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VI. Transformation and Binding of Protein in Soil
PROTEIN TRANSFORMATION IN SOIL
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
MICHAEL J. LOLL AND JEAN-MARC BOLLAG
detected in soil, but these compounds do not seem to be a large proportion of the
total amino acid content and may even be artifacts in some instances (Stevenson,
1956; Bremner, 1965). The presence of peptides in humic acid was indicated by
Piper and Posner (1968) and by Sowden (1966). Peptide-like compounds from
soil have been found to react with phenylisothiocyanate and fluorodinitrobenzene, two compounds that characteristically react with the terminal groups of
proteins (Sowden, 1966). Because of the heterogeneity of soil material, conclusions about such findings can only be speculative, as it is not known how soil
constituents may interfere with reactants for proteins. Classical tests for protein
content (i.e., the Lowry and Biuret methods) have given low values for the
peptide composition of humic acids, but soil polymers may serve to mask protein
from detection by conventional means.(Ladd and Butler, 1966). The vast amount
of research performed in soil enzymology also lends support to the notion that
proteins exist in a stabilized form in soils, but clear data are lacking.
Because of their cation-exchange capacity and relatively large surface area,
clays act as absorbents of positively charged organic material. Association with
clay confers upon proteins some resistance to degradation (Ensminger and
Gieseking, 1942; Pinck and Allison, 1951; Pinck et ul., 1954; McLaren, 1954b;
Lynch and Cotnoir, 1956). The effect of clay is attributed to its inhibition of
proteolytic enzymes, its shielding of protein from enzymatic attack, or a combination of the two processes. In general, bentonite-protein complexes are more
resistant to hydrolysis than are kaolinite- and illite-protein complexes (Lynch
and Cotnoir, 1956; Estermann et al., 1959). Bentonite has a higher cationexchange capacity than kaolinite and illite, and more protein is adsorbed to
montmorillonitic materials. X-Ray diffraction shows that proteins can form interlamellar monolayers in montmorillonite and that increasing the amount of
protein added to clay suspensions results in the formation of bilayers (Talibud e n , 1950).
Protein is best shielded by a clay lattice when it forms a single layer on the clay
surface, a monolayer produced when the weight of the protein in the complex is
roughly 8% of that of the montmorillonite. The upper portions of protein multilayers are more susceptible to degradation by proteolytic enzymes (Pinck et al.,
1954). As excess protein in the clay structure is hydrolyzed, the layers contract
and may help to protect the thin coating of adsorbed protein (Estermann et al.,
1959). Aggregates of lysozyme and bentonite or kaolinite are less vulnerable to
decomposition when dried and then rewetted (Estermann et ul., 1959); this
treatment may wash superfluous protein out of the matrix. The conformation of
the protein in a clay complex either speeds up or slows down its hydrolysis.
PROTEIN TRANSFORMATION IN SOIL
Denatured lysozyme is broken down more quickly in combination with kaolinite
than is the native globular form in a free or complexed state (McLaren, 1954b).
Inorganic compounds other than clay may prolong the survival of proteins in
soil. Free iron and aluminum oxides can be chelated by organic compounds
(Greenland, 1965) and seem to prevent decay, perhaps by changing the shape of
the organic substrate and making it less suitable for enzymatic attack. The amino
groups of humus seem to be involved in chelating metal ions, forming complexes
that help to stabilize clays (Brydon and Sowden, 1959).
Montmorillonite and other soil minerals inhibit biodegradation but do not stop
it entirely. Species of Pseudomonas, Flavobacterium, and Bacillus exude proteases that can penetrate clay layers (Estermann and McLaren, 1959; Estermann
et al., 1959; Marshman and Marshall, 1981). McLaren and Estermann (1956)
theorized that proteases may be adsorbed to kaolinite and can move over it, in
some fashion, to react with substrates. Marshman and Marshall (1981) investigated the growth of protein-decomposing bacteria on clay-adsorbed proteins, and
from their data proposed a model based on four assumptions:
1. Protein binds to clay matrices at sites that are available to proteolytic
organisms as well as to sites that are not available to them
2. Protein binds strongly to both sites and is not readily released from them
3. Protein prefers to react with unavailable sites
4. The amount of protein bound to the two types of sites is determined by the
nature of the proteases present.
The problem of inhibition has led soil scientists to examine the factors which
affect the bonding of a protein or proteinase to clay. Proteins bind to clays very
quickly, and 90% of the protein added to mineral systems may be adsorbed in
only 3 minutes (McLaren, 1954b; Armstrong and Chesters, 1964). Reaction sites
on clays include not only the interlamellar surfaces but also the edges of the
platelets (Harter and Stotzky, 1973), depending on the peptide type and concentration. Adsorption may be in mono- or multilayers (Talibudeen, 1950). The
primary mechanism of binding is ionic and results from the cation-exchange
capacities of soils (Ensminger, 1942; Ensminger and Gieseking, 1939, 1941).
Amino, imidazole, and protonated carboxyl groups on proteins compete with
inorganic ions for sites on the CECs of clays (Ensminger, 1942; Armstrong and
Chesters, 1964; Schnitzer et al., 1980; Stefani and Sequi, 1978). Hydrogen
bonding may also be involved in the formation of protein-mineral aggregates
(McLaren, 1954a; McLaren e? al., 1958; Albert and Harter, 1973). It is possible
that the stresses induced by adsorption denature proteins as they attach to the clay
surface (Talibudeen, 1950), but this is a matter of some contention.
Maximal adsorption is sometimes observed when the pH of the clay suspension is equal to the isoelectric pH of the protein (McLaren, 1954a; Armstrong
and Chesters, 1964; Albert and Harter, 1973). At pH values above the isoelectric
MICHAEL J. LOLL AND JEAN-MARC BOLLAG
pH, the protein is negatively charged and repulsed from the mineral matrix. At
pH values below the isoelectric value, the protein is positively charged, and
although a greater affinity for the clay would be expected, this is not necessarily
the case. In fact, adsorption decreases with lower pH, a phenomenon variously
ascribed to competition between protein molecules and hydrogen ions for exchange sites (McLaren et al., 1958) and charge density effects. As the pH drops,
the number of positive functional groups on a peptide increases and fewer molecules are needed for satisfying the clay’s negative charges (Armstrongand Chesten, 1964). Protein adsorption to kaolinite, according to Albert and Harter
(1973), is not dependent on pH, and bonding in this instance must be nonionic.
Harter and Stotzky (1971) criticized the importance of the influence of pH in the
formation of protein-mineral complexes. First, they pointed out that even at pH
values above the iwelectric pH of a peptide there are still some positively
charged groups on a protein that are available for reaction with clays. Second,
they mentioned that the pH at the surface of a clay is not always the same as that
of the overall clay suspension, and the surface pH may be more favorable for
binding. More important factors governing adsorption appear to be protein molecular weight and the ion population on the clay surface. Proteins with higher
molecular weights are more likely to be adsorbed than those with low molecular
weights and evidently have a certain competitive advantage. The number of
positively charged functional moieties has a certain secondary influence, explaining discrepancies in the adsorption of materials of different weight. Catalase, for example, has a molecular weight more than twice that of casein
(25 1,OOO versus 121,0oO), yet the difference in their respective affinities for clay
is less than a factor of two. Casein probably has more positive charges at low pH
than does catalase, and proportionately less casein is required for saturation
(Harter and Stotzky, 1971).
Lysozyme adsorption has been related to the valence or ionic potential of
cations on the cation-exchange capacity and decreased with an increase in the
size of the ionic radius of the saturant. Ovalbumin adsorption on the same clay
was affected more by the pH of the suspension. Protein adsorption was found to
be highest for clays homoionic to hydrogen and decreased in the order H > Na >
Ca > A1 > La > Th. Proteins seem to compete better with monovalent ions for
clay exchange sites as divalent and trivalent cations are bound more strongly
(Harter and Stotzky, 1971). Only hydrogen ions behave differently. The presence of salts in clay suspensions discourages peptide adsorption and can desorb
bound organic matter (Harter and Stotzky, 1971).
B . POLWHFNOLIC-PROTEIN
Lignins, tannins, and melanins are common plant and fungal metabolites
found in leaf litter, crop residues, and soil and are the major components of
PROTEIN TRANSFORMATION IN SOIL
humic acids. Phenolic polymers such as these adsorb and react with proteins,
stabilizing them against enzymatic digestion. As with clays, stabilization is the
probable consequence of protease inhibition and protection of the substrate from
In 1932 Waksman and Iyer discovered the inhibitory effect of lignin upon
proteolysis and proposed that most of the organic nitrogen of soil was present in a
lignoprotein “humic nucleus.” Since that time tannins (Davies et al., 1964;
Basaraba and Starkey, 1966; Benoit et al., 1968; Lewis and Starkey, 1968),
melanin (Kuo and Alexander, 1967), humic acids (Lynch and Lynch, 1958), and
humic acid analogs (Verma ef al., 1975; Verma and Martin, 1976; Martin et al.,
1978; Martin and Haider, 1979) have been proven to retard protein breakdown. It
is now clear that the nature of soil nitrogen is quite complicated. This complexity
is compounded by the variety of interactions phenols and proteins undergo.
There are several mechanisms by which protein-phenol complexes are formed,
and the Occurrence of any one is regulated by certain environmental conditions.
Hydrogen bonding takes place between proteins and phenols and is probably a
rather common event in litters and soils. The bond which forms between substituted amides and phenolic hydroxyl groups is very strong and no doubt is
important in the production of resistant complexes. Strong hydrogen bonds also
occur with carboxyl groups (Loomis and Battaile, 1966; Ladd and Butler, 1975).
Hydrogen bonding would explain the widespread observation that tannin-protein
complexes synthesized at low pH are more resistant to decomposition than complexes produced at high pH (Davies et al., 1964; Basaraba and Starkey, 1966;
Benoit et al., 1968). It has been noted that hydrolyzable tannins do not provide
protein with as much protection as condensed, more aromatic ones do, and this
may be because of intermolecular hydrogen bonding within hydrolyzable tannins. Tannins of this kind would be less likely to bind to proteins (Ladd and
Butler, 1975). Acid forest soils with relatively large amounts of condensed
tannins would probably be the best areas for the formation of stabilized proteins
(Basaraba and Starkey, 1966).
Covalent bonding is another widespread mode of reaction in soil and is responsible in part for the origin of humic and fulvic acids. Proteins, peptides, amines,
and amino acids react with phenols, giving rise to polymeric products. These
oxidations can be strictly chemical or mediated by phenolase enzymes (Loomis
and Battaile, 1966; Taylor and Battersby, 1967; Ladd and Butler, 1975). Catechol forms a p-aminohydroquinone with primary amines in the presence of a
chemical oxidant (Mason, 1955). Theis (1945) has hypothesized that proteins
react with quinones via meta linkages between the ring and the free amino groups
of the peptide backbone or of lysine. Glycine does not combine with phenols as
quickly as glycylglycine does, and the tripeptide reacts fastest of all, in what is
referred to as the peptide effect (Mason, 1955). The bond between the aromatic
ring and the protein amino group is thought to be the most impervious to enzymatic action (Ladd and Butler, 1966; Robert-Gero et al., 1967), although a species
MICHAEL J. LOLL AND JEAN-MARC BOLLAG
of Achromobucter has been isolated that can utilize N-(0-carboxypheny1)glycine.
The same species, however, was unable to metabolize N-phenylglycine, N-knitrophenyl)glycine, or N-@-hydroxypheny1)glycine. Clearly the susceptibility
to degradation is a function of other moieties (Ladd, 1964). Covalent reactions
may also occur between free amino groups and humic acid carboxyls and other
Ionic interactions between organic compounds occur in soil between basic and
acidic functional groups. Associations of this kind are highly pH dependent and
are similar to mineral cation exchange. Ladd and Butler (1971) and Butler and
Ladd (1969) implied that the amino groups of enzymes react ionically with
humic acid carboxyls. The activity of the enzyme can be greatly altered by this
Finally, roots are capable of adsorbing proteinaceous matter. Some proteins
can penetrate the outer spaces of barley roots, which have a negative charge
(McLaren et al., 1960). Whether plants can actually utilize native proteins is
unknown, but roots may be another “shelter” against enzymatic hydrolysis.
Covalent and coulombic processes are always at work in soil and it would not
be surprising if a protein were bound to organic and inorganic matter by two or
three mechanisms at the same time. The heterogeneity of bonding is one reason
that proteins are stabilized in soil. A battery of enzymes would be needed to
destroy the various linkages between peptides and soil components, and this is
why protein or portions of protein can remain intact in soil.
VII. ECOLOGICAL AND AGRONOMIC IMPORTANCE
OF PROTEIN TRANSFORMATION
Protein transformations in soil have a considerable influence on soil ecology,
agriculture, and public health. From an ecological point of view, protein is
important, as discussed previously, in its contribution to soil structure and
through its reactions with clays and naturally occurring phenols during humus
formation. Apparently a number of extracellular microbial enzymes are stabilized by their complexation with soil components and can survive conditions
inimical to intact cells (Ladd and Butler, 1975). Some of these enzymes act for
long periods in degrading the basic constituents of plant and animal residues.
Others such as ureases, amidases, and phenoloxidases transform fertilizers and
alter pesticides and their intermediates and thus have agricultural significance.
PROTEIN TRANSFORMATION IN SOIL
Protein mineralization affects public health because of its role in the biodegradation of industrial and sewage wastes (Gray and Biddlestone, 1974). In
addition, the tendency of protein to bind with soil substituents could pose problems for sanitation. It is feared that the protein coats of enteric viruses are sorbed
to clays in sludge-amended soils. The sorbed viruses may later be released
(especially if there is an increase in the ionic strength of the soil solution) and
could contaminate the soil, crops, and groundwater (Harter, 1975; Seidler et al.,
The most important function of protein in soil is as a nitrogen source. In the
years to come more emphasis will probably be placed on protein function in this
respect. With increasing petroleum prices, crop fertilization becomes a more
expensive proposition; to offset cost increases, it is probable that farmers will use
more organic fertilizer and will apply agricultural practices that enhance the
availability of nitrogen from protein. For instance, pesticide treatments and
tillage could be changed so as to facilitate maximum proteolysis by the soil
microflora. This might include the substitution of pesticides which do not interfere in protein metabolism. No-till methods may not be best for the degradation
of proteins and peptides, as proteolysis is an aerobic process. Plowing helps to
increase soil aeration, which is important for the incorporation and transformation of organic fertilizer in soil. In some cases liming acid soils may improve the
degradation of proteinaceous wastes.
Protein transformation affects many agricultural and biological processes.
Therefore, more research on proteolysis and protein transformation in soil is
needed. A better understanding of these problems could help agronomists to
improve and maintain soil fertility through the use of proteinaceous wastes as
The authors thank Drs. Jon K. Hall and Les E. Lanyon for their assistance and helpful comments.
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Alexander, M. 1977. “Introduction to Soil Microbiology,” 2nd ed. Wiley, New York.
Ambroz, Z. 1966. Sb. Vys. Sk. Zemed. Brne Rada Al, 57-62.
Ambroz, 2. 1970. Zentralbl. Bakteriol. Parasitende. Abt. 2 125, 433-437.
Aomine, S.,and Kobayashi, Y. 1964. Soil Sci. P h Nutr. (Tokyo)10, 28-32.
Appleby, J. C. 1955. J . Gen. Microbiol. 12, 526-533.
Armstrong, D. E., and Chesters, G. 1964. Soil Sci. 98, 39-52.
Banwart, W. L., and Bremner, J. M. 1976. Soil Biol. Biochem. 8, 439-443.
Basaraba, J., and Starkey, R. L. 1966. Soil Sci. 101, 17-23.