Enzymes in the Environment: Activity, Ecology and Applications - Chapter 21 (end) pps

Questions asked during the earlyyears of soil enzyme research dealt with the origin, stabilization, importance in plant nutri-tion, and role of soil enzymes in organic matter turnover..

21 Enzymes in Soil

Research and Developments in Measuring Activities

Reactions in the environment involve chemical, biochemical, and physical processes It

is well known that most biochemical reactions are catalyzed by enzymes, which are teins with catalytic properties Catalysts are substances that, without undergoing perma-nent alteration, cause chemical reaction to proceed at faster rates In addition, enzymesare 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

pro-or as intracellular fpro-orms as part of the living biomass These enzymes are involved in thesynthesis of proteins, carbohydrates, nucleic acids, and other components of living systemsand 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 rangingfrom biology to the physical sciences This is not surprising as enzymes have a centralplace 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 reaching consequences for the living organism It is safe to say that soils would remainlifeless and basically unaltered without enzymatic reactions Within the past five decadesenzymology, the science of studying enzymes, has developed rapidly This field of sciencehas connections with many other sciences and has contributed to our understanding ofmicrobiology, biochemistry, molecular biology, botany, soil science, toxicology, animalscience, pharmacology, pathology, medicine, and chemical engineering

far-II ENZYMES IN SOILS

The first known report on enzymes in soils was presented by Woods at the 1899 AnnualMeeting of the American Association for the Advancement of Science in Columbus, Ohio(1) However, little significant progress occurred in the area of soil enzymology until the1950s This was mainly due to a lack of appropriate methodologies and understanding ofthe true nature of enzymes Although Sumner first isolated urease in crystalline form from

jack bean (Canavalia ensiformis) meal in 1926, for which he received a Nobel Prize, this

field of biochemistry took several decades to mature Questions asked during the earlyyears of soil enzyme research dealt with the origin, stabilization, importance in plant nutri-tion, and role of soil enzymes in organic matter turnover Many of these questions remain

Soil can be thought of as a biological entity (i.e., a living tissue with complex chemical reactions) (11) Soil contains free enzymes, immobilized extracellular enzymesstabilized by a three-dimensional network of macromolecules, and enzymes within micro-bial cells Each of the organic and mineral fractions in both bulk soil and the rhizospherehas a special influence on the total enzymatic activity of that soil (12,13)

bio-Enzymes are protein catalysts, and physicochemical measurements indicate thatenzyme-catalyzed reactions in soils have lower activation energies than non-enzyme-cata-lyzed reactions and, therefore, have faster reaction rates (14–17) Enzymes in soil aresimilar to enzymes in other systems, in that their reaction rates are markedly dependent

on pH, ionic strength, temperature, and the presence or absence of inhibitors (8,18)

of their large biomass, high metabolic activity, and short lifetimes, which allow them toproduce and release relatively large amounts of extracellular enzymes in comparison toplants 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 andsodium nitrate at 22°C and found that bacterial numbers increased almost 2-fold in 36hours, accompanied by a 3.2-fold increase in phosphatase activity However, the new

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 Ithas 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 wasdemonstrated that sterile corn and soybean roots could exude acid phosphatase into asolution 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 werereleased 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 soilswas 43–63% (average⫽ 52%) and 11–62% (average ⫽ 44%), respectively The inhibition

of a similar amount of partially purified acid phosphatase from wheat (Triticum aestivum)

germ was 88–95% (average⫽ 92%) Inhibition by steam-sterilized soils was less thanthat 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 soilincreased as the quantity of soil added was increased from 0.1 to 1.0 g No such increase

in inhibition was observed with sterilized soils Soil extracts also inhibited acid tase from corn roots and wheat germ, but this inhibition was most likely due to inorganic orheat-resistant organic compounds, because sterilized and nonsterilized soil extracts yieldedsimilar results.

phospha-Although most enzymes introduced into soil are rendered inactive, a small age of active enzyme protein may become stabilized in the soil Kinetic studies indicatethat the activity of clay-enzyme complexes formed in soil is greatly reduced, but not totallyeliminated (32,33)

percent-Plants are able to synthesize many enzymes These enzymes, added to soil as plantresidues, may remain active Phosphatase activity in soil has been observed to be associ-ated with intact cell walls of plant tissue, with cell wall fragments, and with amorphousorganic material (34,35) Thus, it is not surprising that the type of vegetation added tosoil can greatly affect soil enzyme activity Plants also influence soil enzyme activity byindirect means Enzyme activity is considerably greater in the rhizosphere of plants than

in ‘‘bulk’’ soil, and this increased activity is due to either a specific flora or the plant root,

or most likely, to the relationship between both (4) Another indirect influence of plants

on enzyme activity is the increased number of microorganisms present upon addition ofplant litter to soils Examples of plants affecting soil enzyme activities can be found inthe review chapters in the book Soil Enzymes edited by Burns (2) and inChapters 4and

6of this volume

B States or Locations of Enzymes in Soils

The term state of enzymes in soils has been used by Skujins (4,5) to describe the non whereby enzymes exist in soils Characterization of the state of an enzyme in soilentails the attempt to describe the location and microenvironment in which it functionsand the way the enzyme is bound or stabilized within that microhabitat (36) As indicated

phenome-by Burns (37), the activity of any particular enzyme in soils is a composite of activitiesassociated with various biotic and abiotic components Burns envisaged 10 distinct catego-ries of enzymes in soils, ranging from enzymes associated with proliferating microbial,animal, and plant cells (located in the cytoplasm, periplasmic space, on outer surface orsecreted) to extracellular enzymes associated with humic colloids and clay minerals.Extracellular enzyme accumulation in soils was clearly demonstrated by Ramirez-Martinez and McLaren (38), who reported that the amount of phosphatase activity in 1

g soil was equivalent to 1010bacteria or 1 g of fungal mycelia If we assume that nonproteinsoil components do not catalyze hydrolysis of organic P compounds in soils, then it can

be concluded that a certain portion of phosphatase activity in soils is no longer associatedwith living tissue

Several theories have been proposed to explain the protective influence of soil onextracellular enzyme activity The dominant mechanisms of enzyme immobilization andstabilization (Fig 2) have been summarized by Weetall (39) These include microencapsu-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 sorbed to montmorillonite was stabilized against microbial attack Haig (41) also foundthat acetylesterase activity in a fine sandy loam soil—fractionated into sand, silt, andclay sizes—was associated primarily with the clay fraction McLaren (42) observed thatkaolinite adsorbed trypsin and chymotrypsin, and Mg-bentonite was shown to adsorb pep-

ad-Figure 2 Schematic representation of methods of immobilizing enzymes (From Ref 39.)

sin and lysozyme (43) This adsorption occurred rapidly and was 90% complete after2–3 min The mechanisms by which clay minerals bind proteins are not always clearlyunderstood 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-proteinsuspension They interpreted this result as evidence that a cation-exchange adsorptionmechanism was occurring

More recently, Ruggiero et al (10) have summarized a large amount of work thathas 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 conditionmakes it difficult to extrapolate results obtained by using relatively pure clay mineralssaturated 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 withthis 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 stablethan urease added to soils He concluded that organic soil constituents protect ureaseagainst microbial degradation and other processes, leading to decomposition or inactiva-tion Since then, numerous studies have supported this conclusion by showing that enzymeactivities 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, whichwas free of clays (confirmed by x-ray analysis), contained urease activity Supportingresults indicating that enzyme activity is associated with humus–enzyme complexes havebeen 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

difficult to determine Work by Simonart and associates (56) suggests that hydrogen ing may be only a minor factor in enzyme stabilization in soils By using phenol to breakhydrogen bonds, they were able to dissolve only a small amount of proteinaceous material.Enzymes may be bound to organic matter by ionic-bonding mechanisms Butlerand Ladd (57) proposed that enzyme–organic matter complexes are formed through theformation of amino–carboxyl salt linkages Such complexes, however, should be easilybroken by many of the extraction reagents (i.e., urea and pyrophosphate) used to removeactive enzyme materials from soils The small yields of active enzyme materials that havebeen extracted from soils indicate that ionic-bonding mechanisms may be only partiallyresponsible for enzyme stabilization (58–60) However, Burns et al (61) extracted approx-imately 20% of the original soil urease activity by using urea (urea hydrolyzed subse-quently by the extracted urease) The clay-free precipitate has urease activity that was notdestroyed by the addition of the proteolytic enzyme pronase The native soil urease wasthought to be located in organic colloidal particles that contained pores large enough toallow water, urea, ammonia, and carbon dioxide to pass freely, but small enough to excludepronase.

bond-A clear hypothesis explaining enzyme stabilization by means of covalent attachmenthas yet to be proposed Ladd and Butler (45) suggested that the linkage of soil quinones

by nucleophilic substitution to sulfhydryl and to terminal andε-amino groups of enzymeproteins may lead to active organoenzyme derivatives, provided these groups do not form

a part of the active site of the enzyme

One hypothesis that has as yet received little attention is that enzymes exist in soils

as glycoproteins Malathion esterase, extracted from soils by Satyanarayana and Getzin(62), was thought to be a glycoprotein because of the following evidence: (1) amino acidsconstituted only 65% of the purified enzyme; and (2) a carbohydrate-splitting enzyme,hyaluronidase, enhanced the catalytic effect of the esterase, presumably by loosening thecarbohydrate shield and allowing the protein core to gain easier access to the substrate.The evidence gained by incubating the esterase with hyaluronidase suggested that the

carbohydrate–protein linkage occurs through N-acetylhexoseamine–tyrosine bonds

May-audon et al (63) drew similar conclusions when they observed that diphenol oxidaseactivity was not affected by pronase alone, but was destroyed when incubated in the pres-ence of both lysozyme and pronase

In soils, a strong association exists between clay and humus Each does not rately influence enzyme stabilization; rather, Paul and McLaren (64) postulated, a three-dimensional network of clay and humus complexes exists in which active enzyme becomesincorporated (Fig 3) A study by Burns et al (51) supported this hypothesis when theyobserved that a bentonite–lignin complex protected urease from degradation much moreeffectively than did bentonite alone

sepa-C Stability

Most of the information available on the stability of enzymes in soils is derived fromwork on urease, acid phosphatase, and arylsulfatase The first evidence that soil enzymesare more stable than those added to soils was obtained by Conrad (46) in his work onurease; Conrad concluded that organic matter in soils protect-enzymes (urease) againstmicrobial degradation Support for this conclusion has been provided by numerous studiesshowing that enzyme activities are significantly correlated with organic C in surface soilsand soil profiles (8,50) Further evidence supporting this conclusion is provided by work

Figure 3 Model for soil enzyme location and activity consisting of enzyme embedded in, andperhaps 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 oforganomineral complexes (13,65)

III ROLE OF CHEMISTRY IN ENZYME ACTIVITY MEASUREMENT

One of the fundamental requirements of enzyme measurements is a thorough ing of the reactions involved, quantitative extraction of the product(s) released, and asuitable analytical method for measuring quantitatively the extracted compound There-fore, knowledge of analytical chemistry and chemical kinetics are essential in soil enzymeresearch In addition, because soils contain both organic constituents and mineral compo-nents, a thorough understanding of the potential reactions between the substrate, and moreimportantly the product released, and the soil constituents is a prerequisite for any methodsdevelopment

understand-The detailed study of an enzyme reaction in soils involves characterization and surement, if possible, of several properties, some of which cannot be obtained for enzyme

mea-in soils One, therefore, has to rely on the biochemical literature for the mea-information quired

re-1 Protein properties: Even though it is difficult to extract and purify enzyme

proteins from soils, information about the enzyme molecular weight, isoelectricpoint, electrophoretic mobility, and stability to pH, heat, and oxidation can beobtained from the biochemical literature Some of these properties can be ob-

tained from direct experiments by using soil samples, as has been demonstratedfor 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 toknow 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 suchactivity

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; ficity for substrate; nature of the chemical structure; and specificity to inhibitors

speci-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 strate combination cannot be determined, other properties, however, can be.They include the activation energy of an enzyme-catalyzed reaction, affinity ofthe enzyme for its substrate, Michaelis–Menten constant, effect of pH on affinity

enzyme–sub-of the enzyme for its substrate, affinities for inhibitors, inhibitor constants, andcompetition of inhibitors with the substrate

The temperature dependence of the rate constant, at a temperature belowthat which results in activation of the enzyme activity, can be described by the

Arrhenius equation (k ⫽ A ⋅ exp (⫺Ea/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 ⫽ (⫺Ea/2.303 RT) ⫹ log A), allows calculation of Ea by plotting log of the initial rate of the reaction vs 1/T The slope of the resulting

line is equal to⫺Ea/2.303R The enthalpy of activation (∆Ha) can be calculated

from∆Ha ⫽ Ea ⫺ RT.

6 Kinetics: Characterization of the kinetic parameters of the Enzyme–substrate

reaction is important because anyone who is concerned with catalysis in soil ismost certainly concerned with the velocities of chemical reactions (chemicalkinetics) The usual way to follow an enzyme-catalyzed reaction is by measuringthe amount of reactant remaining or the product formed By contrast, most ki-netic models are formulated in terms of rates of reaction Traditionally enzymekinetic 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 reactioncurve 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, whichreact with a single enzyme The Michaelis–Menten equation does an excellentjob of describing this type of kinetics and there are several assumptions thatare made when applying this equation to soil systems The enzyme reaction isexpressed by the following equation:

E⫹ S 53k1

k2

ESErk3 E⫹ PThe assumptions made in deriving the Michaelis–Menten equation are as fol-lows: (1) The rate of reaction of the enzyme-catalyzed system changes from

Table 1 Parameters of Linear Equations Describing Inhibition of Enzyme–SubstrateInteractions

Linear competitive Km/Vmax(1⫹ [I]/Ki) 1/Vmax ⫺1/Km(1⫹ [I]/Ki)Linear noncompetitive Km/Vmax(1⫹ [I]/Ki) (1/Vmax)(1⫹ [I]/Ki)

Linear uncompetitive Km/Vmax (1/Vmax)(1⫹ [I]/Ki)

first-order to zero-order kinetics; (2) enzyme (E ) reversibly binds with substrate

(S) to form an intermediate enzyme–substrate (ES) complex, which then breaksdown to form product (P) Each reaction is described by a specific rate constant:

k1, k2, k3; (3) a steady-state equilibrium between the rate of formation of ESand 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 k3); and (6) Vmaxis achieved when

ES complex concentration reaches a maximum equal to the total enzyme centration: i.e., there is no free enzyme

con-Much of what we know about biological systems is based on more plex enzyme systems that have an inhibitor present For example, it is wellknown that the presence of inorganic phosphate in solution strongly inhibitsphosphatase activity in soils (8) The simplest systems are those in which there

com-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 Theparameters 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 distribuInforma-tion of the enzyme among different speciesand associated with different plant and microbial tissues that are deposited insoils 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 theproduct released markedly affects its extractability from soils and the potential for itsquantitative determination by any procedures or techniques Detailed discussion of enzymespecificity 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 particularatomic structure and configuration of both the substrate and the enzyme There are threetypes 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 Anexample 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

extent This term may also be used to illustrate the different rates of reactions within agiven class The third type is optical specificity, which is a common property of some yeastenzymes, which act on optically active substrate Stereochemical specificity is strikinglyillustrated by the action of glycosidases Maltase hydrolyzes maltose and several otherα-glucosides to glucose but notβ-glucosides Emulsin contains a β-glucosidase, which actsonly onβ-glucosides but not on α-glucosides Both α- and β-glucosidases are present insoils (66) Other similar examples are the d- and l-specific amino acid oxidases (67).

An essential step in enzyme activity measurement requires the availability of cal methods and instrumental techniques for determination of the reaction product formed.Almost all the methods developed by biochemists for enzyme assay are useful as guidesfor assay on enzyme activities in soils, but caution should be exercised to be sure thatthe product formed is determined quantitatively This is because many of the methods arenot compatible with the complex chemical characteristics of the soil system

chemi-V ENZYME PROTEIN CONCENTRATION IN SOILS

Numerous attempts have been made to extract pure enzymes from soils, but in reality thebest that has been achieved is the extraction of enzyme-containing substances and com-plexes (68) The reagents used in the extraction procedures range from water to salt solu-tions or buffers to strong organic matter-solubilization reagents, such as NaOH or sodiumpyrophosphate The extracted activities are usually associated with carbohydrate–enzymeprotein complexes and are often difficult to purify Modern biochemical techniques havebeen used in the purification of the extracted enzymes, but little progress has been made

in obtaining pure enzyme proteins from soils Several of the enzymes extracted from soilscould be present in soils as glycoproteins Although many investigators have demonstratedthat clay-free extracts could be obtained from soils, the major problem appears to be thestrong affinity of the carbohydrate–enzyme complexes for chromatographic columns,which makes the separation difficult It appears that various carbohydrates in soils adsorbthe enzyme proteins and are responsible for their stabilization against denaturation orproteolysis

A Estimation of Concentrations of Enzyme Proteins in Soils

Enzyme activities are associated with active microorganisms because the microbial mass is considered the primary source of enzymes in soils Nevertheless, there is no directcorrelation between the size of the microbial biomass and its metabolic state (69) Oneapproach to estimate the metabolic state of microbial populations in soils is to differentiatebetween intra- and extracellular enzyme activities Among the many attempts that havebeen made to determine the state of enzymes in soils are techniques that employ elevatedand decreased temperatures; antiseptic agents such as toluene, ethanol, Triton X-100, di-methyl sulfoxide; irradiation with gamma rays or electron beams; and fumigation withcompounds such as chloropicrin, methylbromide, and chloromycetin (2,3,8,70,71) None

bio-of these methods can distinguish between intracellular (activity associated with the bial biomass) and extracellular activity (that portion stabilized in the three-dimentionalnetwork of clay–organic matter complexes) (Fig 3), because all these techniques alsodenature the enzyme proteins Another suggested approach is plotting enzyme activityagainst the number of ureolytic microorganisms (in the case of urease) or adenosine tri-

micro-phosphate (ATP) concentration (in the case of phosphomonoesterases) The extrapolation

to zero population or ATP concentration produces a positive intercept, which is assumed

to be the extracellular component of the enzyme activity (72,73)

B Estimation of Active Enzyme Protein Equivalent

Studies in 1998 and 1999 by Klose and Tabatabai (74–76) have estimated the tions of 12 enzyme proteins in soils The averages of the concentrations in 10 Iowa surfacesoils ranged from 0.014 mg protein kg⫺1 soil forβ-glucosidase to 22.5 mg protein kg⫺1

concentra-soil for acid phosphatase (Table 2) These estimates were done by analysis of referenceprotein materials by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by calculation of the specific activity of the reference proteins From thespecific activities of the reference proteins and activities of the enzymes in soils in thepresence of toluene, the enzyme protein equivalents in soils were calculated These calcu-lations were not intended to give an accurate concentration of enzyme proteins in soilsbut instead to provide some quantitation of enzyme protein equivalent Actual concentra-tions of enzyme proteins in soils are undoubtedly much greater than those calculated be-cause many soil components can inhibit activity, and structural stabilization also leads todecreases in the activity However, the calculations illustrate one reason for the difficultiesencountered in the extraction and purification of enzyme from soils (68) From the resultsreported in Table 2, it is clear that the small concentrations of enzyme proteins in soilseither are denatured during extraction or bond tightly with the soluble carbohydrates, mak-ing their separation very difficult

VI TYPES OF ENZYMES AND SUBSTRATES

To date, soil enzyme studies have been primarily restricted to hydrolases but with tional effort also put into measuring specific types of oxidoreductases and lyases Thisemphasis on hydrolases is understandable because of the need for microorganisms in soil

addi-to degrade a complex variety of substrates in soil Many of these substrates are polymericand can only be degraded by enzymes secreted into soil The fate of the secreted or extra-cellular enzymes is still not well understood, but it is probably safe to assume that most

of them are rapidly degraded by proteases and/or inactivated in soil However, some maybecome immobilized and stabilized in soil through a variety of mechanisms (7) so thattheir activity continues long after they are first introduced into soil Hydrolases are alsorelatively simple enzyme systems, which generally do not require cofactors; have multiplesubunits; and are small in size Thus they are much more resistant to denaturation bytemperature, desiccation, sorption, or other physical factors of the soil environment.The specificity of an enzyme reaction is difficult to assess in soils For example,total cellulase activity in soils may be due to wide variety of extracellular enzymes fromfungi and bacteria including those stabilized by association with the organic and mineralcomponents An accurate description of all enzymes in all locations, for example, thatcontribute to measured cellulase activity is not possible at this time In addition, cellulasesare endocellular or ectocellular (i.e., cleave internal or exposedβ1-4 linkages) and interact

in a synergistic way that would make it very difficult to distinguish between the individualcomponents of ‘‘total’’ cellulase activity

InTable 3, we have described some of the major polymeric substrates that are rally introduced to soil as plant, animal, or microbial products The enzymes involved and

natu-Table 2 Estimated Enzyme Protein Equivalents in Soils

Enzyme protein equivalent (mg protein kg⫺1soil)a

Glycosidasesb Amidohydrolasesc Phosphatasesd Arylsulfatasee

Soil α-Gal β-Gal α-Glu β-Glu l-Asg l-Glu Amid Urea l-Asp Acid-P Alk-P A BHarps 0.031 1.6 4.8 0.021 2.4 1.2 4.2 3.6 8.5 12.2 5.2 9.0 37.6Okoboji 0.038 2.1 4.8 0.018 0.82 0.69 4.3 2.6 3.3 21.5 3.2 7.5 31.4Muscatine 0.018 1.2 3.6 0.014 0.84 0.58 3.6 0.95 2.8 16.3 3.3 7.3 30.7Grundy 0.022 1.1 3.6 0.014 0.56 0.45 2.8 0.87 1.8 25.7 1.8 4.2 17.7Gosport 0.035 2.5 3.9 0.014 0.59 0.57 3.5 1.4 2.8 29.2 1.8 4.6 19.5Clinton 0.033 2.8 4.2 0.019 0.80 0.71 7.3 2.6 2.9 33.5 2.1 5.7 24.1Pershing 0.028 1.4 3.9 0.013 0.35 0.22 2.1 0.95 1.1 34.5 0.97 3.20 13.3Luther 0.010 0.29 1.6 0.005 0.41 0.10 0.65 0.87 0.40 8.8 0.36 0.10 0.42Grundy 0.033 1.5 3.3 0.012 0.37 0.38 3.3 1.4 1.6 21.0 1.1 3.3 13.9Weller 0.020 1.2 3.1 0.011 0.19 0.14 2.0 0.57 0.78 22.7 0.97 1.70 7.2average 0.027 1.56 3.7 0.014 0.73 0.50 3.4 1.6 2.6 22.5 2.1 4.7 19.6

of the purified reference enzyme proteins.

Source: Ref 74.

Table 3 Summary of Some of the Major Polymeric Substrates, the Enzymes Involved,and Techniques Used in Their Assay

Polymeric material Enzymes Involved Assay Methods CommentsCellulose—a crystal- Many cellulases of Measure release of Various pretreatmentsline polymer associ- three main types: 1) glucose using a glu- may be helpful toated with lignin and exocellulohydrolase cose oxidase reac- expose the cellulosehemicellulose (EC 3.2.1.91) tion to enzyme The ma-

(2) endo-1,4-β-d-glu- jor barriers are

asso-Measure release of

p-canase (EC 3.2.1.4) ciation of the

cellu-nitrophenol (many3)β-glucosidase (EC lose with lignin and

substrates available3.2.1.21) These ex- the crystalline struc-

with this ist extracellularly in ture of the cellu-

chromo-phore)

Fluorometric not possible

sub-strates (e.g., ylumbelliferyl)

4-meth-Proteins—a polymer A large variety of pro- The three most com- Not all of the that comprises teinases and pepti- monly used sub- ods for using theseamino acids bound dases most of strates are substrates in soiltogether by peptide which exist extracel- (1) peptide 4-nitroani- have been worked

thi-(2) peptide thioesters oesters provide a(3) peptide derivatives sensitive assay be-

of 7-amino-4-meth- cause they haveylcoumarin high kcat/kmvalues

and low Prepare proteins with

and measure rescence in the solu-tion phase

fluo-Lipids—derived from Lipases of many types Assays can measure Many natural lipid

a large number of including the phos- the organic leaving substrates are notcell membranes pholipases Lipases group or, in the soluble in water

exist extracellu- case of phospholip- and this makes thelarly in soil ids, the phosphate design of an assay

released Methods difficult used include titri- chain phospholip-metric, radiomet- ids that are waterric, colorimetric, soluble can be usedand fluorometric as synthetic sub-procedures strates and choice

Short-of substrate can Short-ten distinguish thedifferent types of li-pases and phospho-lipases

of-Table 3 Summary of Some of the Major Polymeric Substrates, the Enzymes Involved,and Techniques Used in Their Assay

Polymeric material Enzymes Involved Assay Methods CommentsLignin—a nonre- Ligninases,(e.g., phe- Polymeric dyes are Only lignin degradingpeating polymer of nol oxidases, perox- substrates that have fungi can decolor-sinapyl, coniferyl, idases) of fungi are been developed for ize many of theand caumaryl al- the best studied assays of ligni- dyes used to mea-chols that is part of nases Assays gener- sure ligninases Thecell walls and is a ally require days, polymeric dyes aremajor part of soil not hours, to com- inexpensive and sta-humus and more re- plete Different ble, can be obtainedsistant to degrada- dyes that vary in ab- commercially, havetion than most sub- sorbance intensity high purity, are wa-

be used.14C-La- have high beled materials can tion coefficients.also be used

extinc-Chitin, pectin, and Degradation of most Chitinase is most com- Chitin is insoluble Aother polymers in polymers is due to monly assayed by pure form of chitinsoils Chitin is a extracellular en- measuring n-acetyl- can be purchased.mucopolysaccharide zymes as these mol- glucosamine re- Tritiated chitinoften intimately as- ecules are too large lease by using a must be prepared insociated with calcar- to be taken into mi- spectrometric pro- the laboratory, andeous shell material crobial cells Pec- cedure when chitin the amount of tri-Pectin polymers are tinases (especially) is incubated with tium that remainschains of predomi- and chitinases have soil Tritiated chitin in solution after cen-nantly 1,4-linked-α- various forms can also be pre- trifugation is a mea-d-galacturonic acid pared and used as a sure of chitinase ac-

tradi-tionally assayed byusing a viscosity re-duction and by mea-suring reactionproducts by a vari-ety of chemical andbiochemicalmethods

the techniques used to measure these enzymes are summarized Detailed accounts of theprocedures for individual enzymes assay can be found in a book chapter by Tabatabai (8)and a manual edited by Alef and Nannipieri (77)

VII MEASUREMENT METHODS

Techniques to measure enzyme activity in soils are primarily derived from the biochemicalliterature However, because the soil system is generally much more complex than manysystems studied by biochemists, most of these methods and techniques require modifica-tions Advances in our scientific understanding of many subjects is directly linked to ourability to develop methods to measure what we are attempting to study For many years,

progress in soil enzymology was hampered by a lack of standard methods and development

of new methods was difficult because of the great complexity of soils There have beenmany advances in analytical capabilities, but application of these new procedures to soilenzymology has not kept pace The reader is referred to the series Methods in Enzymologyfor up-to-date accounts Major published works related specifically to describing methods

of soil enzymes include those by Alef and Nannipieri (77) and Tababatai (8) The factorsthat limit advances in soil enzyme research are related to our inability (1) to separateextracellular from intracellular enzyme activity, (2) to extract and purify enzymes fromsoil, and (3) to extract the many products of enzyme reactions from soil quantitatively.Depending on whether a decrease in the substrate concentration or an increase in theconcentration of the product released is to be measured, the method selected for quantita-tively following any enzyme reaction may be one of many analytical techniques:

A Spectrophotometric Methods

Many substrates and the products of enzymatic reactions absorb light, either in the visible

or in the ultraviolet region of the spectrum Most often the change in the concentration

of the substrate or the product is followed colorimetrically after extraction from a soilsample incubated with the substrate at specific temperature, pH, and time Here we summa-rize some of the most commonly used methods

Numerous colorimetric procedures for analysis of urea with diacetylmonoxime havebeen developed (78); most of these methods are actually variations of that developed byFearon (79) One of the procedures has been evaluated for the determination of urea (ex-

tracted from soils with 2 M KCl-phenylmercuric acetate) with a reagent containing

diac-etylmonoxime and thiosemicarbazide in a boiling water bath for 30 min (80) The sorbance of the chromogen complex is measured at 550 nm The main disadvantages ofmost of the procedures available for colorimetric determination of urea are (1) lack ofsensitivity, (2) lack of linearity at low urea concentration, (3) lengthiness of procedure,(4) low precision, and (5) instability of some of the reagents or the chromogen compound.Because of these problems, attempts have been made to automate the development ofcolor (81,82) Caution should be exercised in using this method, however, because nobuffer is used to control the pH of the incubation mixture

ab-Following are examples of methods involving colorimetric determination of theproducts released in assays of arylsulfatase and arylamidase activities in soils

Arylsulfatase (EC 3.1.6.1) is the enzyme that catalyzes the hydrolysis of organicsulfate ester (R⋅ O ⋅ SO3⋅ ⫹ H2O→ R ⋅ OH ⫹ H⫹⫹ SO4 ⫺) This enzyme has beendetected in plants, animals, and microorganisms, and it was first detected in soils by Taba-tabai and Bremner (83) This enzyme hydrolyzes a number of organic sulfate esters

Among those p-nitrophenyl sulfate, 4-nitrocatechol sulfate, and phenolphthalein sulfate

have been tested as substrates for this enzyme in soils