Enzymes in the Environment: Activity, Ecology and Applications - Chapter 8 potx

Specific Enzymatic Activities Although an interminable array of enzymes involved with carbon and nitrogen metabolismcould be evaluated in soil and associated ecosystems, only a limited nu

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 ofprocesses associated with the biogeochemical cycles involving nitrogen and carbon Aquick perusal of any basic biochemistry text suggests a nearly endless array of metabolicenzymes that catalyze the reactions necessary for energy transformation and cell replica-tion and survival Indeed, on a larger scale, ecosystem stability and sustainability (termsfrequently linked to native and managed systems, respectively) rely nearly in toto on afoundation of a functional microbial community, including the complexities of intermedi-ary metabolism of the diverse soil microbial population Fortunately, in analyzing thestatus of current research relating to this topic, a relatively limited number of nitrogenand 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 beenlinked 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 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 soilsystems (i.e., enhance our capacity to discern proper soil system stewardship), it would

ame-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 tomanagement and reduction of the impact of many organic and some inorganic pollutants.Worldwide implications of soil-based carbon and nitrogen processes are exemplified bytheir impact on global greenhouse and ozone depleting gas production and consumption.For example, selection of cultivation methods can have a significant impact on carbondioxide production from microbial respiration as well as reduction of atmospheric carbondioxide 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

(nitrification and denitrification effects) as well as by protection and creation of wetlands(4,7,39,57,85,122).

It is against this backdrop of the major environmental relevance of the enzymes ofnitrogen and carbon cycling processes that this chapter is presented The utility of soilenzyme activities as indicators of soil quality and in monitoring of the effects of soilpollution is presented elsewhere (14,34,60,116,131) and inChapters 15,16, and17 Thegeneral objective of this chapter is to highlight the current status of our understanding ofsoil carbon and nitrogen processes and the properties of the soil system that controlsactivity of the enzymes catalyzing these nitrogen and carbon transformations

A General Metabolic Considerations

Enzymes associated with carbon and nitrogen transformations are central to cellulargrowth and energy processes Thus, it is logical to conclude that any enzyme involved incellular 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 nitrogentransformation 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 asignificant portion is intrinsically more resistant to biodegradation because of its chemicalstructure Additionally, substrates generally expected to be more ephemeral may exhibitextended longevity that is due to their physical location within the soil matrix (1,122).Further complications in interpreting or predicting biodecomposition kinetics may arisefrom 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 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 cellularmembrane—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), andchemically limiting (e.g., pH, water-soluble heavy metals) extracellular environment.The emphasis of this presentation is on the current status of basic enzyme studiesinvolved 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 historicalcontext More current examples of the types of reactions studied in soil, considerations

conver-of the implications conver-of the physical structure conver-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 metabolismcould be evaluated in soil and associated ecosystems, only a limited number of enzymicactivities are commonly studied Many of the enzymes are those generally found to existand to express their catalytic activities extracellularly, such as cellulase Others, such as

urease, are found to catalyze reactions both within and outside cells Ideally, enzymeactivities selected as indicators of soil fertility or soil quality should be easily quantifiedand vary with ecosystem type, condition, or degree of human intervention.

Until the more recent era most soil-based research has been directed toward meetingagricultural needs (34,36) Therefore, to a large degree, the historical evaluation of enzymeactivity in soil has been concentrated on quantification of cropping and management ef-fects on activities involved with biogeochemical cycles (e.g., recycling of plant biomassnutrients and nitrogen fixation) or more directly agriculturally pertinent enzyme activities,such as urease As can be concluded from the investigations cited later, the commonlystudied activities involving nitrogen transformations have been associated with ammoniumgeneration (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 withhydroxylation of aromatic rings (e.g., polyphenyl oxidases, laccases), leading ultimately

to either mineralization or humification of the parent compounds; hydrolysis of charides (e.g., amylases, cellulases, xylanases); and a variety of lipases and esterases; plusthe indicator of respiratory activity, dehydrogenase These enzymatic activities haveproved useful for assessment of more general ecological concerns, such as organic mattertransformations in native soil systems, as well as of the effect of human intervention Forexample, in the latter arena, any of the general carbon or nitrogen catabolism enzymes(e.g., cellulase, hydrolases, dehydrogenase) is useful in assessing impacts of recyclingwaste organic matter (e.g., composts, sludges) through soil ecosystems, whereas poly-phenyl oxidases and laccase activity assessments are commonly linked to decompositionand humification of aromatic ring–containing xenobiotic chemicals

polysac-III ENZYME ASSAYS AND THEIR EFFECT

ON DATA INTERPRETATION

The two primary questions that must be addressed in assessing carbon and nitrogen bolic processes in soil are, How can the activity be quantified and what is an appropriateassay method? and How does the activity vary both in a relatively defined system in thetest tube as well as in the more complex, heterogeneous environment of the soil? A primaryproperty 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 reactantsand 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

meta-to either of the preceding questions are nontrivial when considering a soil ecosystem,especially when dealing with those enzymatic activities most closely linked with cellularenergy and nutrient management

The characteristics of the enzyme reaction must clearly be understood (i.e., reactionsubstrates, products, optimal conditions, and activity curves), but more important are theproperties 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 associatedwith 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

the soil sample itself prior to quantification, must be prevented As is documented in thefollowing section, there is a delicate balance between the amount of enzymatic activityand enzyme molecules, cellular metabolic state, and substrate (or inducer) level A slightchange in the soil physical structure can result in a significant change in quantity of enzyme

in a soil sample in an assay mixture as a result of induction of new activity or enzymerepression This variation in enzyme activity could result from liberation of substrate,inducers, or inhibitors from the soil matrix by disruption of the soil structure Thus, appro-priate design of a study of soil enzymes must include an appreciation of not only the basictraits of the enzyme reaction itself but also the ways that the soil properties may alter themeasured activity

Presentation of specific assay methods for the various enzymes commonly quantified

in soil samples can be found inChapter 21 Nonetheless, consideration of some of thegeneral factors associated with the physical status of the enzyme and the state of the cellsproducing 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 ofany assessment of enzymatic activity is to relate the amount of activity to properties orconditions of the site from which the sample was collected Furthermore, current questionsrelating to appropriate soil stewardship necessitate sufficient understanding of the variabil-ity of enzyme activity with soil properties to allow prediction of the relationship betweenenzyme 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 atelucidating 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 activeare altered (121,122) Two examples of changes that can affect the metabolic status ofthe enzyme-producing cells are soil oxygen tension and the availability of the carbon andenergy source Oxygen concentration in soil is generally controlled by its diffusion ratefrom 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 thelarger soil aggregates Disruption of soil aggregates through the mechanics of soil samplecollection (as well as by the common practice of sieving the soil in preparation for enzymeassays) alters this distribution of aerobic and anaerobic microsites and affects microbialmetabolism accordingly Additionally, much of the native soil organic matter is physicallyprotected from access by microbes and their enzymes That is, the organic material isphysically 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 physicalstate 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 ofexisting activity through microbial replication may occur Thus, an altered microbial com-munity and its associated enzyme activities are necessarily created by the simple act ofsample collection At least a minimal change in soil enzyme activity, particularly thatcentral 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) extracellularenzymes are not likely to be greatly changed by this process

Soil sampling procedures may also affect the activity associated with mineralization

of xenobiotic contaminants This is especially true in aged, contaminated soils where theaccessibility 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 andtheir 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

Generally, assay procedures for soil enzymes are designed to prevent increases inmicrobial numbers in enzyme levels during the assay Thus many assay protocols recom-mend the use of growth inhibitors (e.g., toluene, mercaptoethanol, sodium azide, radiationsterilization, antibiotics) or utilization of an assay time that is insufficient for microbialgrowth 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 freshlycollected 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 soilare 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 insoil 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 isprovided by studies of denitrification rates Quantification of the kinetics for appearance

of new enzyme, albeit from enzyme induction or microbial growth, has been used toestimate nitrous oxide reductase activity in field soils A common means of estimatingdenitrification is to inhibit nitrous oxide reductase activity with acetylene Thus, all of thenitrate denitrified accumulates in the reaction vessel as nitrous oxide Smith and Tiedje(114) observed three-phase reaction kinetics for this process when quantifying nitrousoxide 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 tion of new enzyme by induction of enzyme synthesis in preexisting cells In the thirdphase 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 estimatingnative nitrous oxide reductase enzyme levels in soil samples the duration of the initialphase of the process has been evaluated by several investigators It is reasonable to assumethat 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

produc-of the presence produc-of O2 or already maximized through optimal conditions—therefore nofurther growth or induction of the population or activity may occur), as would the occur-

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 preexistingdenitrification activity in soils Similarly, Dendooven and Anderson (27) found that denovo synthesis of nitrite reductase started in their system 5 hours after imposition of anoxicconditions and that of nitrous oxide reductase after 16 hours Other procedures that areuseful in evaluating preexisting or indigenous nitrogen–oxide reductase activity in nativesoil 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 thatthe 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 apotential activity in that it is likely that the nitrogen oxide substrate does not exist atsaturating 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 thatthe laboratory assessments are based on maximizing the interaction between enzyme andsubstrate 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 aresult of a variety of soil properties affecting the efficiency of interaction of the substrateand enzyme molecules In other words, a portion of the enzyme molecules existing in thefield soil may not be actively engaged in catalyzing their requisite reaction or may betransforming 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 ofthe 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 delineateenzyme 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 organicsubstances 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 withclay minerals or soil organic matter, and soluble in the aqueous phase as free enzyme orenzyme–substrate complexes Most commonly quantified soil enzyme components as-sayed are the activities contained within living cells, bound to soil organic matter, orsoluble in the soil interstitial water Additionally, soil enzyme may be associated withclay minerals or occluded within soil aggregates The consideration of the inclusion ofenzymes within soil aggregates is rarely taken into account because enzyme activities areusually measured in soil suspensions in the laboratory This practice ensures maximumrates of enzyme–substrate interaction and adheres to basic enzyme assay principles whentotal enzyme within a system is considered A future concern may be to evaluate in greater

detail the impact of heterogeneity in location within the soil system on the portion of theenzyme activity that is expressed in situ To reiterate, these distribution considerationsrelated to enzymes of nitrogen and carbon cycling in soil affect the total quantity of activityexpressed as well as the rate of the reactions—thereby controlling overall ecosystem andpopulation dynamics.

The general spatial variability of microbes, enzymes, and their activities in soil haslong 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 ofreadily metabolized fixed carbon resources Thus, these enzymatic activities are highest

in regions of native biomass production or inputs (e.g., rhizosphere) or in soils receivingorganic 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 andkinetics of reactions associated with organic pollutant decomposition or the effects ofmanagement decisions relating to the sustaining or improving of soil quality From theforegoing, 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 nitrogenmetabolism For example, Kandeler and Murer (68) noted that increased soil aggregation

in a conventionally tilled agricultural soil converted to grassland was accompanied bysignificant increases in dehydrogenase, protease, and xylanase activities Conversely, re-turning the soil ecosystem to conventional agricultural management caused a decline inthe elevated enzymatic activities

The distribution of enzymes involved in carbon and nitrogen transformation withinthe 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 withlevels 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 (⬎200-µm fraction) containing the decomposingmaize straw (117,118) Association of individual enzymes with specific size fractionsrelates in part to their interaction with fresh organic matter and the degree to which theactivity is linked to humic acids (i.e., humic acid stabilized enzymes) (95,118) Theserelationships between organic matter sources and enzyme activities support a hypothesisthat any soil management procedures that encourage the maintenance or development ofsoil aggregates optimize plant biomass production Therefore, since the primary source

of energy for the soil microbial community and substrates for the associated enzymes isthe carbon fixed by the plants, the soil microbial biomass and their associated intra- andextracellular 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 organicmatter, 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

enzyme protein when in the sorbed state (clay micelles) or covalently bound to soil humicacid Additionally, it must be remembered that in a multicomponent system the activityquantified 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 moleculesand their compliance with Michaelis–Menten kinetics may be at times coincidental.

A primary consequence of the physical location of enzyme molecules in soil is itsimpact 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 Vmaxand the efficiency of the transformationare environmentally controlled Analysis of the kinetics of a reaction can be used to showoccurrence 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 kineticparameters 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 thereaction in situ but not the kinetics of a purified enzyme Therefore, enzyme propertiesassessed 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 surfacearea and high cation exchange capacity) and kaolinite (low surface area and low cationexchange capacity) on diffusion of endoglucanase andβ-d-glucosidase in soil The kaolin-ite had no effect (i.e., binding) on enzyme diffusion, whereas the bentonite significantlyreduced (bound the enzyme) mobility Thus, these studies showed that movement of anenzyme 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 enzymeefficiency

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 notedpreviously involving kaolinite and bentonite already suggests that there is considerabledisparity in properties of different clay minerals Because of the high variability in claymineral quantities and types among various soil types (12), generalizations regarding therole of clay in expression of activities of the enzymes of the carbon and nitrogen cyclesare 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 cationexchange capacity The effects of these properties and their variation with clay type andenzyme have been documented with a variety of enzymes (104,121,122) Examples of thisanalysis of nitrogen and carbon metabolism associated enzymes are provided by studies ofurease 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 withmontmorillonite 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

type Invertase was stabilized by association with the clay surface in that resistance toheat was increased in the sorbed state In a similar study with urease (51), using the sameclay minerals, the heat stability of the sorbed enzyme was reduced, as were the Michaelis

constants, Vmax, and K m Lai and Tabatabai (74), in an evaluation of the sorption of jackbean

urease on kaolinite and montmorillonite, found that the K mvalues of the sorbed enzymewere 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 usuallycovalently 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 enzymewas 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 byconformational 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 enzymeproperties 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 thatthe adsorption parameters of the enzyme and polysaccharide varied with sodium chlorideconcentration of the reaction mixture, suggesting that the nature of the interaction betweenthe enzyme and the polysaccharide involved electrostatic associations rather than covalentlinkages Variation in the kinetic parameters and stability of the enzyme were used toassess the effect of association of the sugar polymer on the accessibility of the enzymeand its conformation The bound extracellular enzyme exhibited increased stability with

time and to heat denaturation compared to the soluble enzyme The similarity of the Vmax

values between the two enzyme forms suggests that little conformational change in theenzyme structure had occurred but accessibility of the enzyme to its substrate was altered,

as indicated by variation in the K mvalue

Similar studies have evaluated the effect of humification of proteins on their matic activities Examples using enzyme involvement in carbon and nitrogen transforma-tions include the effect of bonding ofβ-d-glucosidase to a phenolic copolymer of l-tyro-sine, pyrogallol, or resorcinol (108) and of linking of urease to tannic acid (49,52) Sarkarand Burns (108) found that their copolymers had several properties in common with those

enzy-of native soil humic acid–enzyme complexes (E4/E6ratios; carbon, hydrogen, nitrogen,and sulfur ratios; and infrared [IR] spectra) A lowering of the efficiency by polymerization

was shown by both reduced Vmaxand increased K mvalues Association of the copolymerwith bentonite resulted in a complex that was resistant to protease and much more stablethan the native enzyme Similarly, Gianfreda and associates (52) found that inclusion offerric ions and aluminum hydroxide species with tannic acid in forming organomineralurease 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 lar state (urease) and one more closely related to the microbial cell (β-d-glucosidase) can

extracellu-be stabilized with soil organomineral complexes with activity levels sustained at a level

that allows the enzyme to continue to contribute to ecosystem carbon and nitrogen 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 concludedthat the total impact of the enzyme on overall ecosystem function was extended by theinteractions with the abiotic soil constituents This dissociation of enzyme activities fromliving cell is a factor that must be considered in assessing the utility of assessing soilenzymatic activities as an indicator of soil status or quality.

dynam-The foregoing analysis of the properties of enzyme reactions leads to the conclusionthat a number of questions need to be addressed before applying basic enzyme kineticsanalyses to studies of soil ecosystem function, such as environmental impact or soil qualityassessments, or ultimately before predicting and measuring the outcome of soil ecosystemmanagement decisions Soil enzyme activities generally can be anticipated to followMichaelis 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 kineticsmay 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 enzymeaccessibility 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 productgeneration may reflect Michaelis–Menten kinetics, the kinetics of the reaction for theecosystem 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 aresult of its great heterogeneity (compared with the environment of a test tube with adefined 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 andhumification of the enzymes), as well as variations in the degree of saturation of theenzyme molecules with substrate and the distribution of the catalysts and reactions withinthe 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 carbondioxide evolution from labeled parent compound to assess mineralization capacity Thus,the kinetics for the overall mineralization processes as estimated by radio-labeled carbondioxide production from the parent compound in soil reflects the totality of the processesoccurring between entry of the parent molecule into the ecosystem and generation of themineralization products Therefore, a variety of zero-order (substrate levels are generallysaturating the available enzymes) and first-, second- or mixed-order models are used todescribe 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 detailedanalyses of this aspect of soil enzymology are found elsewhere (2,122)

ACTIVITY IN FIELD SITUATIONS

A consideration of the status of studies of carbon and nitrogen transformations in soilcould be initiated by the question, Why study soil enzymes? In attempting to answer this

question, five general areas of endeavor, some more novel, others strongly linked to thehistory of soil enzyme research, are apparent These are (i) determination of the basiclevels of various activities in native ecosystems—including methods development (i.e.,basic enzyme studies); (ii) provision of an understanding of an essential soil process (e.g.,denitrification, nitrogen fixation, humification); (iii) elucidation of basic soil ecosystemproperties; (iv) quantification of the impact of management on soil ecosystem function;and (v) assessment of the impact of anthropogenic activities on a system (i.e., pollution,climate change).

A Basic Enzyme Studies

Reviewed under the topic of basic enzyme studies are research aimed at optimizing thetechniques needed for the study of soil enzymatic activity as well as the application ofthese methods to answer some basic soil science or ecological questions A large variety

of enzyme activities associated with carbon and nitrogen metabolism have been evaluated

in soil These range from the general activity measured by dehydrogenase, through a ety of hydrolases and amidases, to a number of activities directly related to the mineraliza-tion of xenobiotic compounds Typically such studies involve the optimization of a specificassay procedure then the application of the procedure to the study of a limited number

vari-of soils from one or more different ecosystem types A general objective vari-of this type vari-ofresearch is commonly to determine specific environmental factors (e.g., pH, moisture,organic carbon or nitrogen, total soil organic matter) that delimit the extent of the reaction.One major soil biological property of interest in environmental studies is the generalrespiration rate of the biological community Many such studies are based on the quantifi-cation of carbon dioxide evolution If the objective of an assessment of carbon dioxidefluxes from a soil system is to estimate organic carbon mineralization rates, field measure-ments of carbon dioxide fluxes would necessarily be an overestimate This is because thetotal carbon dioxide flux from a soil reflects not only microbial respiration but also plantand animal respiration plus any carbon dioxide produced through chemical processes (e.g.,limestone dissolution) A logical resolution to this appears to be to collect soil samplesand sieve the soil to remove material that contributes a significant portion of the excesscarbon dioxide The assumption underlying this procedure is that any remaining nonpri-mary decomposers in the soil sample would contribute minimal quantities of carbon diox-ide to the total evolved This is a reasonable assumption, but as described earlier otherproblems are created by sieving the soil sample In this example, sieving of the soil in-creases the quantities of soil organic matter available to the microbial cell (121,122) andgives rise to respired carbon dioxide in excess of that likely to be generated in the field.Development of an enzyme-based assay that would be less sensitive to soil samplemanipulation than is the assessment of carbon dioxide flux is desirable As is shown inthe research cited later, dehydrogenase activity has served frequently in that capacity.Dehydrogenase is a nonspecific assay in that it represents the activity of several differentenzymes (122) An electron acceptor, generally triphenyltetrazolium chloride (TTC), isadded to soil as a terminal electron acceptor The colorless TTC is reduced to triphenylfor-mazan (TPF) by cellular respiratory enzymes TPF, a red product, is quantified spectropho-tometrically The quantity of triphenylformazan yielded in the assay is proportional tomicrobial respiration and in some cases to microbial biomass (122) Because of its generalutility for estimating soil respiratory activity under a variety of conditions, the generalprocedure for quantifying dehydrogenase activity is consistently being revised to augment