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V. Environmental Factors Affecting Proteolysis

V. Environmental Factors Affecting Proteolysis

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PROTEIN TRANSFORMATION IN SOIL



365



proliferation of the more proteolytically active microorganisms, assuming that

the proper nutrients are available. Nonproteolytic organisms may be affected by

temperature in an analogous manner. Although it is possible that they could

compete with protein decomposers, it may be that they also act to break down

materials to which proteins are bound, freeing substrate for the proteolytic microbes. Finally, heating could increase the rates of enzymatic and chemical

reactions involved in the decay process. There is, no doubt, an upper limit on

stimulatory phenomena that is determined by the heat tolerance of the soil population and the inactivation temperature of the proteases. As temperatures in

excess of 60°C are required to denature soil proteinases (Mayaudon et al., 1975;

Ladd and Butler, 1972), frequent thermal inactivation in nondesert habitats is

improbable. Temperature regulates decomposition indirectly through its effects

on oxygen tension and moisture. In addition, a number of biological and chemical processes occur coincidently with, and not always because of, the onset of

warmer summer temperatures, and these processes have their effects on the

degradative capabilities of a soil.

B. MOISTURE



Moisture is essential for the mineralization of proteins. Plants and microbes

need water for growth and metabolism. Proteases, in order to function, must be

in aqueous solution or, if bound to a solid surface, at the interface of an aqueous

solution. Complete or partial dissolution of a protein substrate helps in its hydrolysis as more of its surface area becomes accessible to proteolytic enzymes. It is

not surprising, then, that there is a strong correlation between protein hydrolysis

and soil moisture (Kuprevich and Shcherbakova, 1971; Cahenzli and Staffeldt,

1976; Khaziyev, 1977; Klein, 1977). In areas having high temperatures and little

rainfall, conditions typical of deserts and prairie summers, proteolysis is severely

restricted (Klein, 1977; O’Brien, 1978).

C . REDOX CONDITIONS



Most of the current data suggest that the breakdown of protein is facilitated by

the presence of oxygen, although hydrolysis probably goes on at reduced rates

under anaerobic conditions. The proteolytic activity of aquatic organisms has

been shown to decline with decreasing oxygen tension (Sugahara et al., 1974).

Ross and Cairns (1978) found that protein zein decomposed much faster at a

depth of 5 cm than at 15 cm in New Zealand soil, and that the proteolytic

capability of mineral soils in Karelia decreased with depth in the soil profile. Peat

bog soils from the same part of the Soviet Union had hydrolytic activities



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MICHAEL J. LOLL AND JEAN-MARC BOLLAG



throughout their profiles (Katznel’son and Ershov, 1958). This would indicate

the presence of anaerobes in the lower horizons or the permeation of peat soils by

oxygen.

D. SOILPROPERTIES



The type, characteristics, and composition of the soil all influence the behavior of a soil enzyme. Peat bog soils, for instance, have higher proteolytic activities than meadow soils, sod podzols, and iron podzols (Katznel’son and

Ershov, 1958; Kuprevich and Shcherbakova, 1971). The large amount of organic

matter in peat probably acts as a favorable nutrient source for the development of

protein hydrolyzers and stabilizes exocellular proteases. Organic matter content

and protease action are strongly correlated (Kuprevich and Shcherbakova, 197 1;

Ladd and Butler, 1972; Mayaudon and Sarkar, 1974). Proteases are also quite

active in agrillaceous soils (Katznel’son and Ershov, 1958).

Soil reaction is an important consideration in studying proteolysis. Chunderova (1970) was able to raise the relatively low proteolytic activity of acid soils

(pH 4.2-4.8) by liming them. Activity peaked at a pH of 6.3 and did not change

with further increases in pH. In soils infested with Trichoderma viride, fungal

proteinases are functional only at pH values between 5.5 and 6.5; pH 6.0 is

optimum for substrate hydrolysis (Rodriguez-Kabana et al., 1978). The pH has

an effect on the kind as well as the activity of soil proteases. Alkaline proteases

are uncommon or undetectable in acid soils (Ambroz, 1970).

Ladd and Butler (1972) could not correlate proteolysis with soil pH, but they

could link it to other soil properties. The activity of a peptidase specific for Z phenylalanylleucine was related to cation-exchange capacity (CEC), clay content, and surface area, whereas that of a protease specific for benzoylargininamide was correlated with soil surface area, clay content, CEC, total nitrogen,

and organic matter content. Mayaudon et al. (1975) reported a positive correlation between organic matter content and the proteolytic capacity of a soil extract,

and a negative correlation between activity and clay content. These results are

interesting because they indicate that adsorption phenomena between proteases

and organic and mineral substances may take place in soil. Ambroz (1966) has

briefly discussed the affinities of neutral and alkaline proteases for different

clays, pointing out that protein adsorption is more pronounced with montmorillonite and illite, which have high CECs, than with kaolinite, which has a

lower CEC. Reactions with the inorganic fraction of soil decrease, but do not

eliminate, the ability of proteases to attack proteins (GHith and Thomas, 1979).

Protein adsorbed into the clay matrix is better protected from decomposition than

surface-adsorbed protein. It is possible, however, that some smaller proteases

can penetrate the clay layers and hydrolyze proteins and peptides. Allophane at a

pH of 5 is a strong inhibitor of pronase, as are montmorillonite and halloysite to



PROTEIN TRANSFORMATION IN SOIL



367



lesser degrees at the same pH (Aomine and Kobayashi, 1964). Inhibition occurs

even when montmorillonite is partially saturated with protein substrate (Griffith

and Thomas, 1979). Reduction in activity is probably caused by conformational

changes in the enzyme when it binds to clay.

In some cases, soil minerals promote proteolysis. When lysozyme was adsorbed to bentonite and incubated with pure cultures of Pseudomonas or Fluvobacterium species, the bacteria decomposed the complexed protein faster than

the uncomplexed protein. Clay apparently can concentrate protein on its surface,

whereas in solution the substrate is too dilute for rapid hydrolysis (Estermann and

McLaren, 1959). Soil mineral matter can, on occasion, provide sites for enzyme-substrate interactions (McLaren and Estermann, 1956).

E. NATURALLY

OCCURRING

ORGANIC

COMPOUNDS



Polyphenolics, specifically plant tannins and humic acids, are the most studied

organic compounds in terms of their reactions with proteins and enzymes in soil.

Tannins have long been known to lessen enzymatic activity (Goldstein and

Swain, 1965) and exert a depressive effect on proteolysis (Basaraba and Starkey,

1966), either changing the shape of the protease or protecting the substrate from

hydrolysis. Dihydroxphenyalanine (DOPA) melanin, found in fungi, inhibits

protease (Kuo and Alexander, 1967), and humic and fulvic acids may increase or

decrease the potency of proteolytic enzymes (Ladd and Butler, 1969b). The

particular effect depends on the protease in question and the molecular weight

(Butler and Ladd, 1971) and carboxyl content (Butler and Ladd, 1969) of the

humic acid. Pronase, trypsin, carboxypeptidase A, and subtilopeptidase A have

been inhibited by soil humic acids, and this inhibition varied for some of the

proteinases depending on the substrate used (Ladd and Butler, 1969b). The

action of Pronase on peptides was more sensitive to humic acid than was its

action on protein (Ladd and Butler, 1969a). Papain, ficin, and thermolysin have

been stimulated by humic acids, and phaseolain and chymotrypsin were unaffected by similar treatments (Ladd and Butler, 1969b). Butler and Ladd (1971)

theorize that humic substances, especially those with large molecular weights

(greater than 30,000), are rigid, and when they bind to proteases they distort the

enzymes’ structures, thereby changing their reactivities. The binding sites involve the carboxyl groups of the humic acids (Butler and Ladd, 1969).

Plants and animals synthesize their own protease inhibitors for physiological

regulation. Little is known about the survival of these compounds in soil, but it is

possible that they could affect proteolysis. Natural inhibitors are common in

mammals, potatoes, beets, and soybeans (Vogel et al., 1968).

Proteases are subject to autodigestion and may act as their own substrates

(Cunningham, 1965). It is not known whether reaction with soil components



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MICHAEL J. Lou AND JEAN-MARC BOLLAG



prevents this. Calcium stabilizes some proteolytic enzymes (Cunningham, 1965)

and may protect them from self-digestion in soils if that element is prevalent.



Plant roots exude substances that can be used by microorganisms for growth.

As a result, protease activity is higher in the rhizosphere than in the soil outside

of this area, and even more activity is evident on the rhizoplane (Vagnerova and

Macura, 1974~).Conditions at the root surface are optimal for nutrient assimilation by microbes, but this is not the only reason for the superior protease activity

of the rhizoplane. Roots, like clays and humic acids, have exchange capacities

and can bind enzymes, reacting with them electrostatically or actually taking

them up into the root’s free space (McLaren et al., 1960). Sorption reduces the

efficacy of the proteases (Vagnerova and Macura, 1974b), but the amount of

bound protease can be so great that the entire activity of the root surface is well

above that of the surrounding soil. The sorption of proteinases by roots could be

another way in which enzymes are preserved in soil (Vagnerova and Macura,

1974~).

G . ENZYMEACTIVITIES



The physiological processes of plants and nonproteolytic bacteria and fungi

may coincide with the breakdown of protein. There are strong relationships

between protease activity and the activities of sulfatase (Ladd and Butler, 1972),

DOPA oxidase (Mayaudon et al., 1975), invertase, amylase, and catalase (Ambroz, 1970), implying that protein decomposers have a broad range of metabolic

abilities or that environments that favor proteolytic organisms also favor organisms which synthesize these other enzymes.H. AGRICULTURAL

PRACTICES



Farming produces fundamental alterations in the biochemistry of soils, and

planting methods, fertilization, and herbicide application can effect drastic

changes in protease activity. Plowing mineral soils can improve their proteolytic

activity (Romeiko, 1969), and in some cases cultivation decreases the nitrogen

content of a soil (Keeney and Bremner, 1964). Ammonium sulfate and potassium nitrate fertilization may be beneficial for protease synthesis (Bei-Bienko,

1970). Blagoveshchenskaya and Danchenko (1974) saw higher enzymatic activity in crop rotations than in a corn monoculture. When they added nitrogen,

phosphorus, and potassium fertilizers to a soil after manuring, protease activity



PROTEIN TRANSFORMATION IN SOIL



369



increased as compared with soils amended with only mineral fertilizers. Rates of

application of mineral nitrogen over manure had little influence on activity in the

monoculture but increased it by 20% in the rotation. Amending a Belgian soil

with manure diminished the numbers of proteolytic microbes but enhanced proteinase activity (Batistic and Mayaudon, 1978). Sewage sludge, in spite of its

heavy metal content, similarly can aid protein decomposition (Varanka et al.,

1976). Manures and sludges nurture soil microflora, add their own active microbial populations to soil, and may contain extracellular proteases stabilized by

organic matter (Varanka et al., 1976).

I. PESTICIDES



Pesticides often affect helpful as well as harmful organisms in the area of

application. For soil populations, these xenobiotics are beneficial or detrimental

depending on the concentration and type of pesticide, the persistence of the

compound in soil, and the composition and sensitivity of the microbial community. Eptam (S-ethyl-N,N-dipropylthiocarbamate),for example, stimulates soil

proteolysis at low concentrations but is inhibitory at higher levels (Cullimore and

Ball, 1978). The same study showed that bromacil [5-bromo-6-methyl-3 (1methylpropy1)-2,4-(lH,3H)-pyrimidinedione] fosters protein decomposition

whereas MCPA (2-methyl-4-chlorophenoxyacetic acid), diquat (1,l ’-ethylene-2,2’-dipyridylium dibromide), paraquat (1, l ’-dimethyl-4,4’-bipyridylium

ion), dicamba (3,6-dichloro-o-anisic acid), 2,4-D (2,4-dichlorophenoxyacetic

acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), and monuron [3-@-chloropheny1)- 1,1-dimethylurea] all reduce the hydrolysis of proteins. Dinoseb (2,4dinitro-6-sec-butyphenol)decreases the number of protein decomposers in soil

(Wainwright and Pugh, 1973). In Swedish soils, 2,4,5-T slightly promoted the

growth of proteolytic microorganisms (Torstensson, 1974), as did bentazon [3isopropyl- lH-2,1,3-benzothiadiazin-4-(3H)-one-2,2-dioxide]at a concentration

of 1 ppm (Torstensson, 1975). The fungicides thiram (tetramethylthioperoxydicarbonic diamide), verdasan (phenylmercuric acetate), and captan {N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide} in high concentrations

caused increases in soil ammonification (Wainwright and Pugh, 1973), indicating that ammonifying or proteolytic microbes (or both) were stimulated. Malathion (0,0-dimethyl-S-( l ,2-dicarbethoxyethyl)dithiophosphateis not harmful

to species of Bacillus and Pseudomoms (Stanlake and Clark, 1975), two proteolytic genera often found in soil. Proteinase activity is relatively unaffected by

the application of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazene)

(Ghinea, 1964). The clay and sand contents of two Belgian soils appeared to

govern the growth of les germes protiolytiques in the presence of three phenylcarbamates (Bellinck and Mayaudon, 1978). The pesticides encouraged ini-



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MICHAEL J. LOLL AND JEAN-MARC BOLLAG



tial growth in a clay soil, but bacterial development eventually peaked and

declined sharply over time. This pattern was also evident in a sandy soil to a

lesser degree, except that monophenylcarbamateproduced an initial decrease in

the number of protein hydrolyzers. This inhibition was followed by an increase

in growth which gradually leveled off. The growth phase and decline was much

shorter lived in the sandy soil than in the clay soil. Bellinck and Mayaudon

(1978) see this as the result of herbicide absorption by soil colloids with slow

herbicide release for microbial nutrition.

Pesticides occasionally exhibit synergistic phenomena with fertilizers in altering enzymatic operations. Following an initial surge in protease synthesis, soil

treated with both urea and paraquat in the laboratory showed a 42% loss of

activity after 1 month (Murakami et al., 1969). The addition of dalapon and urea

to samples caused an immediate reduction in activity of 11%. Plots in the field

lost 41-56% of their prior activity with dalapon-urea combinations and 46%

activity with the addition of urea and paraquat (Namdeo and Dube, 1973b).

Soil peptidases and proteases are not equally susceptible to fumigation. Peptidases capable of hydrolyzing Z-phenylalanyl leucine and benzoylargininamide

were not affected by methyl bromide, whereas caseinases from the same sterilized soil were markedly inhibited by that compound. The activities of the

enzymes were partially suppressed by chloropicrin and then rebounded, exceeding the activities of untreated controls (Ladd and Butler, 1966). It has been

proposed that the deactivation of caseinase comes from its binding to clay and the

destruction of unbound caseinase. The subsequent recovery of activity resulted

from a microbial flush, which happens when surviving organisms begin to develop on the lysed cell material produced by pesticide treatment. In fact, fumigation

probably releases relatively large amounts of protein from the microbial biomass,

and the dead cells then provide carbon and nitrogen for the few unharmed cells.

This conclusion was supported by a study done by Wainwright and Pugh (1975),

who extracted more free amino acids from soil after, than before, fumigation.



VI. TRANSFORMATION AND BINDING OF PROTEIN

IN SOIL

Protein undergoes different transformations in the soil environment (Fig. 1).

Quantitative studies are practically nonexistent, but a large proportion of the

protein introduced into soil is broken down into peptides, which in turn are

hydrolyzed to amino acids. The amino acids are metabolized by the soil microflora or react with soil constituents. Surviving proteins may react with soil

minerals, lignins, tannins, melanins, and humic acids. The aggregates that proteins form with these materials protect peptides from microbial action and weath-



37 1



PROTEIN TRANSFORMATION IN SOIL

MICROORGANISMS

PLANTS

ANIMALS



PLANT ROOTS



1

-



HUMIC

SUBSTANCES



PROTEINS



t



CLAYS



PEPTIDES



AMINO ACIDS



NH3



FIG. 1. Protein transformation in soil.



ering (Haworth, 1971). Complexed protein is still susceptible to some biodegradation, but decays at a much slower rate than does uncomplexed protein

(Estermann et al., 1959). The evidence for clay and humoprotein complexes is

indirect, and there is still some controversy about whether or not intact protein

can exist in soil over long periods of time. Infrared spectra of some humic acids

and straw extracts show what appear to be protein amide I and amide I1 peaks

which disappear after treatment with 6 N HCI (Goulden and Jenkinson, 1959;

Stevenson and Goh, 1971; Boyd et al., 1980). Acid hydrolysis of soil releases

amino acids, which comprise 20-50% of the total nitrogen content (Bremner,

1949, 1955, 1965; Cheng and van Hove, 1964; Piper and Posner, 1968). Humic

acids contain up to 15% a-amino nitrogen (Piper and Posner, 1968).

Some nonprotein amino acids synthesized by bacteria have been occasionally



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V. Environmental Factors Affecting Proteolysis

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