 g for 5 min; filter through glass-fiber filter. 3. Determine enzymatic activity by using a method described in the other chapters of this book, substituting the soil for the extract. 16–2.3.4 Comments Centrifuging soil extracts at high speed (i.e., >25.000 g) can be more effective than filtering in removing colloidal particles. In any case, filtration is required to remove small fragments with a specific gravity less than 1 g mL−1. Extraction can be performed more conveniently in centrifuge tubes. Toluene and other plasmolytics should not be used when extraction is performed using moist soils, as they can, in principle, cause the release of intracellular enzymes. In addition, as the duration of extraction is short, no microbial production of enzyme should occur. If dry soil is used for extraction, the extract potentially contains intracellular enzymes released on desiccation. Extractions times longer than 1 h should not be necessary as water removes only free, nonadsorbed enzymes. A very small portion (less than 1%) of total soil enzymatic activity is usually extracted. Kandeler (1990) extracted less than 1/1000 of the total enzymatic activity of alkaline phosphomonoesterase in two moist soils using a saturated soil–water paste. However, this was sufficient for determining the kinetic parameters. By contrast, Ladd (1972) extracted with water, from a dried soil, an amount of protease activity comparable to that obtained with a variety of buffers but with a much lower humic content.
16–3 EXTRACTION WITH ACETATE BUFFER 16–3.1 Introduction This method is useful for acidic soils where the use of buffers with higher pH could extract high amounts of organic matter. Vepsalainen (2001) extracted a high percentage of phosphodiesterase, i.e., 16, 23, and 30% of total soil activity by using respectively 0.1, 0.2, or 0.5 M sodium acetate solutions from a highly organic soil. Bollag et al. (1987) found that 50 mM pH 6 acetate buffer was as effective as 50 mM pH 6 phosphate buffer for extraction of peroxidase from a mineral, neutral (pH 6.8) soil. 16–3.2 Principle Sodium acetate is a weak acid that would solubilize small amounts of organic matter. Extraction is performed at pH 5, close to the pK of acetate (4.8), where its buffering power is high and carboxylic groups are mostly not ionized. Given the ability of acetate to interact with the supramolecular structure of humic substances (Piccolo, 2001), it could be hypothesized, though not proven, that this extractant reduces the interactions between soil enzymes and humic substances. It would be expected to have a small amount of enzymes bound to humic substances, weakly held enzymes released from ionic binding, and free enzymes in soil solution. 16–3.3 Assay Method (Vepsalainen, 2001) 16–3.3.1 Apparatus • Beaker, 1 L • Volumetric flask, 1 L
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• Beaker, 100 mL • Volumetric flask, 100 mL • Plastic bottles, 50 mL • Centrifuge tubes, 50 mL • Magnetic stirrer • Magnetic bars • pH meter • End-over-end or reciprocating shaker • Centrifuge, 4000 g • Glass-fiber filters, 0.7 μm 16–3.3.2 Reagents 1. Sodium hydroxide solution, 1 M: Weigh 4 g of NaOH (MW 40.00) into a 100-mL beaker; add about 80 mL of distilled water and stir to dissolve. Transfer the solution to the 100-mL volumetric flask and make up to 100 mL with distilled water. 2. Sodium acetate solution, 100 mM pH 5: Dissolve 6.804 g (50 mmol) of sodium acetate trihydrate (MW 136.08) and 3.0025 g (50 mmol) of glacial acetic acid (MW 60.05) in a 1-L beaker with about 800 mL of distilled water. Adjust the pH to 5 using Reagent 1, then transfer the solution to the 1-L volumetric flask and adjust to 1 L with distilled water. 16–3.3.3 Procedure 1. Weigh 5 g (oven-dry basis) of moist soil in the plastic bottle, add 25 mL of Reagent 2, close the bottle, and place the bottle horizontally in the shaker. Adjust the shaker speed so that the soil suspension is well mixed and shake for 1 h. 2. Transfer the soil suspension into centrifuge tubes and centrifuge at 4000 g for 5 min; filter through glass-fiber filter. 3. Determine enzymatic activity by using a method described in the other chapters of this book, but adding soil extract rather than whole soil to the appropriate reaction mixture. 16–3.3.4 Comments See 16–2.3.4. The pH of the soil–buffer suspension should be checked to verify the value of pH during extraction. Extraction should not be performed over a long time as acetate can be utilized by microorganisms as a substrate for growth. For the use of plasmolytics and dry soil, see Extraction with Water method (16–2).
16–4 EXTRACTION WITH PHOSPHATE BUFFER
16–4.1 Introduction Phosphate buffer is considered to be a mild extractant, therefore it probably extracts soil enzymes loosely bound to organic matter and free enzymes not associated with soil colloids (Kiss et al., 1975; Skujiņš and Burns,1976). Usually both
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the extraction yield and amount of humic substances in solution are higher compared with water extractions but lower compared with pyrophosphate extractions. It has been one of the most used buffers for the extraction of a variety of enzymes (Tabatabai and Fu, 1992). 16–4.2 Principle The phosphate extract is a salt solution and for this method the pH is 7. This can be viewed as a mild extractant that would release low amounts of organic matter but more than water (Ladd, 1972). It would be expected to capture free enzymes but also release loosely bound enzymes. Since this extractant would dissociate both anionic (phosphate) and cationic (Na) molecules, it could be expected that it would displace some enzymes based on ionic reactions. 16–4.3 Assay Method (Hayano et al., 1987) 16–4.3.1 Apparatus As described in section 16–3.3.1. 16–4.3.2 Reagents 1. Hydrochloric acid solution, 1 M: Put about 500 mL of distilled water in a 1-L volumetric flask, then add 83.09 mL of 37% (w/w; specific gravity 1.186) concentrated HCl (MW 36.461). Swirl rapidly, then bring to 1 L with distilled water and mix thoroughly. 2. Sodium hydroxide solution, 1 M: Weigh 4 g of NaOH (MW 40.00) into a beaker; add about 80 mL of distilled water and stir to dissolve. Transfer the solution to the 100-mL volumetric flask and make up to 100 mL with distilled water. 3. Sodium phosphate solution, 100 mM pH 7.0: Dissolve 6.084 g (i.e., 39 mmol) of monobasic sodium phosphate dihydrate, H2NaO4P·2H2O (MW 156.01), and 10.857 g (i.e., 61 mmol) of dibasic sodium phosphate dihydrate, H2NaO4P·2H2O (MW 177.99), in about 800 mL of distilled water. This solution should exhibit a pH of 7.0. Check the pH with a pH meter; if necessary correct the pH with 1 M NaOH (Reagent 2) or 1 M HCl (Reagent 1). Transfer the solution to a 1-L volumetric flask and adjust the volume to 1 L. 16–4.3.3 Procedure 1. Weigh 5 g (oven-dry basis) of moist soil in the plastic bottle, add 50 mL of Reagent 3 buffer, close the bottle, and place the bottle horizontally in the shaker. 2. Adjust the shaker speed so that the soil suspension is well mixed and shake for 1 h. 3. Transfer the soil suspension in centrifuge tubes and centrifuge at 4000 g for 5 min; filter through glass-fiber filter. 4. Determine enzymatic activity by using a method described in the other chapters of this book, substituting the soil for the extract. 16–4.3.4 Comments See 16–2.3.4.
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16–5 EXTRACTION WITH PYROPHOSPHATE 16–5.1 Introduction This extractant has been used to extract protease, catalase, urease, phosphatase, and b-glucosidase in several studies (Nannipieri et al., 1982a; Nannipieri et al., 1982b; Perez-Mateos et al., 1988; Busto and Perez-Mateos, 1995). A further advantage of this extractant is that it reduces microbial activity, which would limit changes to extracted enzymes during the extraction process. At the same time, Nannipieri et al. (1974) showed that pyrophosphate extracted preserved microorganisms that produce urease. This method extracts large amounts of organic matter as shown by Nannipieri et al. (1974). 16–5.2 Principle Pyrophosphate has the ability to complex cations, which favors solubilization of humic substances (Stevenson, 1994), allowing extraction of humus–enzyme complexes (Busto and Perez-Mateos, 1995; Nannipieri et al., 1996). Therefore, the extract is richer both in enzyme activity and in humic substances compared with that obtained with phosphate buffer. 16–5.3 Assay Method (Nannipieri et al., 1974) 16–5.3.1 Apparatus As described in section 16–3.3.1. 16–5.3.2 Reagents 1. Hydrochloric acid solution, 1 M: Put about 500 mL of distilled water in a 1-L volumetric flask, then add 83.09 mL of 37% (w/w; specific gravity 1.186) concentrated HCl (MW 36.461). Swirl rapidly, then bring to 1 L with distilled water and mix thoroughly. 2. Sodium hydroxide solution, 1 M: Weigh 4 g of NaOH (MW 40.00) into a 100mL beaker; add about 80 mL of distilled water and stir to dissolve. Transfer the solution to the 100-mL volumetric flask and adjust to 100 mL with distilled water. 3. Sodium pyrophosphate solution, 100 mM pH 7.0: Dissolve 22.194 g of dibasic sodium pyrophosphate, H2Na2O7P2 (MW 221.94), in about 800 mL of distilled water. Stir the solution and bring the pH to 7 by adding NaOH (first about 10 M, then 1 M). Transfer the solution to 1-L volumetric flask and adjust the volume to 1 L. 16–5.3.3 Procedure 1. Weigh 5 g (oven-dry basis) of moist soil in the plastic bottle, add 50 mL of Reagent 3, close the bottle, and place the bottle horizontally in the shaker. 2. Adjust the shaker speed so that the soil suspension is well mixed and shake for 1 h. 3. Transfer the soil suspension into centrifuge tubes and centrifuge at 4000 g for 5 min; filter through glass-fiber filter. 4. Determine enzymatic activity by using a method described in the other chapters of this book, substituting the soil for the extract.
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16–5.3.4 Comments See 16–2.3.4. Extraction times longer than 1 h can increase extraction yield; however times longer than 2 h are not necessary, as extraction yield increases slightly (Nannipieri et al., 1980).
16–6 EXTRACTION WITH TRITON X-100 AND BOVINE SERUM ALBUMIN 16–6.1 Introduction A variety of reagents, ranging from mild extractants like salt solutions and buffers at neutral pH to strong organic matter solubilizing reagents (e.g., NaOH and sodium pyrophosphate) have been used to extract enzymes from soil (Tabatabai and Fu, 1992). A high extraction yield usually has been obtained only under conditions that brought humic substances into the solution (Nannipieri et al., 1996). This would suggest that a significant amount of the enzymes extracted would be extracted as enzyme–humus complexes. To enable the separation of enzymes from humic substances, Fornasier and Margon (2007) tested nondenaturing detergent extractants. This research showed that extraction of arylsulphatase and acid and alkaline phosphomonoesterase with sodium pyrophosphate (0.14 M, pH 7.1) yielded extracts with a low enzymatic activity. Similarly, Tris–HCl (50 mM, pH 7.5) gave a very low extraction yield (
