11 Interaction of Enzymes with Soil Mineral and Organic Colloids

Published 1986

11

Interaction of Enzymes with Soil Mineral and Organic Colloids R. G. Bums l

It is common practice in the study of soil biology to collect samples without regard for their location within a soil profile. The samples are then homogenized following the removal of plant debris and macrofauna, sieving, and air drying. As a result, soil for experimental purposes assumes a homogeneity that may be totally misleading. For example, in bulked samples such measurements as percent organic carbon, cation exchange capacity, viable microbial count, or phosphatase activity, will represent an average of a number of extremely diverse environments within the soil profile. A cursory investigation of soil profiles reveals that in fact they have a complex spatial heterogeneity. Thus total counts, species diversity, estimates of microbial activity, and physicochemical properties will vary according to which horizon is sampled. This is not surprising because each horizon is different-a difference defined by organic matter level, textural status, water and mineral content, soil atmosphere and so on, and determined by macroecological factors such as climate, vegetative cover, and parent rock composition. Therefore, over a distance of 1 to 2 m there may be as many as a dozen distinctly different environments with regard to biological activity. An experimental approach to applied soil microbiology and biochemistry based upon an acknowledgement of the discontinuity within a pro£lle, satisfies certain objectives. For instance, it is useful to know the capacity of a soil at various depths for organic matter degradation, plant nutrient solubilization, destruction of a potential pollutant, and retention of a fertilizer. It may even be possible to conduct this type of investigation in situ, thus mollifying those who believe that the only relevant studies are those carried out in the field. However, even this approach takes no account of the many microenvironments in which soil microorganisms dwell (Stotzky, 1980; Burns, 1979, 1980). 'Reader in microbiology, Biological Laboratory, Univ. of Kent, Canterbury, Kent, CT2 7NJ, UK. Copyright © 1986 Soil Science SOciety of America, 677 S. Segoe Rd., Madison, WI 53711, USA. Interactions of Soil Minerals with Natural Organics and Microbes, SSSA Spec. Pub. no.l7.

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430

In order to do this, we must recognize another order of complexity altogether-a soil environment in which gradients are measured not in meters (profiles), centimeters (horizons) or even millimeters (aggregates) but in micrometers and nanometers.

THE SOIL MICROENVIRONMENT

11-1

Figure 11-1 shows, diagrammatically, a soil microenvironment and is composed of:

1. A solid phase-the predominantly negatively charged, high unit surface area, clay and organic colloids. 2. An aqueous phase-the soil water existing in various physical states and containing a large number of organic and inorganic solutes. 3. A gaseous phase-of fluctuating composition and very different from the above soil atmosphere. 4. The microflora-principally bacteria and fungi carrying a variable but predominantly negative charge. 5. Interfaces-including solid/liquid and liquid/gas interfaces having distinct properties of their own. It is important to remember when discussing soil microenvironments that the various phases and interfaces do not have constant dimensions or properties in time or in space. A study of Fig. 11-1 will suggest some rather specific consequences for microorganisms residing in soil microenvironments.

Q--_

Water film

",.,.

--------

--

/

/ A

'b' ,-+ nlr rolre

(H 2 PO' NO

I

_

+

Prolein+

-

Peslieide+ __ -

=

z +

+

--

Microbial cell

\ N0 3 \ "

Fig. 11-1. Diagrammatic representation of a soil microbial microenvironment containing clay and organic colloids, adsorbed organic and inorganic cations and repulsed anions, The diagram represents an area of approximately 8 /Lm2,

INTERACTION OF ENZYMES

431

1. Clay and organic colloid surfaces attract and therefore concentrate (vis-a-vis the aqueous phase) numerous organic cations which are potential substrates for, or inhibitors of, microbial growth. In contrast, anionic organics generally tend to be repelled from colloid surfaces and must present different problems to microorganisms which use them as growth substrates. The consequences of organic substrate sorption for an adjacent microbial cell will depend on: the strength of cation retention at the colloid surface; how much and what part of the macromolecule is involved in sorption and how much is exposed; and the ability of the microbial species to remove and assimilate adsorbed substrate. 2. Ammonium, the end product of aerobic N mineralization, is strongly concentrated at colloid surfaces. The consequences of this phenomena for local Nitrosomonas populations are readily apparent whilst the result of nitrification, that is anionic and repulsed nitrite and nitrate, suggests that different problems face Nitrobacter and those organisms using nitrate as a N source. 3. Magnesium and phosphate availability will have a profound effect on the formation of teichoic and teichuronic acids and therefore the properties of Gram-positive cell walls in relation to cation binding, bacteriophage attachment, sensitivity to antibiotics, and other functions attributed to cell wall polyphosphates. 4. Perhaps one of the most intriguing properties of soil microenvironments (and certainly one of the most far-reaching) is that due to the accumulation of H+ at clay and organic surfaces. This will give rise to a surface pH which may be two to three units more acid than that of the aqueous bulk phase barely 100 nm away (McLaren & Skujins, 1968). Furthermore, the magnitude of this pH effect will be accentuated by microbial metabolism and proton release. One can only speculate as to the consequences of this microenvironment property, but possibilities include: 1. The setting up of microenvironment pH gradients which may influence proton motive force and transport processes. 2. The solubilization of some inorganic nutrients and the precipitation or adsorption of others. 3. Different rates of enzyme-substrate interaction at acidic surfaces compared with those distant from that surface. 4. Changes in the composition and ionogenic properties of microbial cell walls. With regard to the last-mentioned possibility, it has been suggested (Burns, 1983) that pH gradients within the microenvironment in which the microbe is immersed may turn what was once electrostatic attraction into electrostatic repulsion-a switch that could be accelerated as metabolism and H+ output declines. Furthermore, self-induced cell surface charge reversal at the isoelectric point (pI) of the cell wall constituents may be just one of the ways that microorganisms respond to nutrient depletion in soil environments-an environment where close and tenacious

432

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association with a surface is important if growth substrates are present, but is a disadvantage when all the substrate has been metabolized. So, microbes, organic substrates and metabolites, and inorganic ions tend to accumulate at charged clay and organic matter surfaces rather than being freely diffusible in the aqueous phase (this can be confirmed by adsorption experiments with potential reactants, the measurement of the electrostatic mobility of microbes and their energy and growth substrates, and microscopic examination of adsorbent surfaces on which biofilms develop). Therefore, the most influential particles are those clay and organic colloids that expand upon hydration to reveal an extensive, highly charged internal surface area (Table 11-1). Indeed, it may be helpful to regard soil as a dense colloidal suspension and to think of the properties of the microenvironment as being dominated by the physicochemical properties of colloids-flocculation, dispersion, ionic gradients, adsorption and desorption phenomena, and so on. Table 11-1. Some properties of soil clays and humic materials (Burns, 1983). Colloid type

Layering

Swelling

Surface area

Cation exchange capacity cmolkg- t

Kaolinites Vermiculites Smectites Humates

1:1 2:1 2:1

Non-expanding Expanding Expanding Expanding

10-50 500-750 700-800 500-800

2-10 120-250 60-130 200-750 (Fulvic acids 500-750) (Humic acids 300-500) (Humins < 300)

Table 11-2. Influence of soil clays and humates on microbial activity (Burns, 1979).

Colloid surface phenomenon

Effect on substrate decay and/or microbial growth (relative to that in absence of clay/humic colloid)

Juxtaposes microbe (or enzyme) and substrate Orients enzyme beneficially relative to substrate Functions as buffer during metabolism Adsorbs inhibitory metabolite Retains water film Concentrates inorganic nutrient Supplies inorganic micronutrient (clay) Protects microbe from predator Inactivates phage Produces soluble substrate (humic-enzyme complex) Adjusts C/N ratios (humic) Allows co-metabolism of adsorbate (humic) Performs abiological decay in a biological sequence Adsorbs microbe (or enzyme) distant from substrate Intercalates substrate = inaccessible to microbe (clay) Incorporates substrate into humic polymer = recalcitrance Inactivates enzyme due to structural changes Masks active site of enzyme Increases viscosity = retards O. diffusion Entraps microbe in colloidal aggregate = limited O.

Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition


433

There has been a large number of experiments which acknowledge the importance of surfaces to soil microorganisms by comparing some aspect of microbial growth or substrate consumption in the presence and absence of soil or soil components (e.g., Bondietti et aI., 1972; Filip et aI., 1972; Martin et aI., 1976; Marshall, 1976; Marshman & Marshall, 1981a, 1981b). However, the precise cause of any observed effect is rarely revealed although many plausible explanations exist (Table 11-2). Examination of Table 11-2 reveals that a large number of the stimulation and inhibitory effects appear to be due to modifications of extracellular enzyme-substrate interactions and it is this aspect on the soil microenvironment which will form the basis of this chapter.

11-2 ENZYMES IN SOIL Enzymes in soil can be separated into a number of categories according to their location within the soil microenvironment. Indeed, the measured activity of a particular enzyme is usually a composite of activities belonging to two or more categories. The various categories have been fully described elsewhere (Burns, 1982a, 1983) and are only summarized here. 1. Enzymes associated with living, metabolically active cells. These enzymes are principally hydro lases and may be cytoplasmic, periplasmic, cell-wall bound or truly extracellular. Their location will be determined, at least in part, by such factors as membrane porosity (viz. some cells "leak"), substrate size and solubility, and the availability of suitable uptake mechanisms. For example, cellulases and proteinases can only function outside of the cell wall whilst urease and ~-D-glu cosidase may be intracellular. Obviously, enzymes involved in the central aspects of metabolism (e.g., glycolysis, oxidative phosphorylation) cannot function outside of the cell. If the relevant component of the microbial population is identified, there should be a good correlation between this category of activity and microbial numbers. 2. Enzymes associated with viable but nonproliferating cells such as resting vegetative cells, bacterial endospores, fungal spores, protozoan cysts and even plant seeds. 3. Enzymes which are associated, at least briefly, with their substrates in enzyme-substrate complexes. 4. Enzymes attached to entire dead cells, cell debris or having diffused away from dead and lysed cells. Many enzymes in this category may have had an original functional location on or within a cell yet may survive for a short period when released into the soil aqueous phase. 5. Enzymes which are more or less permanently immobilized on the soil clay and humic colloids. Expandable clays have a high affinity for enzymes although this is not always synonymous with the retention of catalytic ability. Enzymes associated with soil humates retain their activity for long periods.

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Table 11-3. Choice of assay conditions for the measurement of soil enzyme activities. Conventional enzyme assay

Field conditions

Excess substrate Homogenous substrate Usually soluble substrate Often artificial substrate Buffered at optimum pH pH poised Slurry conditions Often shaken Constant temperature Flora and fauna absent

Usually limiting substrate Heterogenous substrate Often insoluble substrate Natural substrate Soil pH pH may vary Moisture level variable Stationary Temperature variable Flora and fauna present

Maximum potential activity Highly reproducible

Sub maximal activity Extremely variable

Enzymes can, of course, belong to more than one category and change from one to the other with time: periplasmic phosphatases leak from Gram-negative bacteria and remain active in dormant cells; they also show residual activity in dead cells and tissues and may remain active upon association with soil colloids. It is difficult to differentiate between these categories experimentally although with certain pretreatments of the soil sample, intelligently designed assays and, above all, the study of clay-enzyme and humic-enzyme soil extracts, it may be possible to produce a balance sheet. One perennial problem in soil enzymology is in the choice of assay conditions, one school of thought leaning towards classical enzymology; another towards conditions prevailing in the field (Table 11-3). This problem has been discussed frequently (Burns, 1978; Malcolm, 1983) but not resolved. Notwithstanding, by applying an exhaustive characterization of the soil and soil extracts (Stotzky & Burns, 1982) it is possible to state that a large number of enzymes fall into category 5 (above)-that is, they form stable complexes with clays and humates (Table 11-4) and are part of a persistent extracellular enzyme capacity of soil which is independent of the existing microbiota and therefore of the usual forms of regulation and control of exoenzyme synthesis and secretion. The biological significance of the immobilized enzyme component of soil is difficult to assess, although it seems probable that in one sense viable microbial cells contribute only a small portion to the overall activity of many enzymes. For example, total microbial numbers or specific groups of microorganisms may be poorly correlated with activity, or activity may be only marginally reduced by incorporating a biocidal agent into the soil during an assay. Furthermore, the response times of immobilized enzymes to substrates are likely to be more rapid compared with enzymes associated with viable cells which may require an induction or derepression process prior to enzyme production and substrate breakdown. For example, Gibson and Burns (1977) and Burns and Edwards (1980) showed that the rapid degradation of malathion (diethyl mercapto succinate S-ester with O,O-dimethyl phosphorodithioate) was primarily

INTERACTION OF ENZl'MES

435

Table 11-4. Enzymes detected in sterile soil or in clay and/or humic extracts (Bums,1983). Class Oxidoreductases

EC numbert

Recommended name

1.7.3.3 1.10.3.1 1.10.3.2 1.11.1.7 1.14.18.1

Transferases

2.4.1.5 2.4.1.10 2.8.1.1

Hydrolases

3.1.1.1

Urate oxidase Catechol oxidase Laccase Peroxidase Monophenol monooxygenase Dextransucrase Levansucrase Thiosulphate S-transferase Carboxylesterase

3.1.1.2 3.1.1.3

Arylesterase Triacylglycerollipase

3.1.3.1 3.1.3.2 3.1.6.1 3.2.1.1 3.2.1.2 3.2.1.4

Alkaline phosphatase Acid phosphatase Arylsulphatase a-Amylase {:I-Amylase Cellulase

3.2.1.6 3.2.1.8 3.2.1.21

Endo-1,3(4)-{:I-D glucanase Xylanase {:I-D Glucosidase

3.4.--

Peptidases

3.4.-3.5.1.1 3.5.1.2 3.5.1.4 3.5.1.5 3.5.1.13 4.1.1.15

Proteinases Asparaginase Glutaminase Amidase Urease Arylacylamidase Glutamate decarboxylase Tyrosine decarboxylase Aromatic-L-amino-acid decarboxylase L-histidine ammonia lyase

Lyases

4.1.1.25 4.1.1.28 4.3.1.3

Substrate Uric acid Catechol Phenylenediamine Pyrogallo~ chloroanilines Catecho~ pyrogallo~

hydroquinone Sucrose Sucrose Thiosulphate + cyanide Hydroxy-methylcoumarin butyrate, malathion Phenyl acetate 4-Methyl umbelliferone nonanoate p-Nitrophenyl phosphate p-Nitrophenyl phosphate p-Nitrophenyl sulphate Starch Starch Cellulose, carboxymethylcellulose Laminarin Xylan p-Nitrophenyl {:I-D glucoside, cellobiose N-benzoyl L-arginine amide, benzyloxycarbonyl phenyIalanylleucine Casein, gelatine Asparagine Glutamine Formamide, acetamide Urea Propanil Aspartic acid Tyrosine Tryptophan, DOPA L-histidine

t EC number = Enzyme Commission number authorized by the International Union of Biochemistry.

the function of an extracellular esterase associated with extractable humic material. Only in the absence of the humic-enzyme complex did microbial degradation occur and even then only after a 3 to 4 day lag phase. Similarly, the rapid hydrolysis of urea in soil is due to the presence of immobilized extracellular urease even though ureolytic microorganisms are

436

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abundant in most soils. Thus, it appears that in many instances the microflora of soil is of secondary importance in the ability of that soil to respond immediately to a pulse of substrate. Indeed, microorganisms may remain dormant unless substrate levels are sustained at a high level or until stimulated by an effector molecule produced by activities of immobilized enzymes. These and other nutritional strategies for microbial success in the soil microenvironment are discussed later in the chapter. 11-3

CLAY- AND HUMIC-ENZYME COMPLEXES 11-3.1

Clay-Enzyme Interactions

The adsorption of proteins to clay surfaces and the subsequent protection of the adsorbate from decomposition are phenomena that have been known for many years (Ensminger & Gieseking, 1942; McLaren, 1954a, 1954b). Recently, Stotzky (1980) has summarized the characteristics of clays, proteins and their environment which influence the rate and strength of adsorption of proteins and the availability of the adsorbed protein as a C and/or N source for microbial growth. The complexity of the relationships and the difficulties in predicting clayprotein interactions can be gauged by reference to Table 11-5. A further level of difficulty emerges when studying enzymes at clay surfaces because any involvement of its active sites in binding or changes in the tertiary structure of the enzyme will reduce activity or eliminate it altogether, In other words, the protein may be strongly held at and protected by the clay but it no longer displays enzyme activity. Clayenzyme interactions prior to 1975 have been discussed in detail by Theng (1979) and only the more recent observations will be presented here. Most studies (Haska, 1975, Makboul & Ottow, 1979; Pflug, 1982; Ottow et aI., 1983; Ross, 1983) confirm earlier reports that enzyme activities are reduced upon adsorption to clays (Table 11-6) and that the affinity of the enzyme for the substrate usually decreases as does the maxiTable 11-5. Factors influencing the binding of proteins to clay colloids. Clay

Unit surface area Ion exchange capacity Resident surface ions Hydration

Protein

Molecular mass Isoelectric point Number of binding sites Solubility Concentration

pH

Bulk phase Interface Moisture content Ionic strength Ionic composition Viscosity

Soil water


437

mum enzyme reaction velocity (Table 11-7). In other words, Km values increase and Vmax values decrease. Usually highly adsorptive expandable clays, such as montmorillonite, have a more marked effect than kaolinite. However, there are some notable exceptions to any "adsorptiondecline in activity" rule and two such examples are given here. Makboul and Ottow (1979) measured alkaline phosphatase activity in the presence of kaolinite, illite, and montmorillonite. The Vmax values actually increased by 9.2 % in the presence of Ca 2 +-montmorillonite and Km values decreased (4.26 to 3.92 mM) when enzyme was adsorbed to illite. They suggested that an increase in the affinity of the enzyme for the substrate was due to steric modification of the sorbed alkaline phosphatase. Ross (1983), although reporting a consistent decline in the activity of a- and {3amylase and invertase in the presence of clays, noted that kaolinite had a more inhibitory effect on a-amylase than did the more highly adsorptive clays illite and muscovite. There was no obvious relationship between adsorption and inhibition of {3-amylase after 1-h exposure to muscovite, illite, montmorillonite, or kaolinite. The mechanisms whereby enzymes are held at clay surfaces have usually been assumed to include cation exchange but not all observations have supported this view because if it were true, one would expect adsorption to increase as pH values decreased below the isoelectric point of the enzyme. Under these conditions, the protonation of amino and carboxyl groups should give rise to an increase in positive charges and correspondingly more adsorption to anionic clays. However, Hamzehi and Pflug (1981) showed that the adsorption of polysaccharases (i.e., celluTable 11-6. Influence of clay minerals on the activity of enzymest (Ross, 1983). Clay mineral

Invertase

(X·Amylase

{3-Amylase

20t

57

9

27

12 1 1

%

Allophane Muscovite Illite Montmorillonite Kaolinite

33

96

o

o o

4

45

1

t Percent of original activity. Table 11-7. Kinetic constants of urease in the presence of various clay minerals (Makboul & Ottow, 1979). Urease alone Kaolinite Illite Montmorillonite

(50mg) (150mg) (50mg) (150 mg) (50mg) (150mg)

Km(mM)t

Vmax+ (fLg NH, mL -, h-')

6.4 8.3 16.7 8.4 11.9 10.7 18.6

151.4 103.8 79.4 100.0 75.8 73.6 76.9

t The substrate concentration that gives the half maximum rate.

+The maximum rate of catalysis under the given conditions.

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438

lases, amyloglucosidase, pectinase, and a-amylase) was highest at their pI when the enzymes have no net charge. This prompted them to suggest that adsorption may be through van der Waals type forces because, although individual van der Waals forces are regarded as weak, retention may be cumulative if the enzyme is in close contact with the adsorbing surface. Hughes and Simpson (1978) introduced a concept of primary and secondary adsorption of enzymes (arylsulphatase) to kaolinite clay surfaces. Primary surface adsorption, comparable with intercalation of enzymes by expandable clays and due to a combination of ionic, hydrophobic, and van der Waals forces, causes the total inactivation of enzymes which are associated as a monolayer at the clay surfaces. Secondary surface adsorption is the accumulation of enzymes in the vicinity of the clay surface and this "weak" adsorption, which does not result in a loss of activity and can be easily removed by washing, allows the enzyme to retain activity. There are obvious parallels here to theories of the diffuse double layer of inorganic cations as they apply to clay colloids.

11-3.2 Humic-Enzyme Interactions Comparatively few studies have been made of enzyme adsorption to humic matter, probably because of the biochemical heterogeneity, dynamic nature, and physicochemical complexity of soil organic matter. Nevertheless, the idea that enzymes are stabilized by association with soil organic matter is not a new one (Conrad, 1942) and previous studies with stable protein-humic acid complexes are many (Mattson, 1932; Waksman & Iyer, 1932). In recent years, a large number of humic-enzyme complexes have been extracted from soil and their kinetic characteristics (e.g., pH- and temperature-activity profiles, Km and VmaJ in relation to various substrates established (e.g., Burns et aI., 1972; McLaren et aI., 1975; Pettit et aI., 1976, 1977; Ceccanti et aI., 1978; Batistic et aI., 1980; Nannipieri et aI., 1982). The amount of activity varies according to the enzymes, soil type and method of extraction employed, but is generally low « 20 %) compared with that measured in the original soil. Presumably the immobilized enzyme fraction that is most resistant to extraction is that bound to humates which are, in turn, fixed to clays. Extraction techniques using buffers alone are unlikely to remove this humic-enzyme complex-other more rigorous (yet not harmful to the enzyme) methods are needed (e.g., ion exchange, H-bond breaking agents, sonication) in order to extract all the stabilized enzymes. It has been pointed out (Ladd & Butler, 1975) that some extraction methods may produce artifacts, for example simultaneously extracted enzymes and humic acid may co-precipitate to form a complex that was not present in the original soil. These extracts have two properties in common: they all show some measure of enzyme activity; and the activity is extremely resistant to such stresses as storage at high temperatures and proteolytic attack. In


439

addition, many of the now well-known properties of enzymes bound to organic supports (e.g., pH optima shifts, and resistance to solvents, enzyme inhibitors, urea, temperature extremes, etc.) are observed in soil humic-enzyme extracts. Many mechanisms have been proposed to account for the stability of enzymes when in association with soil humic colloids (Ladd & Butler, 1975; Burns, 1978; Maignan, 1982). These have included ion exchange, entrapment within three-dimensional micelles, H bonding, lipophylic reactions, and covalent bonding to humates during synthesis. Although it is not inconceivable that some or even all of these processes are involved, attempts to clarify the relationship between enzymes and humates have been largely unsatisfactory. For example, pH shifts, washing in buffer, (NH4hS04 precipitation, agitation, ion exchange and electrofocussing are only partially (and unpredictably) successful in releasing enzyme activity from humic-enzyme complexes. Indeed, it is not surprising, given the remarkable stability of immobilized enzymes in soil, that enzymes complexed to humic materials are extremely resistant to extraction and subsequent purification using conventional techniques.

11-3.3 Synthetic Humic-Enzyme Complexes An alternative and synthetic approach to understanding the relationship between immobilized enzymes and humic matter was used by Rowell et al. (1973) and has been commented upon by Ladd and Butler (1975). Rowell et al. (1973) prepared p-benzoquinone-trypsin and pbenzoquinone-pronase copolymers as analogues of humic acid-enzyme complexes. Complexed enzymes had measurable, but much reduced activities, yet displayed enhanced thermostability. Significantly, enzymes, added after the aromatic polymer was formed, were comparatively unstable. These authors proposed that covalent and H bonds were important in the binding of the enzymes within the complex and that ionic interactions were insignificant. This type of experiment takes advantage of the fact that many of the phenolic constituents of humus are well known for their ability to autoxidize and respond to enzyme catalysis forming radicals and quinones (Taylor & Battersby, 1967). Radicals may subsequently polymerize through covalent bond formation; qui nones through nucleophilic addition (Martin & Focht, 1977). These artificial polymers resemble naturally occurring humates and will often display considerable recalcitrance when exposed to degradative microorganisms or even incorporated into soil. Using a mixture of aromatics similar to that used by Martin and coworkers (Burns & Martin, 1986) and which are known to protect proteins against biodegradation (Verma et aI., 1975), we attempted to immobilize a variety of enzymes (e.g., urease, /3-D-glucosidase) either during the formation of the phenolic heteropolymer or by mixing and/or exposure to the humic macromolecule after polymerization was complete (R. C.

440

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Burns, unpublished data). None of these methods was particularly successful-that is, enzyme activity associated with the polymer was either very low « 5 % of original activity) or nonexistent. What little enzyme that was adsorbed to the preformed polyphenolic or admixed by agitation was generally unstable (i.e., easily removed by washing in buffer). Not surprisingly, pH had a significant, if somewhat unpredictable, effect on the rate and strength of enzyme adsorption. Enzyme added during the polymerization and condensation steps had little detectable activity even though much of the protein was associated with the complex and difficult to remove. At least four possibilities exist for the lack of enzyme activity in these complexes: unravelling of the tertiary structure such that the bound protein is no longer enzymic; masking of the enzyme's active sites due to copolymer formation; restriction of substrate access (although urea, in particular, has a low molecular mass) due to the entrapment of enzyme in the three-dimensional structure of the humic polymer; and direct inhibition of the bound enzyme by some of the aromatic constituents of the polymer. Later studies identified the fourth possibility as a major factor restricting expression of enzyme activity. This was not surprising in the light of previous reports of enzyme inhibition by a wide range of phenolic substances (Ladd & Butler, 1975). In recently reported work (Sarkar & Burns, 1983, 1984), the inhibitory effect of certain aromatics are quite simply overcome by eliminating them from the polymer, and we found that the polymers containing one or two noninhibitory aromatic moieties are the most suitable. Thus, the process outlined in Fig. 11-2 produced a copolymer of resorcinol (and tyrosine) and i3-D-glucosidase which retained 55% of its original activity and which displayed enhanced stability in comparison with free i3-D-glucosidase. For example, approximately 50 % of the activity was resistant to prolonged exposure to proteinases (noncomplexed enzyme is destroyed in 12 h); the complexed enzyme retained 50 % of its activity after exposure to 75°C for 1 h unlike free i3-D-glucosidase which was rapidly and irreversibly denatured at this temperature. In addition, enzyme-phenolic copolymers incubated in fresh soil were stable for weeks and the soil expressed an increase in activity proportional to the added immobilized enzyme (in one instance the stable enzyme activity was increased almost 30-fold and showed no decline after 4 weeks). In agreement with previous findings (Rowell et al., 1973; Maignan, 1982), the adsorption of enzyme to preformed aromatic polymers did not give significant protection against proteolysis, temperature extremes, and solvents. Furthermore, adsorbed enzyme was easily desorbed by fluctuations in pH and even agitation in water. The polyphenolic-enzyme complexes can also be attached to clays (using cyanuric chloride as a bifunctional reagent) and in some instances this further enhanced stability. It can be seen that this synthetic process attempts to mimic a soil process in which enzymes become covalently bound to qui nones during humic matter genesis, and strongly encourages the view that copolymerization is at least one process contributing to the immobilization of enzymes on soil organic matter. Functional groups of

441


RESORCINOL / PYROGALLOL

H,o,

1""""',-

l

QUINONES

TYROSINE

,o,j

Tmoo_

DIHYROXYPHENYLALANINE (DOPA)

,e-D-GLUCOSIDASE

1

,0,

ENZYME - PHENOLIC POLYMER ( COLLOIDAL )

PHENYLALANINE - 3, 4 -QUINONE (DOPAQUINONE)

021.e-D-GLUCOSIDASE

INDOLE - 5, 6-QUINONE

ENZYME - PHENOLIC POLYMER (PARTICULATE)

Fig. 11-2. Synthesis of ~-D-glucosidase-phenolic copolymers.

enzymes implicated in covalent bonding to organic polymers are numerous and include terminal and basic amino groups, carboxyl groups, sulphydryl groups, phenolic groups of tyrosine, and the imidazole group of histidine. All these may be involved in the stabilization process provided they do not form part of the active site of the enzyme and are not crucial to the retention of its tertiary structure.

11-4 ECOLOGY OF ENZYME-SUBSTRATE INTERACTIONS IN SOIL Soil is generally considered to be an unfavorable environment for microbial activity in contrast to the conditions generally adopted for studies in vitro. Thus, soil microorganisms reside in environments with low available nutrients, constantly shifting hydration and aeration levels, and fluctuating pH. As a consequence, competition between microorgan-

BURNS

442 Table 11-8. Common macromolecular substrates in soil (Burns, 1982b). Macromolecular substrate (origint)

Structure

Cellulose (P, M) Hemicelluloses (P)

,8-(1-4)-D linked glucan ,8-(1-4)-D linked xylan

Pectin (P, M) Starch (P, M) Lignin(P) Chitin (A, M) Proteins and pep tides (A,M,P Lipids (A, M, P) Peptidoglycan (M) Teichoic acid (M) Microbial exopolysaccharides (M)

Glucuronans Galacturonans Xyloglucan Galacturonans a-(1-4) and a-(1-6) linked glucans Polymers of p-hydroxycinnamyl alcohols

Molecule entering cell:l: (known or suspected) Glucose, cellobiose Xylose, xylobiose Glucuronic acid Galacturonic acid Xylose Galacturonic acid Glucose, maltose

Mono-lignols (coniferyl sinapyl and p-coumaryl alcohols), di- and tri-lignols N-acetylglucosamine, ,8-(1-4)-linked Nchitobiose acetylglucosamine Amino acids, low number Polymers of amino acids peptides Glycerols, fatty acids Triglycerides, phospholipids Polymers of N-acetylglucosa- N-acetylglucosamine, Nmine and N-acetylmuramic acetylmuramic acid, amino acid with peptides acids, low-number peptides Polymers of polyol phosphates Glycerol, ribitol, mono- and disaccharides, alanine with saccharides and D-alanine Mono- and disaccharides Mannans, dextrans, levans xanthans, pullulan, alginate

t P = plant; A = animal; M = microbial. :j: Differs with different microorganisms.

isms is intense and there are extreme selection pressures to develop strategies for survival. These problems are acute for soil microorganisms which rely on the products of their extracellular enzymes for soluble C, N, and energy sources. Indeed, much of the organic C and N which enters the soil is of a polymeric nature and as such unsuitable for cell uptake until microbial depolymerases have reduced its molecular mass (Table 11-8). All this would not present a problem if we knew that extracellular enzymes survived in soil for long enough to detect and degrade their exogenous substrates which are not only discontinuous in space, but also in time. However, enzymes added to soil or stimulated within it do not diffuse freely in the soil aqueous phase and generally survive only briefly as active catalysts. Instead, extracellular enzymes are inactivated by adsorption (particularly to clays), denatured by a host of physical and chemical factors (e.g., pH, ionic composition of soil solution), or serve as substrates for proteolytic microorganisms. Therefore, the chances of an enzyme surviving long enough to detect its substrate (if it exists) are remote. Even if these obstacles are overcome, there is a good chance that the microenvironment of the substrate will be unsuitable for catalysis (e.g., pH, hydration level) or the substrate itself (e.g., cellulose) may be complexed with other organic polymers (e.g., pectins, lignin) or fixed to clays and be inaccessible to the enzyme.


443

In the apparently unlikely event of substrate catalysis, the product molecule must diffuse towards the source of the enzyme in order that it may function directly as a growth substrate, as an enzyme inducer, or as a chemoattractant. Of course, throughout its journey, the product or effector molecule, which is biochemically less complex than the original substrate, is also subject to adsorption, nonbiological destruction and metabolism by a host of microbial species. Thus, it would appear that soil microorganisms depending on the activities of their extracellular enzymes have what appear to be insurmountable difficulties. Nevertheless, it is obvious that an enormous volume of macromolecular and insoluble substrates enter the soil each year and that large numbers of microbial species are capable of detecting their presence and utilizing these macromolecules as growth and energy substrates. Therefore, we need to consider the strategies available to microorganisms to enable them to overcome difficulties which include fluctuating levels of growth-limiting, insoluble, nonuniformly distributed substrate in an environment that does not permit the free diffusion or even the survival of extracellular enzymes.

11-4.1

Nutritional Strategies for Soil Microorganisms

Do any of the conventional processes involved in the control of exoenzyme synthesis and secretion suggest an appropriate strategy for microbial response to exogenous substrate in soil? This is a difficult question to answer, as our knowledge of exoenzyme regulation in vitro is limited to a small number of enzymes (e.g., amylases, penicillinase, some proteases) and a few microorganisms (e.g., Bacillus, Neurospora, some yeasts). Detailed reports of exoenzyme regulation in soil are not available. The five mechanisms (constitutive synthesis/induced or derepressed secretion; constitutive synthesis/constitutive secretion; induced synthesis not repressible/ constitutive secretion; induced synthesis repressible/ constitutive secretion; and derepressed synthesis/constitutive secretion) have been discussed at length elsewhere (Cohen, 1980; Burns, 1983). Suffice to say that none of them represents a totally satisfactory strategy, in that they all at some stage depend upon a scavenging and speculative role for exoenzymes. Some elements of these five mechanisms are tenable in certain situations. For example, constitutive synthesis and secretion might be a profitable strategy for microorganisms growing under high nutrient, noncompetitive conditions encountered during the early stages of hostspecific phytopathogenicity. Thus, the pectic enzymes of Erwinia or Fusarium species may usefully be constitutive-that is, always produced, albeit at varying levels. Generally, however, the prolonged absence of substrate in the vicinity of the soil microorganism, in conjunction with continuous enzyme production, would rapidly lead to the death of that microorganism. A mechanism which permitted independent control of synthesis and secretion could represent an economic way of using certain polymeric

444

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substrates in soil. For instance, a polymer such as cellulose or protein will require a number of enzymic transformations before the resulting C or N source can be absorbed and metabolized. Rather than secrete a large number of different enzymes it would be better if the microorganism compartmentalized the various depolymerases in order that they are protected from dilution and activation. Therefore, in a sequence A - B C - D (e.g., cellulose - oligosaccharides - cellobiose - glucose), where A is the extracellular macromolecule and D is the soluble, low molecular mass product that can be taken up and utilized by the cell, only the enzyme converting A to B needs to be truly extracellular. The other enzymes in the sequence would be maintained at a constant level and protected within the various cell compartments (e. g., extracellular polysaccharide capsule, periplasmic space). They might be secreted only if the local concentration of substrate was high enough. Furthermore, the differential porosities of the various compartments suggest that they may act as molecular sieves controlling the passage of products with decreasing molecular weight from one enzyme-containing compartment to another. The success of any strategy based on induction will obviously depend on the presence of an inducer which must be soluble or at least in intimate contact with the microbial cell wall. Considering the insolubility and lack of diffusibility of many substrates, together with the fact that many substrates are not, in themselves, inducers, the disadvantages of induction mechanisms in soil are self-evident. Clearly, of the two induction mechanisms known, that which is subject to repression gives the microorganism more flexibility and is likely to be more efficient-but even then, contact with the inducer is an essential element of the process. There exists the possibility that low levels of constitutive enzymes release the inducer molecule from the exogenous substrate which, in turn, induces higher levels of enzyme synthesis and secretion. This could be described as the "Noah's Ark strategy" whereby the cell sends out its emissaries (i.e., intermittent pulses of enzyme at low concentrations) and awaits the return of a reporter molecule which informs the cell that conditions are favorable (i.e., high substrate levels exist) for exoenzyme synthesis. However, the constitutive enzyme would still be rapidly inactivated and, in the prolonged absence of substrate, the death of the microbial cell is inevitable. Repressed/derepressed synthesis (i.e., no inducer) does not seem a suitable strategy in soil because derepression due to C, N or S catabolite limitation would be the rule rather than the exception. Indeed, in vitro this mechanism may not be any different from constitutive synthesis. Of course, it is probable that species within microbial communities and strains within species have different regulatory mechanisms for the same extracellular enzyme or a range of affinities for the attraction and uptake of effector molecules. Such properties would ensure that a wide range of options were available to a species or to a community. Other possibilities exist for increasing the likelihood of a successful (and efficient) microbial response to substrates in soil and these are considered briefly.

445


1. Enzymes arising from damaged, dead, and lysed cells within a population. This will mean that periodically enzymes are released into the extracellular environment and, even if they fail to react with a substrate, it would not matter to the remaining viable but resting cells. 2. Enzymes only being induced following an accumulation of effector molecules at the cell surface. It has been suggested (Payne, 1980), that effective chemotaxis may be a response to a concentration of bound chemoattractants at the cell wall rather than to individual molecules. 3. Microorganisms creating chemical or physical changes in their immediate environment such that the nonbiological cleavage of a macromolecule occurs giving rise to an effector molecule. These may be less energetically demanding than exoenzyme production and could include pH fluctuations due to low levels of respiration (C0 2 production) or proton extrusion. 4. Enzyme production may also be mediated by external physical and chemical factors, such as pH or ionic composition and concentration. 5. Immobilized soil enzymes may act as stable catalysts for the detection of potential substrates. In the context of this essay, this last possibility will be considered in detail. The major steps in a nutritional strategy involving microbes and immobilized extracellular soil enzymes are depicted in Fig. 11-3. Molecule, S, because of its size, insolubility, association with other substrates, adsorption to soil constituents, or potential toxicity, is unsuitable in its present form as a growth substrate. Moreover, if S is insoluble or adsorbed to a soil surface, a distantly located (in microenvironment terms) microorganism has no way of detecting its presence other than by direct contact following random movement. The immobilized enzyme (soil colloid-enzyme complex), Ee , is protected from inactivation and proteolysis and is certainly not subject to the metabolic control exerted on its microbial counterpart. In other words, Ee is an indigenous constituent of soil capable of responding rapidly to its specific substrate molecule, S. Different enzymes catalyzing the same reaction or the same enzyme existing in different associations with the colloid, may give certain enzymes a

Humic polymer or clay mineral

Microbial cell

- - p -I -- E /

/

/

/

m

Fig. 11-3. A generalized nutritional strategy for soil microorganisms involving clay- and humic-enzyme complexes. Ec, enzyme associated with humic matter or clay; Em' microbial enzymes; S, exogenous substrate; P, product or effector molecule (Burns, 1983).

446 (a)

BURNS

r

jllobiosel

iJ-glucosidase

""

A

IglucOsel~

Glucose utiliser

Humic support

r

(b)

urea~

r

[i§]

/

'"lammonial~ A ~Il

Nitrosomonas sp.

(c)

IstarcN....

/ amylase

..

"

~

___amylase ~

'" R ' ,

cell

Imaltosel~

Starch degrader

Fig. 11-4. Specific examples of a strategy involving clay- and humic-enzyme complexes in the utilization of exogenous substrates by soil microorganisms (Burns, 1983).

range of affinities with regard to each substrate concentration (Nannipieri et al., 1982). The product, P, of the reaction is metabolized directly by an adjacent cell or diffuses to a remote cell. If P is at an appropriate concentration (and therefore indicating the same for S), it may act as an enzyme inducer for the microorganism and/or as a stimulus for chemotaxis. The advantages of P being the trigger or effector molecule is that the microorganism only produces extracellular enzymes if its substrate is present. Let us consider how this strategy could work for microorganisms in a soil microenvironment by using specific examples of enzymes and substrates. Firstly, an example involving soil-i3-D-glucosidase complexes (Fig. 11-4a): this immobilized enzyme is able to catalyze the hydrolysis of cellobiose and has been described in soils and humic extracts (Batistic et al., 1980; Hope et al., 1980). The glucose produced is obviously an excellent C source as well as being a chemoattractant for a great many microorganisms. In addition, more complicated relationship may involve regulation of microbial enzymes through catabolite repression, as well as the acceler-


447

ation of cellulolysis by the removal of an end-product inhibitor (cellobiose) of the cellulases. fj-D-Glucosidase will also release cellotriose and possibly even higher oligomers from cellulose which may act as cellulase inducers in some microorganisms. Urease is well known to form stable complexes with soil humic matter (Burns et aI., 1972; Nannipieri et aI., 1974; Pettit et aI., 1976) and can effect the rapid hydrolysis of urea to CO 2 and NH4. Nitrifying chemoautotrophs such as Nitrosomonas species would obviously benefit from being in close proximity to a stabilized extracellular urease enzyme and, would not need to possess a ureolytic capacity of their own (Fig. 11-4b). Furthermore, released NH4 would be retained at the anionic surface of humates and clays. Other immobilized ammonia-producing enzymes (e.g., asparaginase, amidase, and D-glutaminase) may serve a similar function. Carbon dioxide fixation by autotrophs and its involvement in methanogenesis suggest further relationships between microorganisms and immobilized soil urease. In the third example, (Fig. 11-4c) , which is closest to the model (Fig. 11-3), maltose acts as an inducer for amylase secretion. Therefore, soil immobilized amylase (Pancholy & Rice, 1973) could serve as a stable detector of starch, produce the inducer disaccharide, and initiate the microbial degradation of the substrate. 11-5

A NEW EXPERIMENTAL APPROACH

In an attempt to investigate the relationships between microbes, extracellular enzymes, and soil, we have recently developed a method for the study of exoenzyme activities and microbial growth in model soil environments (Hope & Burns, 1983, 1985). This technique uses "barrierring plates" to follow the rate and extent of diffusion of enzymes. Barrierring plates are made by: (i) removing a central core from an agar plate and adding an enzyme solution to the well; and (ii) removing a ring of agar external to the central well and replacing it with agar less the C source or agar containing various concentrations of soil or soil components. For studying microbial growth across barrier rings, a central microbial inoculum replaces the well. Using two cellulolytic enzymesone definitely extracellular (endoglucanase) and one that can function both intracellularly and extracellularly (fj-D-glucosidase)-we were able to demonstrate the inhibition of enzyme diffusion by bentonite clay, a silt loam soil and a colloidal fraction (clay + humates) of the soil. In contrast, sand and kaolinite clay had no effect on diffusion rates. A study of the radial growth rates of cellulolytic actinomycetes and fungi revealed a more complex relationship. A Streptomyces sp. was inhibited by bentonite, unaffected by kaolinite and sand, and actually stimulated by soil and its colloidal fraction. However, if the organic and inorganic carbon were removed from the soil, growth of the streptomycete was inhibited. Trichoderma viride growth was inhibited by bentonite and sand,

448

BURNS

and somewhat stimulated by kaolin and soil. It is hoped that further research using techniques such as this will shed more light on the relationships between soil surfaces and extracellular enzymes and their substrates. 11-6 SUMMARY AND CONCLUSIONS Microbial activity in soil is strongly influenced by the clays and humates which attract organic substrates, metabolites, inorganic ions, and water films to their surfaces. Thus, smectite clays and colloidal humic matter, with high unit surface areas and high ion exchange capacities, assume an importance far in excess of their percentage contribution to the total soil mass. Extracellular enzymes (along with proteins, peptides, and amino acids) are rapidly sorbed at clay and organic surfaces and this generally results in a partial or total loss of activity. The different mechanisms of adsorption (e.g., ion exchange, H bonding, van der Waals forces), the site of adsorption (e.g., on or within three-dimensional clays and humates, at the face or edge of clay lattices), the nature of the receiving surface (e.g., resident ion population, level of hydration), the properties of the ambient microenvironment (e.g., pH, ionic composition) together with the physicochemical characteristics of the enzyme (e.g., pI, molecular mass), will all influence the residual activity and stability of the complexed enzyme. It is apparent that a significant proportion of the immobilized enzyme in soil becomes associated with the humic fraction, not by adsorption or even entrapment, but by covalent bonding during humic matter genesis. This active and extremely stable complex can be extracted from soil and characterized, and can also be synthesized by enzymatically copolymerizing aromatic constituents of humus (e.g., L-tyrosine, resorcinol, and pyrogallol) and enzymes (e.g., fj-D-glucosidase, urease). Humic-enzyme analogues have many of the properties of their naturally occurring counterparts and can even be implanted into soil where they will produce a sustained elevation in immobilized enzyme activity. A high proportion of the organic carbon and nitrogen entering the decomposer cycle is polymeric, insoluble and has a high molecular mass and the necessity for extracellular enzymes is obvious. However, the conventional mechanisms of extracellular enzyme regulation (Le., constitutive, induced, and derepressed synthesis and secretion), elucidated by the study of pure cultures in vitro, do not suggest successful strategies for exogenous substrate utilization in soil. In this chapter, other strategies were considered, including one which proposes a role for clay-enzyme and, in particular, humic-enzyme complexes. Finally, in the absence of sophisticated probes to study the complex properties of the microbial microenvironment (Stotzky & Burns, 1982), a new technique is presented which may help to shed some light on the function of enzymes in soil microbial ecology.

449


ACKNOWLEDGMENT I acknowledge the support of the Agricultural and Food Research Council, the Natural Environment Research Council, and the European Economic Community, who financed many of the studies discussed in this chapter.

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----, and ----. 1985. The barrier-ring plate technique for studying extracellular enzyme diffusion and microbial growth in model environments. J. Cen. Microbiol. 131:1237-1243. Hughes, J. D., and C. H. Simpson. 1978. Arylsulphatase-clay interactions. II. The effect of kaolinite and montmorillonite on arylsulphatase activity. Aust. J. Soil Res. 16:35-40. Ladd, J. N., and J. H. A. Butler. 1975. Humus-enzyme systems and synthetic, organic polymer-enzyme analogs. p. 143-194. In E. A. Paul and A. D. McLaren (ed.) Soil biochemistry, Vol. 4. Marcel Dekker, New York. Maignan, C. 1982. Activite des complexes acides humiques-invertase: Influence du mode de preparation. Soil BioI. Biochem. 14:439-445. Makboul, H. E., and J. C. C. Ottow. 1979. Alkaline phosphatase activity and the Michaelis constant in the presence of different clay minerals. Soil Sci. 128:129--135. Malcolm, R. E. 1983. Assessment of phosphatase activity in soils. Soil BioI. Biochem. 15:403408. Marshall, K. C. 1976. Interfaces in microbial ecology. Harvard University Press, Cambridge, MA. Marshman, N. A., and K. C. Marshall. 1981a. Bacterial growth on proteins in the presence of clay minerals. Soil BioI. Biochem. 13: 127-134. ----, and ----. 1981b. Some effects of montmorillonite on the growth of mixed microbial cultures. Soil BioI. Biochem. 13: 135-14l. Martin, J. P., and D. D. Focht. 1977. Biological properties of soils. p. 115-169. In L. F. Elliott and F. J. Stevenson (ed.) Soils for management of organic wastes and waste waters. Soil Science Society of America, American Society of Agronomy, and Crop Science Society of America, Madison, WI. ----, Z. Filip, and K. Haider. 1976. Effect of montmorillonite and humate on growth and metabolic activity of some actinomycetes. Soil BioI. Biochem. 8:409-43l. Mattson, S. 1932. The laws of soil colloid behavior: VII. Proteins and proteinated complexes. Soil Sci. 23:41-72. McLaren, A. D. 1954a. The adsorption and reactions of enzymes and proteins on kaolinite. J. Phys. Chern. 58:129-137. ----. 1954b. The adsorption and reactions of enzymes and proteins on kaolinite. II. The action of chymotrypsin and lysozyme. Soil Sci. Soc. Am. Proc. 18: 170-174. ----, A. H. Pukite, and I. Barshad. 1975. Isolation of humus with enzymatic activity from soil. Soil Sci. 119:178-180. ----, and J. Skujins. 1968. The physical environment of microorganisms in soil. p. 3-24. In T. R. G. Gray and D. Parkinson (ed.) The ecology of soil bacteria. Liverpool University Press, Liverpool, UK. Nannipieri, P., B. Ceccanti, S. Cervelli, and C. Conti. 1982. Hydrolases extracted from soil: kinetic parameters of several enzymes catalyzing the same reaction. Soil BioI. Biochem. 14:429-432. ----, ----, ----, and P. Sequi. 1974. Use of 0.1 M pyrophosphate to extract urease from a podzol. Soil BioI. Biochem. 6:359-362. Ottow, J. C. G., H. E. Makboul, and J. C. Munch. 1983. Effect of pedogenic clay minerals on the kinetics (Km and Vmax) of alkaline and acid phosphatase. Z. Pflanzenernaehr. Bodenkd.146:3-12. Pancholy, S. K., and E. L. Rice. 1973. Carbohydrases in soil as affected by successional stages ofrevegetation. Soil Sci. Soc. Am. Proc.37:227-229. Pettit, N. M., L. J. Gregory, R. B. Freedman, and R. G. Burns. 1977. Differential stabilities of soil enzymes: assay and properties of phosphatase and arylsulphatase. Biochem. Biophys. Acta 485:357-366. ----, A. R. J. Smith, R. B. Freedman, and R. C. Burns. 1976. Soil urease: activity, stability and kinetic properties. Soil BioI. Biochem. 8:479--484. Pflug, W. 1982. Effect of clay minerals on the activity of polysaccharide cleaving soil enzymes. Z. Pflanzenernaehr. Bodenkd. 145:493-502. Ross, D. J. 1983. Invertase and amylase activities as influenced by clay minerals, soil-clay fractions and topsoils under grassland. Soil BioI. Biochem. 15:287-293. Rowell, M. J., J. N. Ladd, and E. A. Paul. 1973. Enzymically active complexes of proteases and humic acid analogs. Soil BioI. Biochem. 5:699-703. Sarkar, J. M., and R. C. Burns. 1983. Immobilization of /3-D-glucosidase and /3-Dglucosidase-polyphenolic complexes. Biotechnol. Lett. 5:619-624. ----, and ----. 1984. Synthesis and properties of /3-D-glucosidase-phenolic copolymers as analogues of soil humic-enzyme complexes. Soil BioI. Biochem. 16:619-625.

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Stotzkv, G. 1980. Surface interactions between clay minerals and microbes, viruses and soluble organics, and the probable importance of these interactions to the ecology of microbes in soil. p. 231-247. In R. C. W. Berkeley et al. (ed.) Microbial adhesion to surfaces. Ellis Horwood, Chichester, UK. ----, and R. G. Burns. 1982. The soil environment: clay-humus-microbe interactions. H. Slater (ed.) Experimental microbial ecology. p. 105-133. In R. G. Burns and Blackwell Scientific Publishing, Ox ord, UK. Taylor, W. I., and A. R. Battersby. 1967. Oxidative coupling of phenols. Marcel Dekker, New York. Theng, B. K. G. 1979. Formation and properties of clay-polymer complexes. Elsevier Science Publishing Co., New York. Verma, L., J. P. Martin, and K. Haider. 1975. Decomposition of carbon-14-labelled proteins, peptides, and amino acids; free and complexed with humic polymers. Soil Sci. Soc. Am. Proc. 39:279-284. Waksman, S. A. and R. K. Iyer. 1932. Contribution to our knowledge of the chemical nature and origin of humus. Soil Sci. 34:43-69.

l.