Extraction of Enzymes from Soil

A cathepsin-like activity was measured in a soil extract in 1954 (Antoniani et al. 1954). However, according to Skujins (1967), Fermi and Sabrama-nian were the first to measure enzyme activities (protease and aminase activities, respectively) in a soil extract. Urease was isolated from a surface forest soil with phosphate buffer at pH 6 and purified in the United States by Briggs and Segal (1963). In Australia, Ladd (1972) determined protease and peptidase activities in several pasture and wheat soil extracts by using 0.1 M Tris-borate buffer at pH 8.1 as an extractant. Then, in the 1970s and 1980s, lyase, oxidoreductase and hydrolase activities were monitored in soil extracts (Nannipieri et al. 1996). Several procedures for the extraction of enzymes from soil have been proposed and they are generally based on the use of salt solutions as extractants. Phosphate, acetate, citrate, tris(hydroxymethyl)aminomethane (tris), tris-borate, borate, etc., have been used to extract enzymes from soil, as reviewed by Shcherbakova et al. (1981), Tabatabai and Fu (1992) and Nannipieri et al. (1996). It was realised that it is not acceptable to purify enzymes extracted from soil because the extracted enzymes can be a mixture of enzymes from different sources (Nannipieri et al. 1996). Indeed, enzymes can originate from a multitude of sources from the variety of microbial species inhabiting soil, and from plants and fungi (Torsvik et al. 1996; Nannipieri et al. 2003). It was suggested that enzyme extraction from soil should give high yields of extracted enzymes to allow the characterisation of the properties of the extracted enzyme complexes (Nannipieri et al. 1974, 1980, 1996). In addition, an efficient extraction procedure for immobilised enzymes in soil should avoid the lysis of microbial cells (and thus the consequent release of intracellular enzymes), the formation of artefacts during soil extraction, such as the adsorption or entrapment of the extracted enzymes within the solubilised complexes, and interaction between the extractant and the inorganic components to give enzyme-like activity (Nannipieri et al. 1996). For example, the citrate can interact with manganese to form complexes showing laccase-like activity as a result of soil extraction (Leonowicz and Bollag 1987). Nannipieri et al. (1974) used sodium pyrophosphate at neutrality to extract ureases from a humic podzol without breaking microbial cells, by considering that this solution had been used to extract organic matter under mild conditions (Bremner and Lee 1949). Sodium pyrophosphate was also used to extract urease from coarse textured solodic, reddish-chocolate clay loam, grey-brown podzolic, red-yellow podzolic and alpine humus soils (Lloyd 1975). The same extractant was used to extract fi-glucosidase and catalase from an Umbric Dystrochrept soil (Perez Mateos et al. 1988; Busto and Perez Mateos 1995), and phosphatase, casein- and benzoylargininamide-hydrolysing proteases from Mollisol, Histosol and Alfisol soils (Nannipieri et al. 1980).

Sodium hydroxide, the common solution used to extract organic matter from soil at alkaline pH values (12-13), was used to extract malathion esterase from a clay-loam soil; the enzyme was then purified by a procedure involving addition of salts (MnCl2), with precipitation by (NH4)2SO4, dialysis and ion chromatography. It was shown that the extracted enzyme was a glycoprotein (Getzin and Rosefield 1971; Satyanarayana and Getzin 1973). However, alkaline conditions should be avoided in studies on enzyme-soil colloid complexes because enzymes are denatured and lysis of microbial cells can occur.

Extensive characterisation of extracted enzymes from soil has been mainly carried out by the groups of Mayaudon in Belgium and Nannipieri and Ceccanti in Italy with the aims of studying properties and state of the organic-enzyme complexes and the processes responsible for their formation (Nannipieri et al. 1996). The two groups used different extraction procedures and thus probably studied different enzyme complexes.

Laccases, polyphenol oxidases, phosphatases, phosphodiesterases, aryl-sulphatases, cellulases, xylanases, ^3-glucosidases, invertases and proteases, extracted by shaking a pasture soil with phosphate-EDTA at pH 7-8 for 1h (Mayaudon et al. 1973; Batistic et al. 1980), were supposed to be fungal glycoenzymes protected against proteolysis by their entrapment in lipopolysaccharides synthesised by Gram-negative bacteria (Mayaudon 1986). It was also hypothesised that these multienzymatic lipopolysaccha-ride complexes were linked through Ca bridges to the humic matrix and their extraction by phosphate occurred because EDTA chelated these Ca ions (Fig. 4.2). According to Mayaudon (1986), the interactions between the fungal enzymes and the bacterial lipopolysaccharides occur after partial hydrolysis of the latter once released into soil environment.

The enzymatic preparations extracted by pyrophosphate were rich in humic matter; these humic-enzyme complexes were fractionated by gel chromatography only after exhaustive ultrafiltration of the soil extract with sodium pyrophosphate at pH 7.1 (Ceccanti et al. 1978). The compounds with molecular weight lower than 10,000 were discarded and the retained material was separated by ultrafiltration into two fractions of molecular weight higher (AI) and lower (AII) than 100,000. Gel chromatography of these two fractions (Ai and Aii) gave three and two urease active fractions differing in molecular weight, respectively (Ceccanti et al. 1978). The gel chromatography of the highest molecular weight fraction (AI) with sodium pyrophosphate as eluent gave three peaks of phosphatase activities and one peak of each protease (casein and benzoylargininamide-hydrolysing proteases) activity, whereas the gel chromatography of the lowest molecular

Fig. 4.2. Possible role of stabilised enzymes (model by Burns above; model by Mayaudon below) in microbial ecology (modified by Burns 1982). P Product; S substrate; Em induced microbial enzyme; E enzymes

fraction (AII) using water as eluent gave one peak of each enzyme activity (Nannipieri et al. 1985).

Characterisation of soil extracts by pyrolysis-gas chromatography (Py-GC) showed that 0.1 M sodium pyrophosphate at pH 7.1 extracted condensed humic substances, glycoproteins, intact or partially decomposed carbohydrates (Bonmati et al. 1988). Soil extracts were active against three different protease substrates: N-benzoyl-l-argininamide, specific for trypsin, N-benzyloxycarbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases, and casein, essentially non-specific.

Characterisation of AI and AII fractions by elemental analysis, Py-GC and isoelectric focusing (IEF) showed that both fractions had a C/N ratio higher than a pure protein and properties similar to humic and fulvic acids ofthe same soil (Ceccanti et al. 1986). In addition, the AI fraction was richer in carbohydrates and in highly condensated humus than the Aii fraction. Total activity of these hydrolases was generally increased after ultrafiltration or gel chromatography, probably as the result of separation of inhibitory humic constituents and enzymes (Nannipieri et al. 1985). Phosphatase and urease active fractions with higher molecular weight were more resistant to thermal denaturation and proteolysis than the enzymatically active fractions with lower molecular weight (Nannipieri et al. 1978, 1982, 1988). It was hypothesised that these data validated the hypothesis by Burns et al. (1972a) who proposed that hydrolases (ureases) were entrapped within or ganic or organo-mineral complexes and surrounded by a humic network with pores large enough to allow the passage of substrates and products of the hydrolytic reaction, but not the passage of high molecular weight compounds such as proteases (Fig. 4.2; Nannipieri et al. 1996).

Usually, studies on enzyme extraction from soil and characterisation of enzyme complexes have mainly focused on hydrolases (urease, phos-phatase, ^3-glucosidase, etc.) for their important role in nutrient cycling and for the extensive bibliography on properties of the respective model complexes such as those with clays (see Chap. 7). It is important to underline that the present knowledge in the field derives above all from studies carried out in the 1970s and 1980s. In addition, it is well established that artefacts can occur during the extraction of enzyme complexes from soil.

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