Significance of the Enzymes Associated with Soil C and N Transformation

Abstract

Soil enzymes play a crucial role in soil organic matter transformation and nutrient cycling. Enzyme productions are the result of soil microbial community expression and their metabolomic requirement. Understanding the presence and activity of the enzymes of C and N cycles in soil may have important implications on ecosystem disturbances and can help to understand the role of C and N cycling in sustainable soil management and sustaining agricultural productivity. Among the biological features, soil enzymes are often used as a reliable index of changes in the soil status as affected by differentiated natural and anthropogenic factors since they are more sensitive to any changes than other soil variables. As was shown in the reviewed literature, interest in the enzyme systems responsible for C and N transformation in soil is currently still high. This chapter presents a brief overview of earlier and recent findings dealing with the most important soil enzymes involved in the soil C and N cycle, such as cellulase, β-glucosidase, urease, invertase, laccase, peroxidase, proteases, and nitrate reductase. The role of these enzymes in soil C and N transformation, as well as possible changes in enzymatic activity as influenced by differentiated factors, was also analyzed. Moreover, still existing limits related to the methodology adopted to assay soil enzyme activities have been discussed. Additionally, one subchapter is devoted to the relationship between gene abundance and enzymatic activity in soil. The contribution of transcriptomics and proteomics in soil enzymology is still poorly developed probably because there are still some methodological problems in soil proteomics. Moreover, the relationship between enzyme activity and the gene expression in soil is an important aim of research. Finally, further research needs and directions concerning the activity of soil C- and N-cycling enzymes are outlined.

1 Introduction

The carbon (C) and nitrogen (N) cycles represent the most important biogeochemical cycles found in terrestrial ecosystems. Carbon and nitrogen account for 95% of the biosphere and are two of six elements (carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur) that are the major components found in plants (Nieder and Benbi 2008; Scharlemann et al. 2014). Most of the organic carbon and nitrogen found in terrestrial ecosystems are related to plant residues and soil organic matter (Schlesinger 1997; Lal 2008), which are mineralized to the simple inorganic forms by a set of extracellular enzymes that are produced mainly by soil microorganisms and less by plant roots (Berg and McClaugherty 2003; Paul 2007).

Soil organic carbon (SOC) represents a significant reservoir of carbon in the global C cycle (Lal 2004). SOC consists of a heterogeneous complex of a wide range of organic materials, including simple molecules (e.g., amino acids, monomeric sugars), polymeric molecules (e.g., lignin, cellulose, nucleic acids, proteins), and plant and microbial residues that consist of simple and composed molecules that are bound together into recognizable cellular structures. SOC is formed mainly from the plant, animal, and microbial residues in various stages of decay (Baldock 2007). Soil organic carbon is one of key drivers of the rhizosphere and bulk soil processes and their functions, which results in better soil structure by influencing aggregate stability, nutrient cycling, and availability as well as infiltration and water storage (Hartemink et al. 2014). The soil SOC provides carbon and energy source for soil microbes and fauna. The content and diversity of the soil microorganisms increase with soil organic carbon increase. Since a lot of transformations in soil are conducted by soil microbial communities, an increase in soil microbial biomass usually enhances plant nutrient availability. The range of soil organic compounds can also promote plant growth and thus enhance plant productivity (Lal 2016; Meena et al. 2016).

All living organisms require nitrogen (N) as a necessary nutrient. In terrestrial ecosystems, N is usually available to plants in a limited range, which results in a strong competition for this element between microbes and plant roots (Vitousek and Howarth 1991). In terrestrial ecosystems, the soil organic nitrogen is mainly derived from the remains of plants and/or microorganisms and less from animals (Kögel-Knabner 2002; Norton and Schimel 2011). Therefore, most of the N entering the soil is in the organic forms, such as proteins, chitin and peptidoglycan, nucleic acids, and other N-containing compounds, all of which first have to be broken down into smaller organic molecules by extracellular depolymerases, which take part in the first step of the organic N degradation (Schimel and Bennet 2004, Geisseler et al. 2010). The small organic compounds that are excreted by enzymes can then be taken up directly or be further decomposed and be taken up by microorganisms as ammonium (Feng et al. 2018). The significant processes in the nitrogen cycle include:

• 1. Nitrogen has to be fixed and then converted into a usable form (NH₃, NO₃ ⁻) before it can be used by organisms.

• 2. Mineralization or ammonification: the conversion of amino acids into ammonia.

• 3. Nitrification: oxidation of ammonium into nitrites and nitrates.

• 4. Denitrification: the conversion of nitrates into atmospheric nitrogen, N₂.

Mineralization and nitrification are the most significant processes in the soil nitrogen cycle since they mediate plant uptake, nitrate leaching, and trace gas emissions (Norton 2008; Norton and Stark 2011). Soil N mineralization is key process by which organic N is converted into inorganic forms such as ammonium (NH₄ ⁺), nitrite (NO₂), or nitrate (NO₃), and it determines the amount of N that is available to plants. In turn, in the process of nitrification, the reduced N (NH₃/NH₄ ⁺ or organic N) is oxidized into NO₂ ⁻ or NO₃ ⁻.

Transformation of carbon- and nitrogen-rich bio-macromolecules that occur in soil is a process which is mainly mediated by a set of extracellular enzymes (EEs) produced by soil microorganisms and plants. The EEs catalyze most of the reactions that are involved in the synthesis and degradation of soil organic matter and assure the supply of the essential energy and nutrient for soil organisms which produce the enzymes (Sinsabaugh et al. 2009; Brzostek and Finzi 2011; Wallenstein et al. 2011). The soil enzyme activities take part in main biological processes related to the SOM quality (e.g., the relative availability of C and N) with the capability of microbes to assimilate nutrients and to use carbon for their own need (Allison et al. 2011). This indicates that changes in the C- or N-cycling enzyme activities may be linked to changes in the availability and/or storage of C and N within the SOM pools (Cenini et al. 2016).

In relation to the above, the aim of this chapter is to point out the present state of our knowledge about the features of the enzymes that take part in soil C and N transformations in relation to other soil properties controlling their function and activity.

2 Potential Role of Soil Enzymes in Maintaining Soil Quality and Fertility

Enzymes are protein-related catalysts whose activities can be measured and quantified in the soil system. Soil enzymes play a key role in the transformation of organic matter and nutrient turnover in a soil ecosystem (Burns 1978; Gianfreda and Ruggiero 2006; Piotrowska-Długosz 2014). The overall soil enzymatic activity consists of various intracellular and extracellular enzymes that are actively secreted by soil microorganisms, such as bacteria and fungi, and they less originate from plants and animals (Gianfreda and Bollag 1996; Rao et al. 2014). With using the current methods in soil enzymology, it is difficult to identify the exact source (origin) of the enzyme as well as the temporal and spatial variability of the enzymatic activity (Gianfreda and Ruggiero 2006). Endoenzymes in the soil are retained in living and proliferating cells, while exoenzymes are produced and secreted by living and proliferating cells, but they act outside of these cells as free enzymes that occurred in a soil solution or as enzymes that remain associated with the external root surface or microbial cell wall. After being secreted outside the cell, enzymes can stay free in a soil solution, or they become rapidly absorbed onto soil mineral and/or organic colloids, mainly clay minerals and humic substances. The activity of free extracellular enzymes occurring in soil solution is rather low compared with those immobilized on soil colloids since they are subjected to many unbeneficial factors shortening their life span (Nannipieri and Gianfreda 1998). Although bound enzymes are resistant to proteolysis and different forms of denaturation (Nannipieri et al. 1996) and absorption protects enzymatic protein against degradation, their activity is significantly lower than that of free enzymes. The activity of absorbed enzymes is, however, the most important part of the overall soil enzymatic activity and is responsible for soil organic matter and nutrient transformation.

Although there are many research and related publications concerning the presence, distribution, and functioning of enzymes in different soil types, measuring their catalytic activity, stability, and changes in their activity caused by various biotic and abiotic agents, etc. (e.g., Burns 1978; Kiss et al. 1998; Burns and Dick 2002; Nannipieri 1994; Tabatabai 1994; Gianfreda and Bollag 1996; Tabatabai and Dick 2002; Gianfreda and Ruggiero 2006; Rao et al. 2014), there are many unresolved questions concerning some topics related to enzymes such as their position in the soil environment; contribution in soil organic matter and nutrient transformation; changes under the influence of natural and anthropogenic factors; possible interaction with organisms that are producers of enzymes in soil; and the elaboration of a universal soil enzyme indicator to access the soil status and health, estimation of the role of soil enzymes in changes of soil environment under the influence of various factors, as well as the determination of the enzymes role in the dynamics of plant nutrient transformation (Burns et al. 2013; Rao et al. 2014). These problems and questions are primarily related to the imperfect methodology that is currently being used in soil enzymology, which is discussed in Sect. 5 below.

It is commonly known that enzymatic activity is a sensitive indicator of soil quality and fertility and can be used for determination of ecosystem responses to management and overall environmental changes as well as sustainability of agricultural ecosystems (Nannipieri et al. 2002; Tabatabai and Dick 2002; Gianfreda and Ruggiero 2006; Varma et al. 2017). Soil enzymes play a key role in the overall process of the transformation of soil organic matter, soil nutrient cycling, and pollutants degradation (Burns 1982; Sinsabaugh et al. 1991). The agricultural importance of soil enzymes has been successively increased since the first statement on soil enzymes was presented many years ago. A positive relationship between the soil enzymes activity and nutrient transformation has been reported in some arable soils. Thus, in a long-term cropping systems using N fertilization, the N-acetylglucosaminidase and arylamidase activity were well correlated with the level of nitrogen mineralization (Ekenler and Tabatabai 2002, 2004; Dodor and Tabatabai 2007). Soil enzymatic activity can also be used as an indicator of soil nutrient availability. Many studies have shown that the enzymatic activity of the P and N mineralization is negatively correlated with the available forms of P and N (Muruganandam et al. 2009; Balota and Dias Chaves 2010; Orczewska et al. 2012, Sherene 2017). Similarly, Allison (2005) reported that N fertilization significantly decreased the activity of proteases and chitinase.

3 Enzymes of Soil Carbon and Nitrogen Transformation: A General Overview

The enzymes related to C and N transformations are the most important in the overall process of soil organic matter transformation and energy flow. The main aim of this chapter is to review the current knowledge of the enzymes involved in carbon and nitrogen cycling in soil. Many reviews (e.g., Burns 1982; Tabatabai and Dick 2002; Gianfreda and Bollag 1996; Gianfreda and Ruggiero 2006) dedicated earlier to soil enzymology are important when considering its historical background. More recent research concerning different aspects of enzymatic reactions in soils have been reviewed with special attention paid to the influence of the physical soil properties on enzymatic activity. Finally, future research needs are also specified. Although many of the enzymes that take part in C and N transformation can be determined in soils, only a limited number of enzymes are usually studied. Many of them, like cellulases, are found to act as extracellular enzymes. Others, like urease, are able to catalyze reactions both endo- and extracellularly (Tate III 2002). Earlier, most of the soil-related studies were subjected to meet the agricultural necessity (Dick and Tabatabai 1992; Dick 1994), and determination of soil enzyme activities have mainly been directed toward assessing the quality and quantity of crops and determination of management influence on the enzymes involving in the biogeochemical cycles (e.g., transformation of nutrients in plant biomass/residues, N cycling, and fixation). Special attention has been paid to urease activity due to its great significance in the urea-hydrolyzing. The most often studied enzyme activities involved in N transformations have been related to ammonium formation (amidases, urease), the hydrolysis of proteins (proteolytic enzymes), the loss of N from soil (nitrogen oxide reductases), denitrification (nitrate reductase), and finally N fixation (nitrogenases). In turn, the most important enzymes associated with transformation of carbonaceous compounds are related to the hydrolysis of polysaccharides (e.g., amylases, cellulase complex, xylanases) and hydroxylation of aromatic rings (e.g., laccases and other polyphenol oxidases), which finally lead to either the mineralization or humification of the initial compounds (Dilly et al. 2007) and different lipases and esterases, which catalyze the hydrolysis of the variety of ester linkages in various substrates. Recently, however, these enzyme activities have been considered for evaluation of more broad and universal ecological anxiety, as the impact of human alterations, not only related to agricultural practice, the organic matter, and nutrients transformations in native soil systems such as old-grown forests or barren lands (Rao et al. 2017). For example, some of the C- or N-cycling enzymes (e.g., cellulase, urease) are advantageous in determining the impact of recycling organic wastes (e.g., compost, sewage sludge) on the soil, while determination of laccase and polyphenol oxidase activity is generally related to the breakdown and humification of the xenobiotics with aromatic rings (Gianfreda and Rao 2004; Gómez Jiménez et al. 2011; Meena et al. 2015b; Piotrowska-Długosz 2017).

3.1 Specific Enzymatic Activities of Soil C Transformation

A great number of enzymes are involved in hydrolyzing of C-containing compounds. This group includes mainly enzymes that break down large organic compounds, such as cellulose (cellulases), starch (amylases), chitin (chitinase), and xylan (xylanase). Other enzymes, such as invertase, α- and β-glucosidases, α- and β-galactosidases, and N-acetyl-β-glucosaminidase, decompose disaccharides and oligosaccharides into simple sugars. Some enzymes involved in C cycling and the reactions they carried out are characterized below and specified in Table 1.

Table 1 Soil enzyme activities involved in C cycling

3.1.1 Cellulase Complex and Glucosidases

The microbial degradation of cellulose, which is the most abundant polysaccharide found in the biosphere, requires the action of at least three groups of enzymes to act synergistically in hydrolyzing the β-1,4 bonds of cellulose to glucose. The catalytic system consists of endo-1, 4-β-glucanase (endocellulase EC 3.2.1.4), exo-1, 4-β-glucanase (cellobiosidase EC 3.2.1.91), and β-glucosidase (Deng and Popova 2011; Phitsuwan et al. 2013). Two glucosidases (EC 3.2.1.20/21) can be identified in soil, α-glucosidase (EC 3.2.1.20) and β-glucosidase (EC 3.2.1.21), which catalyzes the hydrolysis of α-d-glucopyranoside and β-d-glucopyanosides, respectively, and catalyzes the hydrolysis of maltose and cellobiose. Glucosidases are produced by a wide range of microorganisms, animals, and plants (Dodor and Tabatabai 2005). The most often found and determined in soil is β-glucosidase (Deng and Popova 2011; Buragohain et al. 2017), which takes part in the last stage of cellulose decomposition. The most often known reaction carried out by the enzyme is hydrolysis of cellobiose into two molecules of glucose by cleaving the β-glucosidic bonds from the nonreducing terminal ends (Lynd et al. 2002; Jørgensen et al. 2007; Deng and Popova 2011). This process is important since cellobiose is an inhibitor of the cellulolytic enzymes activity (Morais et al. 2004). β-Glucosidase is also responsible for the hydrolysis of β-d-glucopyranoside and many of glycosides, such as phenolic glycosides or flavanone glycosides (Berrin et al. 2003; Acosta-Martinez et al. 2007). Because of wide substrate specificity, the activity of this enzyme is considered to be a good indicator of biomass decomposition in soil (Berrin et al. 2003; Zanoelo et al. 2004). Other known glycosidases are α-galactosidase (EC 3.2.1.23) and β-galactosidase (EC 3.2.1.24), which catalyze the hydrolysis of melibiose and lactose, respectively. These enzymes however not occur in the soil in a significant amount. The importance of glycosidases is related to their participation in soil organic matter mineralization. By hydrolyzing the soil organic carbon and nitrogen compounds, they deliver essential carbon components and nutrients for the growth of heterotrophic microorganisms, thereby increasing soil microbial activity (Dodor and Tabatabai 2005).

3.1.2 Invertase

Invertase (β-d-fructofuranoside fructohydrolase [EC 3.2.1.26]) splits off β-d-fructofuranoside rest from nonreducing end of β-d-fructofuranosides, such as sucrose, raffinose, oligofructose, or inulin (Deng and Popova 2011). The preferable substrate for invertase is sucrose, most commonly occurring in plants soluble sugar, which consists of a molecule of glucose and fructose (Jin et al. 2009). Together with cellulase complex, invertase activity is responsible for the disintegration of plant litter in the soil system (Frankenberger and Johanson 1983; Datta et al. 2017b). Although the invertase activity is generally associated with the heavy fraction of soil (clay minerals and silt), in soil under grasslands, the invertase activity was partially related to light soil fraction (Ross 1983). The soil invertase activity is used as an index for nutrient transformation, energy metabolism, and pollutant degradation (Nannipieri et al. 1990).

3.1.3 N-Acetyl-β-d-Glucosidase (Chitinase)

Chitin, the second most often occurring in soil’s amino sugar, is an unbranched polymer consisting of N-acetyl-d-glucosamine. The sources of this compound in the soil are exoskeletons of insects and arthropods as well as fungal hyphae (Duo-Chuan 2006; Wongkaew and Homkratoke 2009). The chitinases system taking part in the hydrolyzing of chitin consists of endochitinolytic (chitinase, or β-1, 4-poly-N-acetylglucosaminidase [EC 3.2.1.14]) and exochitinolytic (N-acetyl-β-d-glucosaminidase, NAGase, [EC 3.2.1.52]) enzymes. They hydrolyze the chemical bonds between N-acetyl-d-glucosamine particles (Brzezińska et al. 2009; Deng and Popova 2011), but they differ in the way of action. Chitinase randomly hydrolyzes the 1,4-β bonds in chitin, while NAGase hydrolyzes the terminal, nonreducing ends (Webb 1992; Moss 2010) with the free N-acetyl glucosamine (NAG) as the final product of the reaction (Brzezińska et al. 2009). The availability of the NAGase is differentiated in soils and depends on many factors such as soil physical and chemical properties, microorganism wealth, and different substrate quality and quantity (Sinsabaugh et al. 1992). Many soil organisms, such as bacteria, fungi, and plants, are able to produce chitinases (Duo-Chuan 2006; Sihag et al. 2015). Bacterial chitinases mainly degrade chitin in order to use the reaction products as the source of carbon and nitrogen, while in fungi, this group of enzymes also plays a significant role in cell wall development and structure during the active growth (Adams 2004; Bhattacharya et al. 2007). The synthesis of chitin is induced when other labile carbon and nitrogen sources are lacking. That is why chitin is more abundant in areas with low nutrient content (Hanzlikova and Jandera 1993; Brzezińska et al. 2009).

3.1.4 Xylanase

According to Deng and Popova (2011), the xylanase enzyme system (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) consists of the following enzymes: β-xylanase, esterase, α-l-arabinofuranosidase, β-xylosidase, α-glucuronidase, acetylxylan, and hydroxycinnamic acid esterases. The enzyme group is classified into tenth and eleventh families of the glycosyl hydrolases and catalyzes the endohydrolysis of β-1, 4-xylosidic linkages in hemicellulose, which, in addition to cellulose, is the second most frequently occurring polysaccharide on earth (Anand et al. 1990; Kandeler et al. 1999; Hu et al. 2008). The final products of hydrolysis are xylose, xylobiose, as well as short chains of various oligomers. The xylanase enzymatic complex is mostly produced by fungi under insufficient quantities of available compounds (Kandeler et al. 1999). According to Hu et al. (2008), xylanse complex plays a significant role in the circulation of organic materials and energy in the soils as well as in seed germination and fruit ripening.

3.1.5 Amylases

Amylases (EC 3.2.11/2), together with cellulases and invertase, are the group of enzymes that are responsible for the rate and course of the decomposition of plant material in soil (Pancholy and Rice 1972). The amylase system includes endo- and exo-amylases that synergistically hydrolyze starch (Deng and Popova 2011; Yadav et al. 2018). Endo-amylases, commonly known as α-amylases, hydrolyze the α-1,4-glycosidic linkages in random. The products of this reaction are dextrins, oligosaccharides, and finally monosaccharides, like glucose. In turn, exo-amylases consist of β- and γ-amylase and hydrolyze the same bonds but solely from the nonreducing ends of the starch molecule, thereby releasing β-maltose and β-d-glucose (Webb 1992; Deng and Popova 2011). Among amylases, the most active in soil is β-amylase which catalyzes the degradation of the so-called heavy fraction of organic material (the large size, recalcitrant carbon) than the light fraction (the small size, rapid transformation) (Ebregt and Boldewijn 1977; Ross 1983). Amylases also occur in plants as intracellular enzymes and can be liberated into soil together with plants residues. Similarly, to other enzymes, amylases are mainly produced by microorganisms, especially by bacteria and fungi (Ebregt and Boldewijn 1977). There are amylases found in some environments with unbeneficial conditions such as acidophilic, alkalophilic, and thermoacidophilic areas (Ebregt and Boldewijn 1977). It was reported that amylase activities in the soil, similar to other extracellular enzymes, were repressed by the presence of clay minerals. Thus, a considerable decrease in the β-amylase activity measured in three different clay fractions collected from soil and from surface layers of two soils from tussock grasslands was found. In these studies the effect of clay minerals on the decreasing of α-amylase activity was in the following sequence: muscovite < allophane < illite < montmorillonite (Ross 1983). In soils that have a higher carbon amount, amylase was more active as compared to the activities of some other enzyme (Pancholy and Rice 1973; Balota et al. 2004).

3.2 Specific Enzymatic Activities of Soil N Transformation

The cycling and fate of N in terrestrial ecosystems are critical for many aspects of environmental quality. That is why the enzymes taking part in nitrogen transformation are important in controlling N in this type of ecosystem and for possible use in assessing soil quality and/or degradation (Kandeler et al. 2011). Various enzymes involved in N cycling differ in their response to environmental change like N deposition and N addition (Hungate et al. 2007, Enowashu et al. 2009). That is why it is suggested to test some enzymatic indicators (e.g., proteases, urease, enzymes taking part in ammonium oxidation and denitrification) in order to observe the most important processes in nitrogen transformation. N-transforming enzymes can be divided into extracellular depolymerases (proteases, chitinases, and peptidoglycan hydrolases) that are involved in the decomposition of the main polymers of organic materials entering the soil and enzymes that take part in N mineralization (urease, amino acid oxidase) (Geisseler et al. 2010; Ashoka et al. 2017). Some important enzymes involved in N cycling and their reactions are presented in Table 2.

Table 2 Soil enzyme activities involved in N cycling

3.2.1 Extracellular Depolymerases

The group of extracellular depolymerizing enzymes breaks down the complex of organic plant materials and microbial residues into smaller, soluble subunits that can be taken up by microorganisms. Based on the chemical composition of the main sources of organic residues in soil, the most important extracellular depolymerases involved in the hydrolysis of N-containing molecules are proteases, chitinases, and peptidoglycan hydrolases (Geisseler et al. 2010; Kandeler et al. 2011).

3.2.1.1 Proteolytic Enzymes

The protein degradation (proteolysis) is a significant process in N transformation in different ecosystems since it is believed to be a limiting step of N mineralization in soil (Weintraub and Schimel 2005) due to the much slower primary phase of protein mineralization compared to amino acid mineralization (Jan et al. 2009). Earlier, Ladd and Jackson (1982) have discussed the role of protease activities in the process of N mineralization, while the concept of the N mineralization-immobilization in soil system has been reviewed later by Nannipieri and Eldor (2009). Extracellular proteolytic enzymes hydrolyze protein polymers and polypeptides into smaller peptides and finally into amino acids. Some researchers proposed the assay of protease activity as a good way to measure N depolymerization, as proteases are the most responsible for supplying bioavailable N (Schimel and Bennet 2004).

Approximately 30–40% of overall organic nitrogen in soil are derived from proteins and polypeptides (Jones et al. 2009). The main source of various soil proteases is microbes and plants. Among soil microorganisms, the most effective in excretion of proteases are bacteria, such as Pseudomonas, Streptomyces, and Bacillus. Also, fungi, like Penicillium, Pythium, and Aspergillus, secrete numerous proteolytic enzymes, which activity is especially significant in releasing available nitrogen in conditions of its deficiency (Kudryavtseva et al. 2008). Proteolytic enzymes are usually categorized according to the type of reaction they catalyze, the molecular structure, and the type of functional group in the active site (Rotanova et al. 2004; Landi et al. 2011; Dadhich and Meena 2014). Thus, exopeptidases catalyze the hydrolysis of the terminal amino acids of the protein structure, whereas endopeptidases (proteinases) catalyze the hydrolysis peptide linkages between amino acids occurring inside the polypeptide chains. The exopeptidases hydrolyze peptide bonds on both ends of the peptide chains. The exopeptidases involved in removing one, two, and three amino acids from the N-terminal end of the chain is named aminopeptidases, dipeptidyl-peptidases, and tripeptidyl-peptidases, respectively (Landi et al. 2011). As regards the catalytic function, the following carboxypeptidases can be specified: cysteine-type carboxypeptidases, serine-type carboxypeptidases, and metal-carboxypeptidases (Table 3).

Table 3 Classification of proteases (peptidases) EC 3.4. according to the EC nomenclature (Webb 1992)

The neutral metalloproteases and serine proteases (SUB) are mostly involved in protein decomposition in agricultural soils that was showed by the selective inhibition of various proteases of bacterial origin (Watanabe et al. 2003; Vranova et al. 2013). Endopeptidases are recognized due to the chemical character of the groups that are responsible for their hydrolytic functions (Landi et al. 2011). According to Kalisz (1988) and Page and Di Cera (2008), four different groups of endopeptidases can be distinguished: aspartic-, cysteine-, serine-, and metalloendopeptidases. Trypsin and subtilisin are two significant serine endopeptidases, and thus trypsin is a particular enzyme that breaks down peptides at arginine and lysine amino acids, while subtilisin has a broad spectrum of activity and hydrolyzes the peptide bonds in various peptide amides. Most metalloproteases require some metals (e.g., zinc and cobalt) for their catalysis. A lot of research has been devoted to study endopeptidases, and their pH optimum (e.g., neutral, acidic, and alkaline) has been determined. Moreover, the extracellular proteolytic enzymes generally reveal a wide specificity toward the substrate and can break down a lot of various proteins (Kalisz 1988). Proteolytic enzymes also act intracellularly and are responsible for metabolism regulation and protein transformation within the cells. The protein turnover is crucial for cells to adapt to new environmental circumstances, particularly in a case of nutrients deficiency (Kalisz 1988). Godlewski and Adamczyk (2007) discussed the problem of proteases secretion by plant roots. They stated that different species and cultivars of the same plant growing in a culture medium could vary in the levels of proteolytic activity, which may indicate that they differ in the capacity to excrete proteases. Measuring the proteases activity at different values of culture medium reaction (pH) has pointed out that the produced proteolytic enzyme activity was the highest at pH = 7. The production of proteolytic enzymes varies within root systems; thus, in the apical parts of the root, proteases are more intensively secreted as compared with the mature section of the root. Increased activity of proteases was found in roots of transgenic plants (Eick and Stöhr 2009).

3.2.2 Enzymes Involved in N Mineralization

In the process of depolymerization, the high molecular weight N-containing polymers are breaking down into simplest compounds, like amino acids, amino sugars, or nitrogenous bases. This process is often considered to be a limiting step in soil nitrogen mineralization (Jones et al. 2009; Kemmitt et al. 2008; Wallenstein and Weintraub 2008). Afterward, ammonium is released from those monomers. These two steps are carried out by primarily microbial-derived extracellular enzymes (Burns et al. 2013; Yadav et al. 2017b). Extracellular enzymes degrade complex, N-containing compounds, such as protein, nucleic acids, and cell wall components (Myrold and Bottomley 2008). All these enzymes carry out the hydrolysis of native N compounds or those added to soil and have been used to assess changes in arable soils under different management practices, such as organic and mineral fertilization, tillage practices, and crop rotation (Hallin et al. 2009, Sinsabaugh et al. 2015). Amidohydrolases (e.g., urease, l-asparaginase, l-glutaminase, and amidases) activity is significant in the process of depolymerization of aliphatic and aromatic nitrogen compounds occurring in soil organic matter (Monreal and Bergstrom 2000). Peptidoglycan breaks the linkages between N-9 acetylmuramoyl and amino acids in the cell wall glycopeptides and thus playing the main function in the transformation of microbial biomass nitrogen (Tabatabai et al. 2010). The range of glycosidases are involved in the hydrolysis of amino sugar polymers that are a significant constituent of microorganism’s cell walls. In turn, the enzyme N-acetyl-β-d-glucosaminidase (NAGase) carries out the hydrolysis of the N-acetyl-β-d-glucosamine from the terminal, nonreducing ends of the chitooligosaccharides. This enzyme also takes part in the catabolism of soil chitin polymer (Tabatabai et al. 2010). Chitin and chitodextrins, which are the major constituents in fungi organisms, are substrates for chitinase activity (Alef and Nannipieri 1995).

The relationships between the indicators of nitrogen mineralization and some amidohydrolases activity were shown by Tabatabai et al. (2010). The authors revealed that the activities of some hydrolytic enzymes (e.g., asparaginase, amidase, urease, and glutaminase) were markedly related to the content of nitrogen mineralized at 30 °C. The correlation coefficient values were between 0.35 and 0.61 for l-glutaminase and l-asparaginase, respectively. Similarly, the activities of l-glutaminase and l-asparaginase were notably related to overall nitrogen mineralization in the study of Muruganandam et al. (2009). Moreover, a significant correlation between the range of hydrolytic activities (e.g., urease l-asparaginase, l-glutaminase, amidase) and N mineralization level was presented in other studies (Khorsandi and Nourbakhsh 2007; Xue et al. 2006). Some other authors in turn (Senwo and Tabatabai 1996; Tabatabai et al. 2010; Muruganandam et al. 2009) found that arylamidase, an enzyme which catalyzes the hydrolysis of amino acids from N-terminal peptide chains, as well as from amides and/or arylamides, was significantly related to nitrogen mineralization process. The obtained correlation coefficients ranged between 0.61 (p < 0.001) and 0.77 (p < 0.005). These data indicated that the activities of some enzymes involved in N mineralization can be used as significant indicators of this process.

3.2.2.1 Urease

There are a lot of studies concerning the soil urease activity because of the importance of urea as a nitrogen fertilizer (Glibert et al. 2006). Urease (EC 3.5.1.5) catalyzes the hydrolysis of urea into two moles of ammonia and one of carbon dioxide. This process is crucial in regulating the N supply to plants after urea fertilization. Urea enters the soil also as a result of the transformation of urine excreted from mammals. Moreover, urea comes from the degradation of the amino acid arginine and of uric acid, which is excreted by birds, reptiles, and insects (Mobley and Hausinger 1989). The wide range of bacteria species, yeasts, fungi, algae, and plants are the main sources of urease activity in soil environment (Mobley and Hausinger 1989; Follmer 2008). Although urease can be released constitutively from some organisms, the production of this enzyme is most commonly regulated by the presence of nitrogen, and its production is inhibited when the producing organism grows in the environment with the sufficient concentration of a suitable N source, like ammonium ions. On the contrary, the presence of urea and some other nitrogen sources activated production of the enzyme (Mobley et al. 1995). It was found that the urease activity in soil was mostly extracellular and was immobilized by soil mineral and organic colloids. According to Pettit et al. (1976) more or less 60% of the urease activity determined in different soils was bound extracellularly, while Klose and Tabatabai (1999) estimated the extracellular proportion of urease activity to be 46%.

A better understanding of the dynamics of urease activity might identify a more effective way of managing N fertilizers (Balota and Chaves 2010; Kumar et al. 2017). Therefore, it is important to detect the set of natural and anthropogenic factors that can modify/reduce/increase the effectiveness of this enzyme activity in an ecosystem. Some of these factors are soil organic matter and nutrient content, agricultural practices such as tillage, mineral and organic fertilization, crop rotation, soil depth, soil pollution with heavy metals, PAHs, soil waste amendments, and weather conditions such as temperatures and rainfall (Yang et al. 2006; Yadav et al. 2017a; Datta et al. 2014). Thus, it has been found that urease activity was very sensitive to higher amounts of heavy metals (Yang et al. 2006). Since urease activity becomes greater with progressive temperature, the fertilizer urea should be applied at a time the temperatures are the lowest. Then the energy of activation is lower, and the loss of nitrogen by the volatilization is minimal. A better understanding of urease properties and its activity would be helpful in urea fertilizer application, particularly in the areas with high rainfall, flooded, and irrigated fields (Bakshi and Varma 2011).

3.2.2.2 Arginine Deaminase Activity

The ammonification of arginine (AA), one of the basic protein amino acids, appears to be a common process in microorganisms (Alef and Kleiner 1986; Singh and Kumar 2008). Arginine ammonification level is significantly correlated with the soil microbial biomass content and other biochemical properties (Alef and Kleiner 1987; Singh and Singh 2005). Ammonification is an important initial stage of organic matter mineralization when proteins and other organic compounds containing amino groups are decomposed by proteolytic enzymes to amino acids which are further deaminated to ammonium ion NH₄ ⁺(Bonde et al. 2001, Lin and Brookes 1999). Bonde et al. (2001) also proposed that arginine ammonification activity provided an index of gross N mineralization in agricultural soils, as this enzyme activity was well correlated with the average rates of gross N mineralization, and there is a similarity between the seasonal variations of gross N mineralization and arginine ammonification activities. The amount of ammonium produced depends on the C/N ratio of the amino acid, with high ammonium production at a low ratio (Ginésy et al. 2017). The arginine deaminase activity is strongly related to respiration and correlated significantly with the carbon content of the soil but is poorly related with soil pH, ammonia content, percentage clay, or the number of microorganisms (Pandey and Singh 2006). Arginine deaminase activity was significantly high in natural, especially forest soil, compared to agricultural soils, thereby indicating continuously higher N inputs in forest stands (Singh and Kumar 2008). The arginine deaminase activity was differentially affected by heavy metals and other pollutants (Kandeler 1996; Guo et al. 2009). The AA activity was markedly stimulated after chlorpyrifos seed and soil treatment. This enhancement might have been due to an increase in the fungal and Actinomycetes population after chlorpyrifos application, which might be using this insecticide as an energy source for microorganisms (Hussain et al. 2009). The same results were obtained by Singh and Singh (2005) for diazinon seed and soil management. The AA activity was found to be greater in the control soil samples in comparison to the samples that had been treated with acetamiprid, where at the enzyme activity decreased over 20% shortly after the pesticide was used (Singh and Kumar 2008; Meena et al. 2015a). Recently, the influence of seasonal changes and forest management on the arginine ammonification was determined in the surface, the organic horizon of some spruce forests (Holik et al. 2017). The authors concluded that the AA was the highest in the soil with the most favorable conditions, such as high water content, a generally lower concentration of ammonium N, and a higher population density following a thinning operation.

3.2.2.3 Arylamidase

Arylamidase (a-aminoacyl-peptide hydrolase, EC 3.4.11.2) is the enzyme that catalyzes the separation of amino acids from the N-terminal end of peptides, amides. The enzyme was found in plants, animals, and microorganisms (Hiwada et al. 1980). Although arylamidase activity is important in the beginning stages of the soil amino acids mineralization, not much recent information is available about this enzyme in soil (Muruganandam et al. 2009). Understanding of the role of arylamidase in soil N cycling and the factors (i.e., soil properties, trace elements liming, tillage, and crop residues management practices) that affect the activity of this enzyme will aid in making decision important for the fertility, productivity, and sustainability of soils. Arylamidase activity was highly influenced by tillage and residue placements, and the greatest arylamidae activity was found in treatments of chisel/mulch, moldboard plow/mulch, and no-till/double mulch, whereas the lowest activity was observed in treatments of moldboard plow/normal and no-till/bare (Acosta-Martinez and Tabatabai 2000).

3.2.2.4 Amidase

Amidase (acylamide amidohydrolase, EC 3.5.1.4) is responsible for the production of ammonia (NH₃) and carboxylic acid through the hydrolysis of amides (Fraser et al. 2013). The production of ammonia is an important process in the N cycle. Amidase activity can be increased in the presence of C, but excess C can lead to both an N and P limitation (Allison et al. 2011). Amidase activity has been positively correlated with high molecular weight amide substrates (propionamide) but not with the low weight substrates (formamide) (Fraser et al. 2013). Amidase is secreted by many of microorganisms, plants, and animals, including bacteria Rhodococcus (Nawaz et al. 1994) and Bacillus (Thalenfeld et al. 1977) genera. Fungal production of amidase was noted in the Aspergillus (Benke 1979) and Fusarium (Reichel et al. 1991) genera. The wide variety of substrates that are available to amidase indicates the diverse nature and prevalence of these enzymes. The irreversible inhibition of amidase activity by organophosphate insecticides, such as fenitrothion and trichlorfon, has been described by Rasool et al. (2014).

3.2.2.5 Nitrate Reductase (NR) Activity

The dissimilatory nitrate reductase catalyzes the first step of the nitrification process by reducing nitrate (NO₃ ⁻) to nitrite (NO₂ ⁻) (Singh and Kumar 2008; Verma et al. 2015a). Most of the NO₃ ⁻ applied to the soil as fertilizer is taken by plants, leachedordenitrified. Not many of this NO₃ ⁻ is transformed to ammonium (NH₄ ⁺) by the assimilatory nitrate reductase (ANR) coming from soil microbes (Abdelmagid and Tabatabai 1987; Singh and Kumar 2008). This assumption is based largely on the results showing that, contrary to dissimilatory reduction of NO₃ ⁻ to ammonium ions, the assimilatory reduction of NO₃ ⁻ is greatly repressed by NH₄ ⁺concentration (McCarty and Bremner 1992) and that the amount of NH₄ ⁺ in soil is usually higher than that needed to inhibit the activity of ANR (Fu and Tabatabai 1989; Šimek et al. 2002; Martens 2005). In the literature, there are different opinions as regards the ways of the inhibition of soil ANR activity by NH₄ ⁺ions. Some researchers have stated that the negative influence of ammonium ions on the assimilatory nitrate reductase activity is caused by the presence of NH₄ ⁺itself and does not depend on the production of ammonium ions by soil microorganisms (Martens 2005), while other studies have concluded that the inhibitory result of NH₄ ⁺ on the soil ANR activity is caused by the occurrence of the glutamine as a result of NH₄ ⁺ assimilated by soil microorganisms (McCarty and Bremner 1992).

4 Genes Encoding the Mineralization Enzymes in Soil

The contribution of transcriptomics and proteomics in soil enzymology is still poorly developed probably because there are still methodological problems in soil proteomics. Linking enzyme activity to gene expression in soil is a challenging task. Detection of the specific enzyme activities does not identify the microbial species directly involved in the measured process, leaving the link between the composition of the microbial community and the production of key enzymes poorly understood (Nannipieri et al. 2002; Krasek et al. 2006; Colloff et al. 2008; Wallenstein and Weintraub 2008). Enzymes in soil may be intracellular or extracellular, wherein these extracellular enzymes are usually absorbed into soil organic (humic substances) and mineral (clay minerals) colloids. Enzymes in the soil can originate mainly from microorganisms but also from plants and animals. Assuming that it is possible to assess that an enzyme comes from microorganisms, there are commonly a lot of microbes that are able to produce the same enzyme. Additionally, there are frequently varied gene-coding enzymes that are able to catalyze the same or similar reactions, thus leading to some functional excess, which increases the ability of the enzyme-producing organism to adapt and cope with a diversity of environmental conditions (Naessens and Vandamme 2003).

Although detecting the presence of selected genes in soil is now possible, only some studies have focused on the connection between gene abundance and enzymatic activity in soil. The chitinase (E.C. 3.2–1.14) activity in soil was the first to be compared using the respective enzyme-encoding genes. An attempt was made by Metcalfe et al. (2003) to cover the whole process of gene expression, chitinase secretion, and its determination in brown forest soil. The community structure was assessed by extracting DNA and cloning and sequencing the PCR products with the application of the primers for A chitinases (bacteria, fungi, virus, animals, and some plant enzymes) for 18 family groups. The activity was measured by either the weight loss of chitin or via an assay using 4-methylumbelliferyl-(GlucNAc)2, which was higher in the soil that had been treated with sludge and was related to many species of actinobacteria. The analysis of the sequence revealed greater changes in the community structure in the case of the sludge and lime treatments.

The degradation of the lingo-cellulose complex is important in maintaining nutrient cycling. A lot of studies have been dealing with enzymes taking part in these processes or their responsible genes, but no many researchers tried to join the enzyme activity to specified gene products. Bogan et al. (1996a) determined the transcripts of the lignin peroxidase genes (lip A-lip J) of Phanerochaete chrysosporium in soil treated with anthracene at the dose of 400 ppm. Following the mRNA extraction, the occurrence of the lip gene transcript was found and determined using competitive RT-PCR. The lip proteins were extracted from the soil, cleaned, and used to a nitrocellulose membrane. The western blotting technique was accomplished using the monoclonal antibodies to Phanerochaete chrysosporium LiP H8. Later, Bogan et al. (1996b) were capable to find the transcripts of nine lip genes, even in non-sterile soil taken from a polluted site. In soil microbiology, laccase-encoding genes have primarily been used to study the structural and functional diversity of fungi (Theuerl and Buscot 2010). The diversity of fungal laccase-encoding genes was greater in the surface layer than in the deeper soil layers, and there was a great deal of variability in the surface soil (Luis et al. 2004, 2005). The presence of the laccase-encoding genes of basidiomycetes (DNA was extracted, amplified, and cloned with a final sequencing) changed during different seasons, whereas the laccase activity of the phosphate extracts of the soil remained constant through the years (Kellner et al. 2009). Cañizares et al. (2012) related the first link in the disclosure and expression of encoding genes coming from bacterial β-glucosidase (βgluF2/βgluR4 primers) to the relative enzyme activity in soil treated with long-term management practices and found that these genes were overexpressed in the tilled soils, which was probably the response of the bacteria to stress. However, only 50% of the amino acid sequences were matched by the database sequences that were retrieved, which indicates the presence of soil bacteria that have unknown β-glucosidases.

Recent progress in detecting extracellular peptidase-encoding genes and characterizing the diversity of the urease-encoding genes in soil bacteria has supported that molecular methods targeting some of the key enzymes may help us further understand the microbial community active in soil N mineralization (Fuka et al. 2008a). Various proteolytic genes have been determined in the soil, including serine peptidases (sub), alkaline metallopeptidases (apr), neutral metallopeptidase (npr), as well as aspartic protease (pep Aa, pepAb, pep Ac and pep Ad) (Fuka et al. 2008b; Veening et al. 2008). Sakurai et al. (2007) showed that the structure of the genes expressing bacterial populations (apr- and npr) was the most significant in characterizing the total protease activity in soil. The latest advances on the sub and npr genes in cultivation soils have demonstrated that those genes were various and spatially differentiated in soil (Fuka et al. 2008b; Mrkonjic Fuka et al. 2009). Some authors have suggested that the sub, apr, and npr genes plentifulness is frequently related to the obtainable proteolytic activity (Fuka et al. 2008a). A wide range of soil organisms including various populations of bacteria, fungi, as well as plants are able to produce enzyme urease, which hydrolysis is urea into ammonium and carbon dioxide (Tabatabai et al. 2010). Urease-encoding genes (ureC) have been found in a range of soil ammonia-oxidizing archaea (AOA) and bacteria (AOB) (Lu et al. 2012; Lu and Jia 2013). Metagenomic-based studies displayed that the ureC gene was widely arranged in various soils and was remarkably positively related to other genes of N-transforming enzymes, such as amoA and gdh (gene coding the enzyme glutamate dehydrogenase) (Yang et al. 2013, 2014). However, there are confined results related to the variety of the ureC genes in soil microbial populations (Singh et al. 2009).

Genes of proteolytic enzymes (e.g., bpr or aprE) are diversely expressed in different populations of soil microorganisms (Veening et al. 2008). Many environmental factors including carbon, nitrogen, phosphate and calcium concentrations, soil pH, moisture and temperature, the quality and quantity of available substrates sugars, salicylic acid, plant hormones, flavonoids, amino acids, and selected antibiotics increase gene expression (Maunsell et al. 2006; Shivanand and Jayaraman 2009; Verma et al. 2015b; Molaei et al. 2017a, b). The secretion of proteolytic enzymes by soil microbes is constitutive (they are still present in the cells) or inducible (their synthesis is stimulated by the presence of an appropriate inductor) as dependent on various growth stages (Burns 1982). The highest expression of genes coding proteolytic enzymes in bacteria and fungi may be in the initial constant period of their growth, throughout the exponential growth stage, or for the time of the late lag period (Allison and MacFarlane 1990).

5 Methodology of the C- and N-Cycling Enzymes: General Overview and Data Interpretation

Catalase and peroxidase were the first enzymes which activity was determined in soil more than 100 years ago (Skujins 1978). Since that time, many enzyme activities have been found and determined in the soil. However, the amount of enzymes in soil is significantly higher than those that have been determined because of many possible sources of enzymes occurring in soil (microorganisms, plants, fauna) (Ladd 1985). Additionally, by using many assays we are not able to identify the specific enzyme activity; e.g., during the measurement of casein-hydrolyzing activities, there is no possible to identify the specific bonds that are hydrolyzed or products that are released. Many methods of enzyme activities determination in soil have been developed by Tabatabai and coworkers (Tabatabai and Bremner 1969, 1970; Tabatabai and Singh 1976). Later, at least three books devoted to the methodology of soil enzymes were published (Alef and Nannipieri 1995; Scinner et al. 1995; Dick 2011).

Enzymes in soil are willingly determined since the assays are generally simple, accurate, sensitive, and relatively rapid. A range of enzyme activities and a large number of samples can be analyzed over a period of a few days using small quantities of soil. Changes in enzyme activities depend not only on variations of gene expression but are also affected by environmental and anthropogenic factors (Nannipieri 1994; Tabatabai 1994). Therefore, the determination of enzyme activities in soil requires the effective extraction followed by the exact assessment of the remaining substrate or the products formed during the reaction. Because most of the assays for assessment of the resulting product or decreasing substrate are based on colorimetric measurements, it is recommended to use buffers, which generally do not release organic matter from the soil. Interference by the color development originating from soil organic matter constituents is commonly known during colorimetric measurement. Every soil enzyme assay has its own optimum conditions, such as the suitable substrate concentration, the defined buffer pH, and the temperature of the incubation. At a substrate concentration that exceeds the value that limits the reaction rate, the incubation time should warrant a linear decrease of the substrate or product release and ought to be as short as possible in order to show a quantifiable part of the activity. Long-time incubation should be avoided due to the possible microbial proliferation and growth, the activities of intracellular enzymes, and the synthesis of new enzymes (Burns 1978; Burns and Dick 2002).

Current methods in soil enzymology allow us to determine rather the potential than in situ activity. This is due to the fact that the incubation conditions are selected in order to guarantee the highest rate of substrate conversion. Additionally, in enzyme assays, we use soil slurries to limit the spreading restriction. Thus, the assay conditions used in laboratories are dissimilar from those that occur naturally in soil, where pH, temperature, and moisture are rarely optimal and change very often, and the substrate concentration is usually in low concentration (Burns 1978).

The main problem in interpreting measurements of enzyme activities is to distinguish among many components contributing to the overall activity (Burns 1982; Nannipieri 1994; Gianfreda and Bollag 1996). The activity of any particular enzyme in soil depends on enzymes that can have different locations (living cells, dead cells, cell debris, soil solution, adsorbed by inorganic colloids and associated in various ways with humic molecules) (Datta et al. 2017a). In addition, abiotic transformations, the so-called enzyme-like reactions, can contribute to the overall activity (Gianfreda and Ruggiero 2006). The significant fractions of soil enzymatic activity are intracellular enzymes that are present in cells of plants, animals, and microorganisms. Taking into account that visible plant and animal residues are removed during the soil preparation before enzyme determination and that enzymes which have been liberated from dead cells are quickly degraded by microorganisms or inhibited by various unbeneficial factor occurring in soil environment, it can be assumed that enzymes in living and proliferating microbial cells are the most crucial part of intracellular enzymes found in soil. Thus, assessment of the intracellular enzyme activities can provide significant knowledge about the functional diversity of soil microorganisms. As regards the extracellular localization, the free enzymes are assumed to be short-lived as compared to enzymes associated with soil colloids (Burns 1982; Ladd 1985; Nannipieri 1994). With the currently used methodology, it is not possible to separate between enzyme activities originated from different locations. The following factors restrict advances in soil enzymology: (1) inability to isolate extracellular from intracellular enzymatic activity, (2) inability to extract and purify enzymatic proteins from soil, and (3) lack of suitable methods to extract quantitatively products of enzyme reactions occurring in soil (Tabatabai and Dick 2002). Another problem that is connected with the methodology of soil enzymes is the lack of standardization. The methodologies adopted for soil enzyme measurement are not universal and create difficulty in comparing soil enzyme research. Differences between substrates, assay conditions, incubation times, and detection methods (Marx et al. 2001; Burns et al. 2013) contribute to differences in the enzyme readings. The various experimental conditions that are used to determine soil enzyme activities are presented in Table 4.

Table 4 Methods for detecting selected activities of N-cycling enzymes in soil

Generally, there are two groups of methods used for soil enzymatic activity determination. One group is based on measuring of the substrate concentration decreases, and the other group is related to an increase in the concentration of the product released during the reaction. Techniques most often used in soil enzymology are presented below or in Tables 4 and 5.

Table 5 Methods for detecting selected activities of C-cycling enzymes in soil

• A. Colorimetric/spectrophotometric methods are based on the absorption of light (visible or ultraviolet) by the substrates and/or products released during the enzymatic reactions. The amount of the substrate remaining after reaction or product extracted from soil sample after incubation with an appropriate substrate and buffer, that are incubated at a specific temperature, pH, and time, is determined using colorimeter (or spectrophotometer). Most of the C and N cycle enzymes can be determined using these methods (Tabatabai and Dick 2002).

• B. Distillation – titration methods are used to determine the amidohydrolases, such as l-asparaginase, l-glutaminase, l-aspartase, amidase, and urease. These methods are based on the incubation of the soil with the proper substrate, and proper pH buffer after which the NH₄ ⁺ produced is assayed. After incubation, the enzymatic reaction is stopped by adding 2MKCl that contains Ag₂SO₄. The defined volume of the obtained mixture is distilled with MgO, and the NH₃ that is released is collected in boric acid that contains the appropriate indicators and is titrated with standard H₂SO₄. The detailed methodology was presented earlier by Tabatabai (1994).

• C. Fluorescence methods – some enzyme activities in soils have been detected using fluorimetric techniques. One of the earliest techniques of this kind was that proposed by Pancholy and Lynd (1971) for soil lipase activity. The reaction was based on the hydrolysis of the nonfluorescent butyryl ester of 7-hydroxy-4-methylcoumarin to 7-hydroxy-4-umbelliferone, the highly fluorescent compound. The fluorescence methods have been used for assaying the β-glucosidase activity in peat (Freeman et al. 1995) and for assaying β-glucosidase, β-cellobiase, β-N-acetylgalactosaminidase, and β-xylosidase in soil (Darrah and Harris 1986). Very important in these methods is to use the small amount of soil sample (milligrams) or to determine the capacity of the soil to adsorb the fluorogenic compounds that are liberated to correct the assay results.

• D. A microplate fluorimetric method was proposed by Marx et al. (2001) (as a modification of the earlier used fluorimetric techniques) to determine the activity of the hydrolytic enzymes and was based on the application of the methylumbelliferyl (MUB) substrates. Following incubation for a defined period of time at the desired temperature, MUF is quantified by terminating the enzymatic reaction by adding 0.5 M NaOH. The concentration of MUF is measured by a computerized microplate fluorimeter and is expressed as the micromoles MUF that are released per kg⁻¹ soil h⁻¹ (Deng et al. 2011). This method offers increased sensitivity and the possibility to estimate the kinetic parameter of the enzyme reaction. If it is successful, the advantages of this methodology are (1) speed of operation (less than 1 h), (2) simultaneous analysis of a large number of samples, (3) simultaneous use of a range of MUB conjugates, (4) measurement under standard conditions, and (5) automatic calculation of reaction rates. This method, which requires only milligram quantities of homogenous soil samples, has been used to measure the activities of β-d-glucosidase, β-d-galactosidase, N-acetyl-β-d-glucosaminidase, β-cellobiase, and β-xylosidase in a sandy loam and a silty clay loan soil (Marx et al. 2001). Modified, faster, microplate methods for the high-throughput determination of β-glucosidase were discussed recently (Hoehn 2016). The evaluation includes the use of an automated pipetting system and sonication, as well as a reduction in the number of analytical replicates, which permits a higher sample throughput suitable for service laboratory use.

6 Conclusions and Future Challenges

Based on the literature, it can be concluded that interest in the enzyme systems responsible for C and N transformation in soil is currently still high. The problem with the enzyme activities in soil is however related to the imperfect methodologies, which do not allow to measure the actual soil functioning. The methods of soil enzyme activities determination have the following limitations: (1) the methods do not differentiate between the constituents that contribute to the overall soil enzymatic activity, (2) they measure the potential rather than actual enzyme activities, (3) they do not separate the real enzymatic activity from the so-called “enzyme-like” activities, (4) there is no methods standardization, and (5) the currently used methods prevent to detect the origin of the soil enzymes (Gianfreda and Ruggiero 2006). That is why the development of the better assays of enzyme activity in soil is required.

Future research should also investigate if the enzymes are actively expressed and if, after expression, they catalyze the proper reactions without inhibition. The other area in soil enzymology should be devoted to the development of the new methods suitable to assess the genetic potential and gene expression, as well as the direct enzyme activity, showing the potential activity in the soil system. This study field includes research on the shift in the genes that codes for individual enzymatic proteins and the determination of soil factors that influence the expression of the specific enzymes (Sect. 5, Krasek et al. 2006). There is a great need to explain the connection between the genetic diversity and microbial community structure and functioning (Suenaga 2011; van Elsas and Boersma 2011; Meena et al. 2014). Since all of the enzymes have their relative genes, they are an ideal base for research on the relationship between microbial specification and particular processes occurring in the ecosystem. In this regard, progress in the development of “omics” technologies such as proteomics and transcriptomics give a great, although not yet proven, potential to explain a lot of aspects of the regulation of the functioning and ecology of soil enzymes.