8 Microbiology and Enzymology of Carbon and Nitrogen Cycling Robert L. Tate III Rutgers University, New Brunswick, New Jersey I. INTRODUCTION The title of this chapter brings to mind the diversity and essentiality to living systems of processes associated with the biogeochemical cycles involving nitrogen and carbon. A quick perusal of any basic biochemistry text suggests a nearly endless array of metabolic enzymes that catalyze the reactions necessary for energy transformation and cell replica- tion and survival. Indeed, on a larger scale, ecosystem stability and sustainability (terms frequently linked to native and managed systems, respectively) rely nearly in toto on a foundation of a functional microbial community, including the complexities of intermedi- ary metabolism of the diverse soil microbial population. Fortunately, in analyzing the status of current research relating to this topic, a relatively limited number of nitrogen and carbon catabolic enzymes have served as indicators of the metabolic status or activity of the soil biological community. Justification of studies of carbon and nitrogen cycling enzymes has frequently been linked to agricultural systems, but associations with soil management in general, as well as reclamation concerns in particular, are becoming more common. With the need to ame- liorate the impact of past anthropogenic intrusion into terrestrial systems through appro- priate management as well as the desire to preclude or minimize future damage to soil systems (i.e., enhance our capacity to discern proper soil system stewardship), it would be ideal if a clear understanding of carbon- and nitrogen-based processes were attainable. Carbon and nitrogen cycling not only are essential processes for the maintaining, transfor- mation, and flux of essential elements and energy in the biosphere, but are also crucial to management and reduction of the impact of many organic and some inorganic pollutants. Worldwide implications of soil-based carbon and nitrogen processes are exemplified by their impact on global greenhouse and ozone depleting gas production and consumption. For example, selection of cultivation methods can have a significant impact on carbon dioxide production from microbial respiration as well as reduction of atmospheric carbon dioxide loading (35,71). Similarly, quantities of nitrous oxide (both a greenhouse and an ozone-depleting gas) evolved from terrestrial systems are affected by fertilizer use Copyright © 2002 Marcel Dekker, Inc. (nitrificationanddenitrificationeffects)aswellasbyprotectionandcreationofwetlands (4,7,39,57,85,122). Itisagainstthisbackdropofthemajorenvironmentalrelevanceoftheenzymesof nitrogenandcarboncyclingprocessesthatthischapterispresented.Theutilityofsoil enzymeactivitiesasindicatorsofsoilqualityandinmonitoringoftheeffectsofsoil pollutionispresentedelsewhere(14,34,60,116,131)andinChapters15,16,and17.The general objective of this chapter is to highlight the current status of our understanding of soil carbon and nitrogen processes and the properties of the soil system that controls activity of the enzymes catalyzing these nitrogen and carbon transformations. II. NITROGEN AND CARBON TRANSFORMATION PROCESSES A. General Metabolic Considerations Enzymes associated with carbon and nitrogen transformations are central to cellular growth and energy processes. Thus, it is logical to conclude that any enzyme involved in cellular metabolism must be present in soil. Furthermore, quantities of the enzyme present in a particular soil and the reaction kinetics should reflect the basic metabolic properties of all cell systems. However, the utility of assessing quantities of carbon and nitrogen transformation enzymes in soil for describing overall system function is more complicated. The environment within which the enzymatic transformations occur is a complex array of sand, silt, and clay particles intermixed with a diverse array of organic substances. Some of the organic matter is readily available to and transformed by soil enzymes, but a significant portion is intrinsically more resistant to biodegradation because of its chemical structure. Additionally, substrates generally expected to be more ephemeral may exhibit extended longevity that is due to their physical location within the soil matrix (1,122). Further complications in interpreting or predicting biodecomposition kinetics may arise from the limited water solubility of the potential substrate. Frequently, only a small portion of the organic complex in soil is water-soluble (121). Because of the necessity of conver- sion of the water-insoluble energy resources to a form that can enter the cell and be metab- olized, the cell must produce enzymes that function outside the confines of the cellular membrane—beyond the relatively safe environment of the cell. Thus, our concept of car- bon and nitrogen transformation in soil must include an evaluation of the sorptive (e.g., clay interactions), physically adverse (e.g., temperature and moisture variations), and chemically limiting (e.g., pH, water-soluble heavy metals) extracellular environment. The emphasis of this presentation is on the current status of basic enzyme studies involved in carbon and nitrogen transformations in soil. A number of excellent reviews (17,33,36,113) that are available on this topic are useful when considering its historical context. More current examples of the types of reactions studied in soil, considerations of the implications of the physical structure of the soil ecosystem on enzyme activities, and future research needs are examined herein. B. Specific Enzymatic Activities Although an interminable array of enzymes involved with carbon and nitrogen metabolism could be evaluated in soil and associated ecosystems, only a limited number of enzymic activities are commonly studied. Many of the enzymes are those generally found to exist and to express their catalytic activities extracellularly, such as cellulase. Others, such as Copyright © 2002 Marcel Dekker, Inc. urease, are found to catalyze reactions both within and outside cells. Ideally, enzyme activities selected as indicators of soil fertility or soil quality should be easily quantified and vary with ecosystem type, condition, or degree of human intervention. Until the more recent era most soil-based research has been directed toward meeting agricultural needs (34,36). Therefore, to a large degree, the historical evaluation of enzyme activity in soil has been concentrated on quantification of cropping and management ef- fects on activities involved with biogeochemical cycles (e.g., recycling of plant biomass nutrients and nitrogen fixation) or more directly agriculturally pertinent enzyme activities, such as urease. As can be concluded from the investigations cited later, the commonly studied activities involving nitrogen transformations have been associated with ammonium generation (amidases and urease), hydrolysis of proteins (proteases), nitrogen fixation (ni- trogenases), and loss of nitrogen from soil ecosystems (nitrogen oxide reductases). Simi- larly, activities involving carbonaceous substances have included those associated with hydroxylation of aromatic rings (e.g., polyphenyl oxidases, laccases), leading ultimately to either mineralization or humification of the parent compounds; hydrolysis of polysac- charides (e.g., amylases, cellulases, xylanases); and a variety of lipases and esterases; plus the indicator of respiratory activity, dehydrogenase. These enzymatic activities have proved useful for assessment of more general ecological concerns, such as organic matter transformations in native soil systems, as well as of the effect of human intervention. For example, in the latter arena, any of the general carbon or nitrogen catabolism enzymes (e.g., cellulase, hydrolases, dehydrogenase) is useful in assessing impacts of recycling waste organic matter (e.g., composts, sludges) through soil ecosystems, whereas poly- phenyl oxidases and laccase activity assessments are commonly linked to decomposition and humification of aromatic ring–containing xenobiotic chemicals. III. ENZYME ASSAYS AND THEIR EFFECT ON DATA INTERPRETATION The two primary questions that must be addressed in assessing carbon and nitrogen meta- bolic processes in soil are, How can the activity be quantified and what is an appropriate assay method? and How does the activity vary both in a relatively defined system in the test tube as well as in the more complex, heterogeneous environment of the soil? A primary property that is intimately linked to the latter question is the kinetics of the reaction. Although a sound assay method based on a clear understanding of the specific reactants and the reaction kinetics of the individual enzyme is essential to provide reliable data, the effect of soil particulates on the reaction properties must also be understood. Responses to either of the preceding questions are nontrivial when considering a soil ecosystem, especially when dealing with those enzymatic activities most closely linked with cellular energy and nutrient management. The characteristics of the enzyme reaction must clearly be understood (i.e., reaction substrates, products, optimal conditions, and activity curves), but more important are the properties of the environment (extra- or intracellular) within which the enzyme functions. Concerns with the possibility of changes in enzymatic activity during sample collection, storage, and analysis are particularly acute when evaluating those activities associated with carbon and nitrogen transformations. A common general objective of enzyme studies is to estimate the quantity of enzymic activity expressed in the native soil site. Thus, changes in activity due to synthesis of new enzyme; either in the reaction mixture or in Copyright © 2002 Marcel Dekker, Inc. thesoilsampleitselfpriortoquantification,mustbeprevented.Asisdocumentedinthe followingsection,thereisadelicatebalancebetweentheamountofenzymaticactivity andenzymemolecules,cellularmetabolicstate,andsubstrate(orinducer)level.Aslight changeinthesoilphysicalstructurecanresultinasignificantchangeinquantityofenzyme inasoilsampleinanassaymixtureasaresultofinductionofnewactivityorenzyme repression.Thisvariationinenzymeactivitycouldresultfromliberationofsubstrate, inducers,orinhibitorsfromthesoilmatrixbydisruptionofthesoilstructure.Thus,appro- priatedesignofastudyofsoilenzymesmustincludeanappreciationofnotonlythebasic traitsoftheenzymereactionitselfbutalsothewaysthatthesoilpropertiesmayalterthe measuredactivity. Presentationofspecificassaymethodsforthevariousenzymescommonlyquantified insoilsamplescanbefoundinChapter21.Nonetheless,considerationofsomeofthe general factors associated with the physical status of the enzyme and the state of the cells producing the enzymes is essential, because both affect the quantity of enzyme detected in the environmental sample and the kinetics of the reaction. Ultimately, the objective of any assessment of enzymatic activity is to relate the amount of activity to properties or conditions of the site from which the sample was collected. Furthermore, current questions relating to appropriate soil stewardship necessitate sufficient understanding of the variabil- ity of enzyme activity with soil properties to allow prediction of the relationship between enzyme and changes in ecosystem conditions, anthropogenically generated or other. A. Soil Sample Collection and Data Interpretation Although, as indicated, considerable effort is expended to assure the accurate measurement of enzyme activity in a reaction mixture, experimental objectives are usually directed at elucidating the activity expressed in a particular soil site. The two values are not necessar- ily equivalent. As soon as a soil sample is collected, the environmental parameters de- termining the amount of enzyme present and the proportion of that enzyme that is active are altered (121,122). Two examples of changes that can affect the metabolic status of the enzyme-producing cells are soil oxygen tension and the availability of the carbon and energy source. Oxygen concentration in soil is generally controlled by its diffusion rate from the atmosphere above the soil into the soil matrix as well as the rate of its consump- tion. This supply/consumption relationship can result in anaerobic microsites within the larger soil aggregates. Disruption of soil aggregates through the mechanics of soil sample collection (as well as by the common practice of sieving the soil in preparation for enzyme assays) alters this distribution of aerobic and anaerobic microsites and affects microbial metabolism accordingly. Additionally, much of the native soil organic matter is physically protected from access by microbes and their enzymes. That is, the organic material is physically occluded within soil aggregates, trapped in soil nanopores, or sorbed onto parti- cle surfaces (1,98,122). Thus, the simple act of collecting a soil sample alters its physical state and likely increases the accessibility of the soil organic matter to enzymes and mi- crobes. As a consequence, induction of new enzymatic activities and augmentation of existing activity through microbial replication may occur. Thus, an altered microbial com- munity and its associated enzyme activities are necessarily created by the simple act of sample collection. At least a minimal change in soil enzyme activity, particularly that central to the metabolism of the microbial cell, by sample collection and storage is inevita- ble. However, it must be noted that the quantities of immobilized (stabilized) extracellular enzymes are not likely to be greatly changed by this process. Copyright © 2002 Marcel Dekker, Inc. Soil sampling procedures may also affect the activity associated with mineralization of xenobiotic contaminants. This is especially true in aged, contaminated soils where the accessibility of organics is often reduced by sequestration (1). During the aging processes (i.e., as the interval between input of the contaminant and sampling), the xenobiotic sub- stances and their metabolites become distributed among soil micro-, macro-, and nano- pores in free and sorbed states. That portion of the chemical retained in interstitial waters of macro- and some micropores is most available for interaction with soil microbes and their enzymes. Therefore, equilibrium solution concentrations dependent upon the seques- tering or sorption of the chemical pollutant can be altered by soil sampling and manipula- tion when the equilibrium is altered through disruption of soil structure and redistribution of soil water (122). Each of these alterations of enzyme activity due to sample collection reflects on the validity of extrapolating the activity detected in the laboratory to that expressed in situ. In each case, the changes may be reduced or minimized by lessening destruction of soil structure during sampling and storing the soil sample under conditions that minimize the potential for microbial growth and enzyme synthesis (commonly at 4°C). Generally, assay procedures for soil enzymes are designed to prevent increases in microbial numbers in enzyme levels during the assay. Thus many assay protocols recom- mend the use of growth inhibitors (e.g., toluene, mercaptoethanol, sodium azide, radiation sterilization, antibiotics) or utilization of an assay time that is insufficient for microbial growth and production of significant quantities of de novo synthesized enzyme (16, 75,113). Although it is reasonable to assume that the level of activity expressed in a freshly collected soil sample is optimized for the in situ conditions, these conditions are not static. Therefore, induction of new enzyme activity can occur when soil conditions change. Ex- amples of evidence supporting the conclusion that enzyme induction occurs readily in soil are provided by studies of l-histidine ammonia lyase (19) and nitrogen oxide reductases (114). Burton and McGill (19) found an increase in l-histidine ammonia lyase activity in soil in the absence of microbial growth when specific inducers of the enzyme were added to soil samples. An additional example of the importance of enzyme induction in soil is provided by studies of denitrification rates. Quantification of the kinetics for appearance of new enzyme, albeit from enzyme induction or microbial growth, has been used to estimate nitrous oxide reductase activity in field soils. A common means of estimating denitrification is to inhibit nitrous oxide reductase activity with acetylene. Thus, all of the nitrate denitrified accumulates in the reaction vessel as nitrous oxide. Smith and Tiedje (114) observed three-phase reaction kinetics for this process when quantifying nitrous oxide production in freshly collected soil samples incubated under controlled conditions in the laboratory. In the first few hours nitrous oxide production results from the activity of preexisting enzyme. This is followed by a transition period that results from the produc- tion of new enzyme by induction of enzyme synthesis in preexisting cells. In the third phase new enzyme activity results from the increased enzyme levels provided by an in- creased population density of active denitrifiers. Because of its critical nature in estimating native nitrous oxide reductase enzyme levels in soil samples the duration of the initial phase of the process has been evaluated by several investigators. It is reasonable to assume that the duration of each of the three phases varies with ecosystem type and status. Differ- ences in the metabolic status of the denitrifier population vary (e.g., inactive as a result of the presence of O 2 or already maximized through optimal conditions—therefore no further growth or induction of the population or activity may occur), as would the occur- Copyright © 2002 Marcel Dekker, Inc. rence of indirect inhibitors of the denitrification process (e.g., excessively high or low pH or temperature) that limit or slow growth and enzyme induction. Luo and associates (79) recommend an incubation period of not longer than 5 h at 20°C for estimating preexisting denitrification activity in soils. Similarly, Dendooven and Anderson (27) found that de novo synthesis of nitrite reductase started in their system 5 hours after imposition of anoxic conditions and that of nitrous oxide reductase after 16 hours. Other procedures that are useful in evaluating preexisting or indigenous nitrogen–oxide reductase activity in native soil samples include gamma sterilization of the soil (75) and incorporation of chloram- phenicol into the assay mixture (28–30,94). The assumption in all of these studies is that the initial rate observed in the incubated fresh soil sample represents that enzyme’s pres- ence in the soil prior to collection from the field. The activity is still considered to be a potential activity in that it is likely that the nitrogen oxide substrate does not exist at saturating concentrations in the field site. B. Relationship of Laboratory Enzyme Activity to Enzyme Expression in Field Soils Of concern when relating the laboratory generated data to field situations is the fact that the laboratory assessments are based on maximizing the interaction between enzyme and substrate. Thus, something approaching total activity is usually assessed in the laboratory, whereas in the field the interaction of the enzyme and its substrate may be reduced as a result of a variety of soil properties affecting the efficiency of interaction of the substrate and enzyme molecules. In other words, a portion of the enzyme molecules existing in the field soil may not be actively engaged in catalyzing their requisite reaction or may be transforming the substrate at a suboptimal rate. Therefore, the enzyme activity expressed in the laboratory assay must be assumed to be maximal (given the defined conditions of the assay) until demonstrated otherwise. Enzyme activities measured in the laboratory are ‘‘potential’’ activities. C. Control of Expression of Enzymes in Soil Microsites Two forms of interaction between the enzyme and its physical environment can delineate enzyme function within a soil microsite: occlusion within a living cell, cell debris, or even a soil aggregate and sorption or binding to soil minerals or non-water-soluble organic substances. Thus, manipulation of a soil sample that disturbs native associations (e.g., disrupts aggregates or fractures cells) or alters the equilibrium between sorption and de- sorption results in reaction rates that differ from those of the native environment. As was described in detail by Burns (17,18), enzymes exist in a variety of states in soil: that is, in growing or nongrowing microbial cells, cell debris, associated with clay minerals or soil organic matter, and soluble in the aqueous phase as free enzyme or enzyme–substrate complexes. Most commonly quantified soil enzyme components as- sayed are the activities contained within living cells, bound to soil organic matter, or soluble in the soil interstitial water. Additionally, soil enzyme may be associated with clay minerals or occluded within soil aggregates. The consideration of the inclusion of enzymes within soil aggregates is rarely taken into account because enzyme activities are usually measured in soil suspensions in the laboratory. This practice ensures maximum rates of enzyme–substrate interaction and adheres to basic enzyme assay principles when total enzyme within a system is considered. A future concern may be to evaluate in greater Copyright © 2002 Marcel Dekker, Inc. detail the impact of heterogeneity in location within the soil system on the portion of the enzyme activity that is expressed in situ. To reiterate, these distribution considerations related to enzymes of nitrogen and carbon cycling in soil affect the total quantity of activity expressed as well as the rate of the reactions—thereby controlling overall ecosystem and population dynamics. The general spatial variability of microbes, enzymes, and their activities in soil has long been appreciated (92). This variation in activity is accentuated in the enzymes associ- ated with carbon and nitrogen metabolism because of their strong linkage with inputs of readily metabolized fixed carbon resources. Thus, these enzymatic activities are highest in regions of native biomass production or inputs (e.g., rhizosphere) or in soils receiving organic wastes (e.g., composts or biosolids) and generally correlate significantly with lev- els of native soil organic matter (10,113). Macrosite variability is of interest in assessing general ecosystem nutrient dynamics, but considerations at a microsite level may be more useful in determining the means and kinetics of reactions associated with organic pollutant decomposition or the effects of management decisions relating to the sustaining or improving of soil quality. From the foregoing, it could be concluded that increases in soil aggregation would result in a decline in soil carbon and nitrogen transformations. Generally, this is not observed (121). In fact, management of soil in a manner that increases soil aggregate formation usually results in a stimulation of the microbial and enzymatic activity associated with carbon and nitrogen metabolism. For example, Kandeler and Murer (68) noted that increased soil aggregation in a conventionally tilled agricultural soil converted to grassland was accompanied by significant increases in dehydrogenase, protease, and xylanase activities. Conversely, re- turning the soil ecosystem to conventional agricultural management caused a decline in the elevated enzymatic activities. The distribution of enzymes involved in carbon and nitrogen transformation within the soil profile and aggregates reflects a central dogma of soil enzymology: that activities of carbon and nitrogen metabolizing enzymes measured in a soil sample correlate with levels of soil organic matter and readily available organic matter. For example, activities of xylanase, invertase, and protease have been found to be stimulated in the detritosphere (the soil litter interphase) (67). In another study, xylanase and invertase levels were ele- vated in the soil particle-size fraction (Ͼ200-µm fraction) containing the decomposing maize straw (117,118). Association of individual enzymes with specific size fractions relates in part to their interaction with fresh organic matter and the degree to which the activity is linked to humic acids (i.e., humic acid stabilized enzymes) (95,118). These relationships between organic matter sources and enzyme activities support a hypothesis that any soil management procedures that encourage the maintenance or development of soil aggregates optimize plant biomass production. Therefore, since the primary source of energy for the soil microbial community and substrates for the associated enzymes is the carbon fixed by the plants, the soil microbial biomass and their associated intra- and extracellular activities are in turn optimized by the improved soil management. D. Distribution of Enzymes in Soil and Enzyme Kinetic Parameters Reactions catalyzed by enzymes in soils, including those complexed to clays or organic matter, can be anticipated to follow Michaelis–Menten kinetics. In the case of humic– and clay–enzymes complexes, any divergence in reaction properties is a result of impair- ment of enzyme–substrate interactions due to alteration of the basic conformation of the Copyright © 2002 Marcel Dekker, Inc. enzyme protein when in the sorbed state (clay micelles) or covalently bound to soil humic acid. Additionally, it must be remembered that in a multicomponent system the activity quantified could result from the summation of a number of enzyme types in various loca- tions that catalyze the same reaction with different kinetic constants. Therefore, the resul- tant kinetic parameters are an average of all forms and states of the enzyme molecules and their compliance with Michaelis–Menten kinetics may be at times coincidental. A primary consequence of the physical location of enzyme molecules in soil is its impact on the probability of substrate–enzyme interactions (i.e., free diffusion) as well as the potential for the induction of enzyme synthesis (19). Sorption of extracellular en- zymes with clay and/or humic substances can also alter the efficiency (K m ) of the reaction. Thus, both the total enzyme as reflected in the V max and the efficiency of the transformation are environmentally controlled. Analysis of the kinetics of a reaction can be used to show occurrence of multiple forms and states of an enzyme in soil (see Ref. 122 for discussion of use of Eadie Scatchard plots in this analysis). An example of use of enzyme kinetic parameters to demonstrate occurrence of specific isoenzymes in soil is a study of urease (21) in which K m values were shown to vary between 0.5 and 1.3 M depending on soil type and pH. Thus, enzyme kinetic patterns observed in soil may reflect properties of the reaction in situ but not the kinetics of a purified enzyme. Therefore, enzyme properties assessed in the complex soil sample are described as apparent reaction kinetic parameters. Hope and Burns (63) developed a method of assessing extracellular enzyme diffu- sion in soil that also reveals the variable affinities of enzymes with specific clay minerals— thereby adding a consideration of soil type to any evaluation of enzyme activity variation in soil ecosystems. These workers studied the variable effect of bentonite (high surface area and high cation exchange capacity) and kaolinite (low surface area and low cation exchange capacity) on diffusion of endoglucanase and β-d-glucosidase in soil. The kaolin- ite had no effect (i.e., binding) on enzyme diffusion, whereas the bentonite significantly reduced (bound the enzyme) mobility. Thus, these studies showed that movement of an enzyme molecule from the vicinity of the cell synthesizing it is environmentally controlled. Concern over the effect of this high affinity of some clay molecules for the enzyme proteins on enzyme kinetics was also discussed, especially in the context of extracellular enzyme efficiency. Clay interactions with or effects on biological systems and products (e.g., enzymes) are frequently discussed as if clay properties are relatively uniform. The example noted previously involving kaolinite and bentonite already suggests that there is considerable disparity in properties of different clay minerals. Because of the high variability in clay mineral quantities and types among various soil types (12), generalizations regarding the role of clay in expression of activities of the enzymes of the carbon and nitrogen cycles are difficult, if not impossible, to derive. Among the foremost of the variable properties of clay minerals affecting interactions with soil enzymes are their surface area and cation exchange capacity. The effects of these properties and their variation with clay type and enzyme have been documented with a variety of enzymes (104,121,122). Examples of this analysis of nitrogen and carbon metabolism associated enzymes are provided by studies of urease and invertase in the early 1990s. Gianfreda and coworkers (50) examined the inter- action of invertase (β-fructosidase) with montmorillonite, aluminum hydroxide, and alu- minum hydroxide–montmorillonite complexes. The sorption of invertase varied with pH of the reaction mixture and the specific clay mineral, most sorption was detected with montmorillonite and least with aluminum hydroxide. Sorption reduced the enzyme activity in general, the proportion of enzyme activity lost due to sorption varied with pH and clay Copyright © 2002 Marcel Dekker, Inc. type. Invertase was stabilized by association with the clay surface in that resistance to heat was increased in the sorbed state. In a similar study with urease (51), using the same clay minerals, the heat stability of the sorbed enzyme was reduced, as were the Michaelis constants, V max , and K m . Lai and Tabatabai (74), in an evaluation of the sorption of jackbean urease on kaolinite and montmorillonite, found that the K m values of the sorbed enzyme were similar to that of free enzyme. E. Enzyme Binding to Soil Humus It has long been appreciated that stable extracellular enzymes occurring in soil are usually covalently linked to humic acid (20,86). For example, Nannipieri and colleagues (86) fractionated urease and proteolytic activity into a variety of molecular weight fractions. The number of molecular weight peaks varied with specific enzymes: that is, the enzyme was fractionated on the basis of the size of humic acid molecules with which it was associ- ated. As with the sorption of enzymes to clay, the enzyme kinetic parameters are altered by any resulting occlusion of the enzyme’s active site by the humic acid molecule or by conformational alterations to the enzyme structure due to changing molecular forces in- duced by the covalent linkage between the two macromolecules (121). Interactions between macromolecules and enzyme proteins may also alter enzyme properties in a soil system. For example, the effect of binding of enzymes to polysaccha- rides on enzymatic activity was evaluated with urease purified from Bacillus pasteurii immobilized on calcium polygalacturonate: a model for mucigel (24). It was noted that the adsorption parameters of the enzyme and polysaccharide varied with sodium chloride concentration of the reaction mixture, suggesting that the nature of the interaction between the enzyme and the polysaccharide involved electrostatic associations rather than covalent linkages. Variation in the kinetic parameters and stability of the enzyme were used to assess the effect of association of the sugar polymer on the accessibility of the enzyme and its conformation. The bound extracellular enzyme exhibited increased stability with time and to heat denaturation compared to the soluble enzyme. The similarity of the V max values between the two enzyme forms suggests that little conformational change in the enzyme structure had occurred but accessibility of the enzyme to its substrate was altered, as indicated by variation in the K m value. Similar studies have evaluated the effect of humification of proteins on their enzy- matic activities. Examples using enzyme involvement in carbon and nitrogen transforma- tions include the effect of bonding of β-d-glucosidase to a phenolic copolymer of l-tyro- sine, pyrogallol, or resorcinol (108) and of linking of urease to tannic acid (49,52). Sarkar and Burns (108) found that their copolymers had several properties in common with those of native soil humic acid–enzyme complexes (E 4 /E 6 ratios; carbon, hydrogen, nitrogen, and sulfur ratios; and infrared [IR] spectra). A lowering of the efficiency by polymerization was shown by both reduced V max and increased K m values. Association of the copolymer with bentonite resulted in a complex that was resistant to protease and much more stable than the native enzyme. Similarly, Gianfreda and associates (52) found that inclusion of ferric ions and aluminum hydroxide species with tannic acid in forming organomineral urease complexes resulted in maintenance of the conformational and enzymatic properties of the free enzyme. These latter studies demonstrate how one enzyme commonly found in an extracellu- lar state (urease) and one more closely related to the microbial cell (β-d-glucosidase) can be stabilized with soil organomineral complexes with activity levels sustained at a level Copyright © 2002 Marcel Dekker, Inc. that allows the enzyme to continue to contribute to ecosystem carbon and nitrogen dynam- ics. Considering that the long-term and heat stability of the enzymes were generally en- hanced in the bound and/or mineral associated forms, it could be reasonably concluded that the total impact of the enzyme on overall ecosystem function was extended by the interactions with the abiotic soil constituents. This dissociation of enzyme activities from living cell is a factor that must be considered in assessing the utility of assessing soil enzymatic activities as an indicator of soil status or quality. The foregoing analysis of the properties of enzyme reactions leads to the conclusion that a number of questions need to be addressed before applying basic enzyme kinetics analyses to studies of soil ecosystem function, such as environmental impact or soil quality assessments, or ultimately before predicting and measuring the outcome of soil ecosystem management decisions. Soil enzyme activities generally can be anticipated to follow Michaelis kinetics, especially intracellular enzymatic activities linked to essential meta- bolic processes. Interactions of extracellular enzymes with soil components result in varia- tion in anticipated reaction rates or efficiencies. Additionally, should the physical condi- tions of the soil dictate, enzyme or substrate accessibility and enzyme reaction kinetics may deviate in toto from that anticipated. This latter situation can occur when the parame- ter quantified is the sum of several enzymatic reactions (e.g., dehydrogenase activity, carbon dioxide evolution from complex organic matter substrates) or when the enzyme accessibility is controlled by processes such as diffusion or by the surface area of non- water-soluble substances. In the latter situation, dissolution or solubilization of the sub- strate may be a nonenzymatic process (122). Even though at the site of action product generation may reflect Michaelis–Menten kinetics, the kinetics of the reaction for the ecosystem in toto would reflect diffusional limitations or other kinetic parameters. Further- more, it is much more difficult to quantify individual enzyme reactions in a soil. As a result of its great heterogeneity (compared with the environment of a test tube with a defined mixture of reactants), the reaction kinetics occurring in the field may reflect en- zyme induction and microbial growth rates, altered reaction kinetics (due to sorption and humification of the enzymes), as well as variations in the degree of saturation of the enzyme molecules with substrate and the distribution of the catalysts and reactions within the soil matrix. These considerations may be particularly important for data interpretation in regard to xenobiotic chemical mineralization since it is not uncommon to use radio-labeled carbon dioxide evolution from labeled parent compound to assess mineralization capacity. Thus, the kinetics for the overall mineralization processes as estimated by radio-labeled carbon dioxide production from the parent compound in soil reflects the totality of the processes occurring between entry of the parent molecule into the ecosystem and generation of the mineralization products. Therefore, a variety of zero-order (substrate levels are generally saturating the available enzymes) and first-, second- or mixed-order models are used to describe these enzyme reactions in soil. In all cases, the reactions approximate the product of the interaction of the enzyme between its substrate and environment. More detailed analyses of this aspect of soil enzymology are found elsewhere (2,122). IV. CARBON AND NITROGEN TRANSFORMATION ENZYMATIC ACTIVITY IN FIELD SITUATIONS A consideration of the status of studies of carbon and nitrogen transformations in soil could be initiated by the question, Why study soil enzymes? In attempting to answer this Copyright © 2002 Marcel Dekker, Inc. [...]... transformations, including dehydrogenase, have been assessed in metal contaminated soils in both laboratory and field studies The metals have a varying impact on enzyme activity, depending not simply on their total concentration in the soil but rather on capacity to interact with the enzyme protein Thus, the direct impact of the cations is affected by soil pH, soil organic matter levels, and interactions with other... Dec, J-M Bollag Determination of covalent and noncovalent binding interactions between xenobiotic chemicals and soil Soil Sci 162 :85 8 87 4, 1997 Copyright © 2002 Marcel Dekker, Inc 27 L Dendooven, JM Anderson Dynamics of reduction of enzymes involved in the denitrification in pasture soil Soil Biol Biochem 26:1501–1506, 1994 28 L Dendooven, JM Anderson Maintenance of denitrification potential in pasture... cycling rates in complex soil ecosystems Both are exclusively intracellular enzymes The results of studies of these enzymes are commonly presented as if a single enzyme following Michaelis–Menten kinetics were being evaluated However, unless the specific substrate for the enzyme activity in question is added to the reaction mixture, the kinetic parameters measured are the result of interaction of all enzymes. .. activity (including a wide variety of extracellular and intracellular enzymatic activities) was provided by an examination of the effect of copper concentrations on carbohydrases, proteases, lipases, and phosphatases in organic soils receiving low levels of copper amendment (82 84 ) In all cases, the enzyme activities were reduced in the presence of the copper As a result of the complexities of the interaction... for quantifying general microbial respiration in a variety of soil ecosystems, including those where inhibitors might be anticipated to present complications for data interpretation Examples of the latter situations include flooded soils (88 ,93), drained and flooded organic soils (119,120), and metal contaminated soils (70) When evaluating the utility of the activities of soil enzymes as indicators or... arylsulfatase, invertase, α-galactosidase, and catalase correlated at the 5% level (42) Of the carbon and nitrogen cycle enzymes, only urease activity did not correlate with the carbon dioxide evolution Frankenberger and Tabatabai (46) found that l-glutaminase activity correlated with amidase, urease, and l-asparaginase activities in their soil samples Similarly, in a study of enzyme activities in rhizosphere... production and removing potentially toxic xenobiotic Copyright © 2002 Marcel Dekker, Inc chemicals from soil interstitial waters through covalent linkage to humic acid Both abiotic and biotic agents in soil catalyze these processes Enzymatic contribution to the process involves formation of free radicals by two common soil enzymes, laccase and tyrosinase The potential role of these enzymes in the humification... cyanobacterial-lichen crusts Biol Fertil Soils 23:362–367, 1996 7 AM Blackmer, JM Bremner Inhibitory effect of nitrate on reduction of N 2 O to N 2 by soil microorganisms Soil Biol Biochem 10: 187 –191, 19 78 8 J-M Bollag Decontaminating soil with enzymes: An in situ method using phenolic and anilinic compounds Environ Sci Technol 26: 187 6– 188 1, 1992 9 H Bolton Jr, JL Smith, SO Link Soil microbial biomass and activity... priori conclusions regarding this activity are that since the general soil microbial community is Copyright © 2002 Marcel Dekker, Inc carbon limited (122) and since the activities of the enzymes associated with carbon and nitrogen transformations are commonly directly linked to levels of microbial activity, then any amendment of a fixed-carbon resource would result in an increase in the relevant enzyme... humification of anilinic and phenolic compounds and reduction of their bioavilability with the passage of time (aging) is sufficient reason to investigate their function in soil further (8, 26,90,125) C Elucidation of Basic Ecosystem Properties Current research about the environmental variability of enzyme activity and how it relates to properties of the soil system is commonly associated with maintaining a highly . During the aging processes (i.e., as the interval between input of the contaminant and sampling), the xenobiotic sub- stances and their metabolites become distributed among soil micro-, macro-,. another study, xylanase and invertase levels were ele- vated in the soil particle-size fraction (Ͼ20 0- m fraction) containing the decomposing maize straw (117,1 18) . Association of individual enzymes. transforma- tions include the effect of bonding of β-d-glucosidase to a phenolic copolymer of l-tyro- sine, pyrogallol, or resorcinol (1 08) and of linking of urease to tannic acid (49,52). Sarkar and