21 Enzymes in Soil Research and Developments in Measuring Activities M. Ali Tabatabai Iowa State University, Ames, Iowa Warren A. Dick The Ohio State University, Wooster, Ohio I. INTRODUCTION Reactions in the environment involve chemical, biochemical, and physical processes. It is well known that most biochemical reactions are catalyzed by enzymes, which are pro- teins with catalytic properties. Catalysts are substances that, without undergoing perma- nent alteration, cause chemical reaction to proceed at faster rates. In addition, enzymes are specific for the type of chemical reactions in which they participate. All living systems, ranging from bacteria to the animal kingdom, from algae and molds to the higher plants, contain a vast number of enzymes catalyzing both simple and complex networks of chemi- cal reactions. Enzymes also are found in ponds, lakes, rivers, water treatment plants, ani- mal manures, and soils and exist either as extracellular forms separated from their origins or as intracellular forms as part of the living biomass. These enzymes are involved in the synthesis of proteins, carbohydrates, nucleic acids, and other components of living systems and also in the degradation and essential cycling of carbon, nitrogen, phosphorus, sulfur, and other nutrients. The study of enzymes, in general, is a subject of interest to many disciplines ranging from biology to the physical sciences. This is not surprising as enzymes have a central place in biology, and life depends on a complex network of chemical reactions facilitated by specific enzymes. Any alterations in the enzyme protein structures might have far- reaching consequences for the living organism. It is safe to say that soils would remain lifeless and basically unaltered without enzymatic reactions. Within the past five decades enzymology, the science of studying enzymes, has developed rapidly. This field of science has connections with many other sciences and has contributed to our understanding of microbiology, biochemistry, molecular biology, botany, soil science, toxicology, animal science, pharmacology, pathology, medicine, and chemical engineering. Copyright © 2002 Marcel Dekker, Inc. II.ENZYMESINSOILS ThefirstknownreportonenzymesinsoilswaspresentedbyWoodsatthe1899Annual MeetingoftheAmericanAssociationfortheAdvancementofScienceinColumbus,Ohio (1).However,littlesignificantprogressoccurredintheareaofsoilenzymologyuntilthe 1950s.Thiswasmainlyduetoalackofappropriatemethodologiesandunderstandingof thetruenatureofenzymes.AlthoughSumnerfirstisolatedureaseincrystallineformfrom jackbean(Canavaliaensiformis)mealin1926,forwhichhereceivedaNobelPrize,this fieldofbiochemistrytookseveraldecadestomature.Questionsaskedduringtheearly yearsofsoilenzymeresearchdealtwiththeorigin,stabilization,importanceinplantnutri- tion,androleofsoilenzymesinorganicmatterturnover.Manyofthesequestionsremain tobeanswered. Awealthofinformationaboutvariousenzymaticreactionsinsoilshasbeencol- lectedsince1950,andtheoreticalapproachesandmethodshavebeendeveloped.Ahistory ofabioticsoilenzymeresearchhasbeenpreparedbySkujins(1),andreviewsofrecent advancesandthestateofknowledgeinthisfieldarepresentedinabookeditedbyBurns (2)andinanumberofbookchapters(3–10)byotherresearchers.Nospecificreview articlehasbeenpreparedontheprogressthathasbeenmadeinthedifferentchemicaland instrumentalmethodsusedinmeasuringenzymeactivitiesinsoils.Thischapterfocuseson thevarioustechniquesusedinassayofenzymeactivitiesofsoils. Soilcanbethoughtofasabiologicalentity(i.e.,alivingtissuewithcomplexbio- chemicalreactions)(11).Soilcontainsfreeenzymes,immobilizedextracellularenzymes stabilizedbyathree-dimensionalnetworkofmacromolecules,andenzymeswithinmicro- bialcells.Eachoftheorganicandmineralfractionsinbothbulksoilandtherhizosphere hasaspecialinfluenceonthetotalenzymaticactivityofthatsoil(12,13). Enzymesareproteincatalysts,andphysicochemicalmeasurementsindicatethat enzyme-catalyzedreactionsinsoilshaveloweractivationenergiesthannon-enzyme-cata- lyzedreactionsand,therefore,havefasterreactionrates(14–17).Enzymesinsoilare similartoenzymesinothersystems,inthattheirreactionratesaremarkedlydependent onpH,ionicstrength,temperature,andthepresenceorabsenceofinhibitors(8,18). A.Sources Bothmicroorganismsandplantsreleaseenzymesintothesoilenvironment(Fig.1).Ithas long been known that ribonucleases and alkaline phosphatase, for example, are excreted by Bacillus subtilis under certain conditions (19,20), and pyrophosphatase and acid phospha- tase may exist extracellularly on the surface of cell walls of Saccharomyces mellis (21,22). A number of bacteria release phosphatases (23) and other microbial extracellular enzymes with important commercial applications, including proteases, amylases, glucose iso- merases, pectinases, and lipases (24). Therefore, it is not surprising that microorganisms are the logical choice to account for most of the soil enzyme activity (25). This is because of their large biomass, high metabolic activity, and short lifetimes, which allow them to produce and release relatively large amounts of extracellular enzymes in comparison to plants or animals. The effect of microorganisms in supplying phosphatase activity to soils, however, seems temporary and short-lived. Ladd and Paul (26) incubated a soil with glucose and sodium nitrate at 22°C and found that bacterial numbers increased almost 2-fold in 36 hours, accompanied by a 3.2-fold increase in phosphatase activity. However, the new Copyright © 2002 Marcel Dekker, Inc. Figure 1 Conceptual scheme of the composition of soil enzyme activities. (From Ref. 1.) activity was rapidly lost and after 21 days, activity had returned to its original level. It has been theorized (25) that the increased activity of phosphatase produced during incuba- tion of soil with glucose and nitrate was largely lost during the microbial proliferation– dying–lysing cycle and that many cycles of microbial activity may be required to obtain a permanent increase in the extracellular level of phosphatase activity. Plants have also been considered a source of extracellular enzymes in soils. Es- termann and McLaren (27) found that barley (Hordeum vulgare) root caps possess phos- phatase activity. Other studies showed that a variety of plants have amidase and urease activities (28) and that sterile corn (Zea mays) and soybean (Glycine max) roots contain acid phosphatase, but no alkaline phosphatase activity (29). In other work (30) it was demonstrated that sterile corn and soybean roots could exude acid phosphatase into a solution that surrounded them. Roots, placed into sterile buffer or water for 4–48 hours, released acid phosphatase into the solution. Greater amounts of acid phosphatase were released into water than into the buffered solution. Major amounts of enzymes introduced into the soil environment by microorganisms or plant roots are inhibited by soil constituents, rapidly degraded by soil protease, or both. Work by Dick et al. (31) showed that when 10 mg corn root homogenate was mixed with 1 g of soil, the inhibition of corn root acid phosphatase and pyrophosphatase by 12 soils was 43–63% (average ϭ 52%) and 11–62% (average ϭ 44%), respectively. The inhibition of a similar amount of partially purified acid phosphatase from wheat (Triticum aestivum) germ was 88–95% (average ϭ 92%). Inhibition by steam-sterilized soils was less than that by nonsterilized soils, suggesting that the observed inhibition was due, at least partly, to heat-labile organic constituents. Also, the magnitude of inhibition by nonsterilized soil increased as the quantity of soil added was increased from 0.1 to 1.0 g. No such increase Copyright © 2002 Marcel Dekker, Inc. ininhibitionwasobservedwithsterilizedsoils.Soilextractsalsoinhibitedacidphospha- tasefromcornrootsandwheatgerm,butthisinhibitionwasmostlikelyduetoinorganicor heat-resistantorganiccompounds,becausesterilizedandnonsterilizedsoilextractsyielded similarresults. Althoughmostenzymesintroducedintosoilarerenderedinactive,asmallpercent- ageofactiveenzymeproteinmaybecomestabilizedinthesoil.Kineticstudiesindicate thattheactivityofclay-enzymecomplexesformedinsoilisgreatlyreduced,butnottotally eliminated(32,33). Plantsareabletosynthesizemanyenzymes.Theseenzymes,addedtosoilasplant residues,mayremainactive.Phosphataseactivityinsoilhasbeenobservedtobeassoci- atedwithintactcellwallsofplanttissue,withcellwallfragments,andwithamorphous organicmaterial(34,35).Thus,itisnotsurprisingthatthetypeofvegetationaddedto soilcangreatlyaffectsoilenzymeactivity.Plantsalsoinfluencesoilenzymeactivityby indirectmeans.Enzymeactivityisconsiderablygreaterintherhizosphereofplantsthan in‘‘bulk’’soil,andthisincreasedactivityisduetoeitheraspecificfloraortheplantroot, ormostlikely,totherelationshipbetweenboth(4).Anotherindirectinfluenceofplants onenzymeactivityistheincreasednumberofmicroorganismspresentuponadditionof plantlittertosoils.Examplesofplantsaffectingsoilenzymeactivitiescanbefoundin thereviewchaptersinthebookSoilEnzymeseditedbyBurns(2)andinChapters4and 6ofthisvolume. B.StatesorLocationsofEnzymesinSoils ThetermstateofenzymesinsoilshasbeenusedbySkujins(4,5)todescribethephenome- nonwherebyenzymesexistinsoils.Characterizationofthestateofanenzymeinsoil entailstheattempttodescribethelocationandmicroenvironmentinwhichitfunctions andthewaytheenzymeisboundorstabilizedwithinthatmicrohabitat(36).Asindicated byBurns(37),theactivityofanyparticularenzymeinsoilsisacompositeofactivities associatedwithvariousbioticandabioticcomponents.Burnsenvisaged10distinctcatego- riesofenzymesinsoils,rangingfromenzymesassociatedwithproliferatingmicrobial, animal,andplantcells(locatedinthecytoplasm,periplasmicspace,onoutersurfaceor secreted)toextracellularenzymesassociatedwithhumiccolloidsandclayminerals. ExtracellularenzymeaccumulationinsoilswasclearlydemonstratedbyRamirez- MartinezandMcLaren(38),whoreportedthattheamountofphosphataseactivityin1 gsoilwasequivalentto10 10 bacteriaor1goffungalmycelia.Ifweassumethatnonprotein soilcomponentsdonotcatalyzehydrolysisoforganicPcompoundsinsoils,thenitcan beconcludedthatacertainportionofphosphataseactivityinsoilsisnolongerassociated withlivingtissue. Severaltheorieshavebeenproposedtoexplaintheprotectiveinfluenceofsoilon extracellularenzymeactivity.Thedominantmechanismsofenzymeimmobilizationand stabilization(Fig.2)havebeensummarizedbyWeetall(39).Theseincludemicroencapsu- lation, cross-linking, copolymer formation, adsorption, entrapment, ion exchange, adsorp- tion and cross-linking, and covalent attachment. Early work by Ensminger and Gieseking (40) provided evidence that protein ad- sorbed to montmorillonite was stabilized against microbial attack. Haig (41) also found that acetylesterase activity in a fine sandy loam soil—fractionated into sand, silt, and clay sizes—was associated primarily with the clay fraction. McLaren (42) observed that kaolinite adsorbed trypsin and chymotrypsin, and Mg-bentonite was shown to adsorb pep- Copyright © 2002 Marcel Dekker, Inc. Figure 2 Schematic representation of methods of immobilizing enzymes. (From Ref. 39.) sin and lysozyme (43). This adsorption occurred rapidly and was 90% complete after 2–3 min. The mechanisms by which clay minerals bind proteins are not always clearly understood. Albert and Harter (44) reported that adsorption of lysozyme and ovalbumin by Na–clay minerals caused an increase in sodium ion concentration of the clay-protein suspension. They interpreted this result as evidence that a cation-exchange adsorption mechanism was occurring. More recently, Ruggiero et al. (10) have summarized a large amount of work that has been done to enhance understanding of the clay–enzyme complex. In soils, clay sur- faces are constantly being renewed or altered by environmental factors, and this condition makes it difficult to extrapolate results obtained by using relatively pure clay minerals saturated with a specific cation to soil conditions. Stabilization of enzymes in the soil environment by soil organic matter, rather than by inorganic components, has also been suggested. Much of the information dealing with this hypothesis has been obtained by studies involving synthetic polymer–enzyme com- plexes (45). Early studies by Conrad (46) showed that native soil urease was more stable than urease added to soils. He concluded that organic soil constituents protect urease against microbial degradation and other processes, leading to decomposition or inactiva- tion. Since then, numerous studies have supported this conclusion by showing that enzyme activities in soils are significantly correlated with organic matter content (47–50). A study by Burns and coworkers (51) found that an organic fraction extracted from soil, which was free of clays (confirmed by x-ray analysis), contained urease activity. Supporting results indicating that enzyme activity is associated with humus–enzyme complexes have been reported (52–55) for protease, phosphatase, tyrosinase, peroxidase, and catalase. Ladd and Butler (45) suggested that enzymes bind to soil humus by hydrogen, ionic, or covalent bonding. The extent that enzymes are bound by each of these mechanisms is Copyright © 2002 Marcel Dekker, Inc. difficulttodetermine.WorkbySimonartandassociates(56)suggeststhathydrogenbond- ingmaybeonlyaminorfactorinenzymestabilizationinsoils.Byusingphenoltobreak hydrogenbonds,theywereabletodissolveonlyasmallamountofproteinaceousmaterial. Enzymesmaybeboundtoorganicmatterbyionic-bondingmechanisms.Butler andLadd(57)proposedthatenzyme–organicmattercomplexesareformedthroughthe formationofamino–carboxylsaltlinkages.Suchcomplexes,however,shouldbeeasily brokenbymanyoftheextractionreagents(i.e.,ureaandpyrophosphate)usedtoremove activeenzymematerialsfromsoils.Thesmallyieldsofactiveenzymematerialsthathave beenextractedfromsoilsindicatethationic-bondingmechanismsmaybeonlypartially responsibleforenzymestabilization(58–60).However,Burnsetal.(61)extractedapprox- imately20%oftheoriginalsoilureaseactivitybyusingurea(ureahydrolyzedsubse- quentlybytheextractedurease).Theclay-freeprecipitatehasureaseactivitythatwasnot destroyedbytheadditionoftheproteolyticenzymepronase.Thenativesoilureasewas thoughttobelocatedinorganiccolloidalparticlesthatcontainedporeslargeenoughto allowwater,urea,ammonia,andcarbondioxidetopassfreely,butsmallenoughtoexclude pronase. Aclearhypothesisexplainingenzymestabilizationbymeansofcovalentattachment hasyettobeproposed.LaddandButler(45)suggestedthatthelinkageofsoilquinones bynucleophilicsubstitutiontosulfhydrylandtoterminalandε-aminogroupsofenzyme proteinsmayleadtoactiveorganoenzymederivatives,providedthesegroupsdonotform apartoftheactivesiteoftheenzyme. Onehypothesisthathasasyetreceivedlittleattentionisthatenzymesexistinsoils asglycoproteins.Malathionesterase,extractedfromsoilsbySatyanarayanaandGetzin (62),wasthoughttobeaglycoproteinbecauseofthefollowingevidence:(1)aminoacids constitutedonly65%ofthepurifiedenzyme;and(2)acarbohydrate-splittingenzyme, hyaluronidase,enhancedthecatalyticeffectoftheesterase,presumablybylooseningthe carbohydrateshieldandallowingtheproteincoretogaineasieraccesstothesubstrate. Theevidencegainedbyincubatingtheesterasewithhyaluronidasesuggestedthatthe carbohydrate–proteinlinkageoccursthroughN-acetylhexoseamine–tyrosinebonds.May- audonetal.(63)drewsimilarconclusionswhentheyobservedthatdiphenoloxidase activitywasnotaffectedbypronasealone,butwasdestroyedwhenincubatedinthepres- enceofbothlysozymeandpronase. Insoils,astrongassociationexistsbetweenclayandhumus.Eachdoesnotsepa- ratelyinfluenceenzymestabilization;rather,PaulandMcLaren(64)postulated,athree- dimensionalnetworkofclayandhumuscomplexesexistsinwhichactiveenzymebecomes incorporated(Fig.3).AstudybyBurnsetal.(51)supportedthishypothesiswhenthey observed that a bentonite–lignin complex protected urease from degradation much more effectively than did bentonite alone. C. Stability Most of the information available on the stability of enzymes in soils is derived from work on urease, acid phosphatase, and arylsulfatase. The first evidence that soil enzymes are more stable than those added to soils was obtained by Conrad (46) in his work on urease; Conrad concluded that organic matter in soils protect-enzymes (urease) against microbial degradation. Support for this conclusion has been provided by numerous studies showing that enzyme activities are significantly correlated with organic C in surface soils and soil profiles (8,50). Further evidence supporting this conclusion is provided by work Copyright © 2002 Marcel Dekker, Inc. Figure 3 Model for soil enzyme location and activity consisting of enzyme embedded in, and perhaps chemically attached to, a humus polymer network in contact with clay particles. Substrates, such as urea, can reach the enzyme by diffusion through pores too small for enzymes to penetrate. (From Ref. 64.) showing that soil enzymes are stable for many months and years in air-dried soils (4). Now it is generally accepted that enzymes in soils are immobilized within a network of organomineral complexes (13,65). III. ROLE OF CHEMISTRY IN ENZYME ACTIVITY MEASUREMENT One of the fundamental requirements of enzyme measurements is a thorough understand- ing of the reactions involved, quantitative extraction of the product(s) released, and a suitable analytical method for measuring quantitatively the extracted compound. There- fore, knowledge of analytical chemistry and chemical kinetics are essential in soil enzyme research. In addition, because soils contain both organic constituents and mineral compo- nents, a thorough understanding of the potential reactions between the substrate, and more importantly the product released, and the soil constituents is a prerequisite for any methods development. The detailed study of an enzyme reaction in soils involves characterization and mea- surement, if possible, of several properties, some of which cannot be obtained for enzyme in soils. One, therefore, has to rely on the biochemical literature for the information re- quired. 1. Protein properties: Even though it is difficult to extract and purify enzyme proteins from soils, information about the enzyme molecular weight, isoelectric point, electrophoretic mobility, and stability to pH, heat, and oxidation can be obtained from the biochemical literature. Some of these properties can be ob- Copyright © 2002 Marcel Dekker, Inc. tained from direct experiments by using soil samples, as has been demonstrated for a number of soil enzymes (8). 2. Structure: Many of the structural features of purified enzymes are useful in classical biochemistry, but for soil enzyme research it is primarily important to know whether the presence of a prosthetic group or special group of metal atoms is required for activity and to know the effect of chemical reagents on such activity. 3. Enzyme properties: It is important to know the nature of the reaction being cata- lyzed; whether a coenzyme is involved, and its nature and mode of action; speci- ficity for substrate; nature of the chemical structure; and specificity to inhibitors. 4. Active center: Knowledge of the nature and composition of the enzyme active center is required. 5. Thermodynamics: Because the exact molecular weights of soil enzymes are not known, several properties such as free energies and entropies of enzyme–sub- strate combination cannot be determined, other properties, however, can be. They include the activation energy of an enzyme-catalyzed reaction, affinity of the enzyme for its substrate, Michaelis–Menten constant, effect of pH on affinity of the enzyme for its substrate, affinities for inhibitors, inhibitor constants, and competition of inhibitors with the substrate. The temperature dependence of the rate constant, at a temperature below that which results in activation of the enzyme activity, can be described by the Arrhenius equation (k ϭ A ⋅ exp (ϪEa/RT ), where k is the rate constant, A is the preexponential factor, Ea is the activation energy, R is the gas constant, and T is the Kelvin (K ) temperature. The Arrhenius equation, when expressed in its log form (log k ϭ (ϪEa/2.303 RT ) ϩ log A), allows calculation of Eaby plotting log of the initial rate of the reaction vs 1/T. The slope of the resulting line is equal to ϪEa/2.303R. The enthalpy of activation (∆Ha) can be calculated from ∆Ha ϭ Ea Ϫ RT. 6. Kinetics: Characterization of the kinetic parameters of the Enzyme–substrate reaction is important because anyone who is concerned with catalysis in soil is most certainly concerned with the velocities of chemical reactions (chemical kinetics). The usual way to follow an enzyme-catalyzed reaction is by measuring the amount of reactant remaining or the product formed. By contrast, most ki- netic models are formulated in terms of rates of reaction. Traditionally enzyme kinetic studies have focused on the initial rates of reactions by measuring tan- gents to the reaction curves (i.e., by measuring the linear portion of the reaction curve at the time the reaction is initiated). In soil, kinetic data have only pro- gressed to the point where we can study simple one-substrate systems, which react with a single enzyme. The Michaelis–Menten equation does an excellent job of describing this type of kinetics and there are several assumptions that are made when applying this equation to soil systems. The enzyme reaction is expressed by the following equation: E ϩ S 5 3 k 1 k 2 ES Er k 3 E ϩ P The assumptions made in deriving the Michaelis–Menten equation are as fol- lows: (1) The rate of reaction of the enzyme-catalyzed system changes from Copyright © 2002 Marcel Dekker, Inc. Table 1 Parameters of Linear Equations Describing Inhibition of Enzyme–Substrate Interactions Inhibitor type Slope Y intercept X intercept No competition K m /V max 1/V max Ϫ1/K m Linear competitive K m /V max (1 ϩ [I]/K i )1/V max Ϫ1/K m (1 ϩ [I]/K i ) Linear noncompetitive K m /V max (1 ϩ [I]/K i )(1/V max )(1 ϩ [I]/K i ) Linear uncompetitive K m /V max (1/V max )(1ϩ [I]/K i ) first-order to zero-order kinetics; (2) enzyme (E) reversibly binds with substrate (S) to form an intermediate enzyme–substrate (ES) complex, which then breaks down to form product (P). Each reaction is described by a specific rate constant: k 1 , k 2 , k 3 ; (3) a steady-state equilibrium between the rate of formation of ES and the rate of degradation of ES is rapidly achieved; (4) total enzyme concen- tration is defined as that in the free state and in the enzyme–substrate complex; (5) the initial rate-limiting parameter is the decomposition of the enzyme–sub- strate (ES) complex to form the product (or k 3 ); and (6) V max is achieved when ES complex concentration reaches a maximum equal to the total enzyme con- centration: i.e., there is no free enzyme. Much of what we know about biological systems is based on more com- plex enzyme systems that have an inhibitor present. For example, it is well known that the presence of inorganic phosphate in solution strongly inhibits phosphatase activity in soils (8). The simplest systems are those in which there is a single substrate, single enzyme, and a single inhibitor (I). In general, the type of inhibition could be one of the following: (1) linear competitive inhibition, (2) linear noncompetitive inhibition, or (3) linear uncompetitive inhibition. The parameters of the linear equations are shown Table 1. 7. Biological properties or role of soil enzymes in metabolic reactions: Informa- tion on the occurrence and distribution of the enzyme among different species and associated with different plant and microbial tissues that are deposited in soils is important. Much of the information can be obtained from the biochemis- try literature. IV. SUBSTRATE STRUCTURE, ENZYME SPECIFICITY, AND ACTIVITY MEASUREMENT Substrate structure has a significant effect on the reaction rate, and the structure of the product released markedly affects its extractability from soils and the potential for its quantitative determination by any procedures or techniques. Detailed discussion of enzyme specificity is beyond the scope of this chapter, but it should be made clear that specificity is one of the most striking properties of the enzyme molecule. It depends on the particular atomic structure and configuration of both the substrate and the enzyme. There are three types of enzyme specificity. The first is absolute specificity, which is rare and describes a reaction in which a single member of a substrate class is attacked by an enzyme. An example is urease. Relative specificity describes a situation in which an enzyme acts pref- erentially on one class of compounds but will attack a member of another class to a certain Copyright © 2002 Marcel Dekker, Inc. extent.Thistermmayalsobeusedtoillustratethedifferentratesofreactionswithina givenclass.Thethirdtypeisopticalspecificity,whichisacommonpropertyofsomeyeast enzymes,whichactonopticallyactivesubstrate.Stereochemicalspecificityisstrikingly illustratedbytheactionofglycosidases.Maltasehydrolyzesmaltoseandseveralotherα- glucosidestoglucosebutnotβ-glucosides.Emulsincontainsaβ-glucosidase,whichacts onlyonβ-glucosidesbutnotonα-glucosides.Bothα-andβ-glucosidasesarepresentin soils(66).Othersimilarexamplesarethed-andl-specificaminoacidoxidases(67). Anessentialstepinenzymeactivitymeasurementrequirestheavailabilityofchemi- calmethodsandinstrumentaltechniquesfordeterminationofthereactionproductformed. Almostallthemethodsdevelopedbybiochemistsforenzymeassayareusefulasguides forassayonenzymeactivitiesinsoils,butcautionshouldbeexercisedtobesurethat theproductformedisdeterminedquantitatively.Thisisbecausemanyofthemethodsare notcompatiblewiththecomplexchemicalcharacteristicsofthesoilsystem. V.ENZYMEPROTEINCONCENTRATIONINSOILS Numerousattemptshavebeenmadetoextractpureenzymesfromsoils,butinrealitythe bestthathasbeenachievedistheextractionofenzyme-containingsubstancesandcom- plexes(68).Thereagentsusedintheextractionproceduresrangefromwatertosaltsolu- tionsorbufferstostrongorganicmatter-solubilizationreagents,suchasNaOHorsodium pyrophosphate.Theextractedactivitiesareusuallyassociatedwithcarbohydrate–enzyme proteincomplexesandareoftendifficulttopurify.Modernbiochemicaltechniqueshave beenusedinthepurificationoftheextractedenzymes,butlittleprogresshasbeenmade inobtainingpureenzymeproteinsfromsoils.Severaloftheenzymesextractedfromsoils couldbepresentinsoilsasglycoproteins.Althoughmanyinvestigatorshavedemonstrated thatclay-freeextractscouldbeobtainedfromsoils,themajorproblemappearstobethe strongaffinityofthecarbohydrate–enzymecomplexesforchromatographiccolumns, whichmakestheseparationdifficult.Itappearsthatvariouscarbohydratesinsoilsadsorb theenzymeproteinsandareresponsiblefortheirstabilizationagainstdenaturationor proteolysis. A.EstimationofConcentrationsofEnzymeProteinsinSoils Enzymeactivitiesareassociatedwithactivemicroorganismsbecausethemicrobialbio- massisconsideredtheprimarysourceofenzymesinsoils.Nevertheless,thereisnodirect correlationbetweenthesizeofthemicrobialbiomassanditsmetabolicstate(69).One approachtoestimatethemetabolicstateofmicrobialpopulationsinsoilsistodifferentiate betweenintra-andextracellularenzymeactivities.Amongthemanyattemptsthathave beenmadetodeterminethestateofenzymesinsoilsaretechniquesthatemployelevated anddecreasedtemperatures;antisepticagentssuchastoluene,ethanol,TritonX-100,di- methylsulfoxide;irradiationwithgammaraysorelectronbeams;andfumigationwith compoundssuchaschloropicrin,methylbromide,andchloromycetin(2,3,8,70,71).None ofthesemethodscandistinguishbetweenintracellular(activityassociatedwiththemicro- bialbiomass)andextracellularactivity(thatportionstabilizedinthethree-dimentional networkofclay–organicmattercomplexes)(Fig.3),becauseallthesetechniquesalso denature the enzyme proteins. Another suggested approach is plotting enzyme activity against the number of ureolytic microorganisms (in the case of urease) or adenosine tri- Copyright © 2002 Marcel Dekker, Inc. [...]... for the nonfumigated soils (except for urease and arylsulfatase, which were based on fumigated samples) on the basis of their activity values and specific activities of the purified reference enzyme proteins b α-Gal, α-galactosidase; β-Gal, β-galactosidase, α-Glu, α-glucosidase; β-Glu, β-glucosidase c l-Asg: l-asparaginase; l-Glu, l-glutaminase; Amid, amidase; urea, urease; l-Asp, l-aspartase d Acid-P,... measure the activities of β-d-glucosidase, β-d-galactosidase, N-acetyl-β-dglucosaminidase, β-cellobiase, β-xylosidase, acid phosphatase, and arylsulfatase in a sandy loam and a silty clay loan soil Marx and coworkers (89) reported that the results confirmed the potential usefulness of this technique and demonstrated the precision of the MUB microplate assay D Radioisotope Methods Among the many enzymes. .. combination of thin section techniques and histochemical and imaging techniques has a long history related to the study of enzymes in soils The techniques have been successfully used in localizing specific compounds in animal and plant tissues (111,112) Early work in visualizing the location of enzymes in soil was reported by Foster and Martin (113) and Foster et al (35) They combined the use of electron... fluorescent 7-hydroxy-4-umbelliferone Other fluorogenic model substrates have been used for the assay of β-glucosidase, phosphatase, and arylsulfatase activities in peat (87) and for assay of β-cellobiase, βgalactosaminemidase, β-glucosidase, and β-xylosidase, arylsulfatase, and alkaline phosphatase activities in soils (88) In all these methods, either a very small amount of the soil sample (in milligrams)... activity of these enzymes, the soil is incubated with the substrate in an appropriate buffer and the NH 4ϩ produced is determined The reaction is stopped by adding 2M KCl containing Ag 2SO 4 Because a simple distillation apparatus is available, normally an aliquot of the incubated soil–solution mixture is distilled with MgO, and the NH 3 released is collected in boric acid containing bromcresol green and. .. because the risk of microbial proliferation increases as the time of incubation increases It is believed that in assay procedures involving short incubation times, toluene inhibits the synthesis of enzymes by living cells and prevents assimilation of the reaction products Toluene has also been shown to be a plasmolytic agent in certain groups of microorganisms in which it apparently induces the release... However, the dose required depends on the soil type, soil moisture, and genus of the organism Copyright © 2002 Marcel Dekker, Inc IX POTENTIAL USEFUL TECHNIQUES IN LOCALIZING ENZYMES PROTEINS IN ENVIRONMENTAL MATRICES Several methods and techniques that are available have potential for detecting and localizing enzyme protein in environmental matrices, including soils A Thin Section Techniques The combination... material Pectin polymers are chains of predominantly 1,4-linked-αd-galacturonic acid and methylated derivatives Degradation of most Chitinase is most com- Chitin is insoluble A polymers is due to monly assayed by pure form of chitin extracellular enmeasuring n-acetylcan be purchased zymes as these molglucosamine reTritiated chitin ecules are too large lease by using a must be prepared in to be taken into mispectrometric... absorbance of the blue azo compound is measured at 700 nm This method, however, is complicated and tedious B Titrimetric Methods Several amidohydrolases are present in soils All are involved in hydrolysis of specific native and added organic N compounds in soils Among these, l-asparaginase, l-glutaminase, l-aspartase, amidase, and urease are the most important in the biogeochemical context In assaying the activity... for the assay of enzyme activities in soils The glass electrode can be used to follow reactions that involve the production of acids, but there are two problems with such approaches The first is that the change in pH during incubation alters the reaction rate of the enzyme The second problem is that the rate of change of pH depends not only on the reaction but also on the buffering poise of the soil and . attached to β-naph- thylamine and p-nitroaniline according to the following reaction (using the amino acid l- leucine as an example): Copyright © 2002 Marcel Dekker, Inc. The β-naphthylamine released. be- of 7-amino-4-meth- cause they have ylcoumarin. high k cat /k m values and low back- Prepare proteins with ground hydrolysis a fluorogenic label and the thiol leav- attached to individ- ing. l-asparaginase, l-glutami- nase, l-aspartase, amidase, and urease are the most important in the biogeochemical con- text. In assaying the activity of these enzymes, the soil is incubated with the