Soil Biochemistry

Volume 6: Volume 6

J.-M. Bollag

• Publisher: Routledge
• Year: 2017
• ISBN:
  9781351416054
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Explores the role of biochemical processes in the soil environment, particularly the activity of microorganisms, and the potential application of those processes to environmental biotechnology. The 11 papers also highlight the application of molecular biology and microbial genetics to soil biology a

1

Enzyme Interactions with Clays and Clay-Organic Matter Complexes

STEPHEN A. BOYD and MAX M. MORTLAND

Michigan State University, East Lansing, Michigan

I. GENERAL ASPECTS OF CLAY-PROTEIN INTERACTIONS

Essentially all of the noncellular protein present in soil is associated with the clay mineral and humus fractions. More than 90% of the nitrogen (N) in the surface layers of soil is present in organic forms. As much as 50% of this nitrogen can be identified as amino acid-N derived from proteins. Obviously, there is a significant component of noncellular proteinaceous material in soil, and its very presence is evidence that it is resistant to microbial decomposition. This resistance is further demonstrated by the fact that only a small portion (<5%) of the soil organic N is mineralized annually [1]. Soils are also known to contain a wide variety of active cell-free enzymes [2], which display remarkable stability [3,4].

The persistence and stability of proteins, including enzymes, in soils are generally attributed to their association with clays and humus. As the result of the complex nature of soil and the association of soil clays and humus, the isolation of discrete clay-enzyme or humus-enzyme complexes for study has proved difficult. However, such complexes can be prepared in the laboratory using purified clays and proteins, and these complexes have been studied in some detail. Much of the work on clay-protein interactions has been detailed in previous reviews [2,5,6] and will not be covered in detail here, except to summarize the general concepts that have emerged from this work. In doing so, representative studies will be cited, but these citations will not be inclusive.

Proteins contain a variety of polar (e.g., C=0, O-H) and ionizable (e.g., NH₂, COOH) functional groups that can interact with clay mineral surfaces. Clay minerals possess a net negative electrical charge that is compensated by exchange cations, such as Ca²⁺ and Mg²⁺, on their surfaces. Proteins possess positive (as well as negative) charges and may become electrostatically bound to clays via cation exchange reactions. The amount of positive charge on the protein increases as the pH decreases, so that higher adsorption vis-à-vis ion exchange would be expected as the pH decreases below the isoelectric point of the protein. However, results from numerous studies have shown that protein adsorption by smectite clays is often greatest at or near the isoelectric point of the protein [7-10]. A decrease in positive charges associated with the protein as the pH increases toward the isoelectric point may require higher uptake to satisfy the negative charges on the clay. Formation of a more compact structure has also been suggested as a possible factor contributing to the increased adsorption of protein as the pH approaches the isoelectric point [5]. Other types of binding mechanisms that may be involved include van der Waals interactions, hydrogen bonding, and ion-dipole interactions with metal exchange ions on the clay surfaces [11].

The earliest investigations into the nature of clay-protein complexes were by Ensminger and Gieseking [12,13]. The reaction of smectite with gelatin and albumin in aqueous media resulted in the intercalation of the proteins, as shown by basal spacings of ~5 nm in these complexes. These studies and others [10,14-16] have provided evidence for the important role of cation exchange in the clayprotein reaction. This evidence included the observation by some workers [12,13] of higher protein adsorption at lower pH values, decreased adsorption resulting from chemical deactivation of the protein amino groups, and more facile exchange of lower-valence exchange cations. Protein uptake has also been shown to be directly related to the amount of exchangeable Na ions released into solution and the cation exchange capacity of the mineral.

Protein uptake by smectite depends on the exchange cation, decreasing in the order H⁺ > Na⁺ > Ca²⁺ > Al³⁺ [9,14,16,17]. Only H^- and Na-smectite have been shown to intercalate protein, presumably as a result of two factors: the relative ease of displacement of H⁺ and Na⁺ via cation exchange and the greater accessibility of interlayer surfaces due to higher swelling. When fully hydrated, smectites saturated with polyvalent ions have only 1 nm between the aluminosilicate sheets, whereas Na-smectite expands to the individual crystallites, thus permitting entrance of larger molecules. Proteins may occupy the interlayer spaces of smectite clays in single or multiple layers. This has been demonstrated in the laboratory, primarily using Na-smectite, which undergoes extensive interlayer expansion in water. Basal spacings reported for protein-smectite complexes range from ~1.4 to greater than 7.0 nm.

An exact description of the relation between basal spacing and the interlayer organization of adsorbed protein has proved difficult. However, it is clear that at least double layers of proteins may be intercalated and that adsorption onto clay surfaces does not always lead to alterations in the conformation of the protein. That clayadosrbed enzymes retain their catalytic activities [6,14,18] is evidence that adsorption does not necessarily result in denaturation via conformation changes (e.g., uncoiling or unfolding) to more random forms. However, some proteins, notably gelatin and pepsin, tend to unfold when adsorbed by smectite [5,15]. Theng [5] concluded that the final conformation of the adsorbed protein depends on the balance between the intramolecular structural forces of the protein and its interaction with the clay surface. It was suggested that proteins with certain tertiary structures, e.g., fibrous proteins, tend to make greater contact with the clay surface and are, therefore, more likely to unfold.

It is important to emphasize that there is no convincing evidence for the presence of interlayer protein in swelling soil clays, although Theng et al. [19] found organic matter within the interlayers of acid soil smectite. The absence of interlayer protein-smectite complexes in soil probably results from several factors, including the predominance of limited-swelling divalent cation (e.g., Ca²⁺)-exchanged clays in soils, the easier accessibility of external surfaces, and the rapid microbial degradation of free protein. Thus, it appears that, in soils, the adsorption of protein by clays is limited to the external surfaces of swelling and nonswelling clays. It is, therefore, unlikely that the stability of protein and enzymes present in soils can be attributed to intercalation in swelling clays.

Proteins are also adsorbed on vermiculite, illite, and kaolinite but in significantly lower amounts than on smectite. For example, the amount of lysozyme bound at a pH near neutrality decreased in the order smectite > vermiculite ≌ illite > kaolinite; the actual amounts of protein adsorbed for these four mineral types were approximately 1.0, 0.2, 0.2, and 0.02 g/g, respectively [10]. Lysozyme appeared to be intercalated by smectite, but adsorption was limited to the external surfaces of vermiculite, illite, and kaolinite. Similar results have been obtained for the adsorption of urease on kaolinite, vermiculite, and smectite [20].

The effect of pH on the adsorption of proteins on kaolinite is different from that on smectite. For kaolinite, the curve relating adsorption to pH typically shows a broad adsorption maximum, which may begin several pH units below the isoelectric point of the protein [7,21,22]. The mechanism of adsorption on kaolinite is primarily ion exchange, and the pH-adsorption curve has been attributed to pH-dependent charges associated with kaolinite and the protein [5].

II. CLAY-ADSORBED ENZYMES

In soils, clays and organic matter can serve as adsorbing surfaces for enzymes in exactly the same fashion as for proteins in general. In this regard, McLaren [23] referred to soil as "a system of humus- and clay-immobilized enzymes." The adsorption of enzymes by clay minerals results in significant changes in enzyme properties, such as pH optimum, stability, activity, and kinetics. These changes are due, in part, to the influence of clay surface properties (e.g., acidity) on enzymes and enzyme substrates and to the fact that catalysis apparently occurs at the mineral-solution interface rather than in the bulk solution.

The apparent pH optimum of clay-adsorbed enzymes is generally displaced one or two pH units to more alkaline values [5,24,25]. This shift in pH optimum to higher values occurs because the Brönsted acidity at the clay surface is significantly greater than in the bulk solution. This effect has been shown clearly by infrared studies of the protonation of ammonia at clay surfaces [26,27]. The difference in acidity between the clay surface and the bulk solution (ΔpH) is generally about one to two pH units [5], although the exact magnitude of this difference depends on such factors as the clay mineral type, exchange cation, and water content [27]. The adsorbed enzyme experiences the acidity of the clay surface, and hence the bulk solution pH needs to be raised above the pH optimum of the enzyme to bring the clay surface to the optimum acidity. As a result, the apparent pH optimum of the adsorbed enzyme (i.e., the bulk solution pH corresponding to maximum activity) increases by an amount equivalent to ΔpH. In contrast to the general observation of a shift in the pH optimum of clay-adsorbed enzymes, Sundaram and Crook [28] found no pH optimum shift for urease adsorbed on kaolinite. Bacteriolytic enzymes immobilized on smectite or unadsorbed showed maximum activity at a similar pH [29].

The stability of enzymes can be affected either positively or negatively by adsorption onto clay surfaces. For example, Garwood et al. [18] observed that glucose oxidase adsorbed on Na-smectite lost significant activity over a 75-hour period at 20°C, whereas little or no denaturation of the free enzyme was observed. Stability increased as the amount of enzyme adsorbed increased. This was attributed to less free mineral surface, which appeared to promote enzyme uncoiling. Coverage of the free mineral surface area by added tetrabutylammonium ion also resulted in higher enzyme stability, supporting the concept that contact with the free mineral surface may promote enzyme denaturation.

The resistance of clay-adsorbed enzymes to enzymatic hydrolysis (proteolysis) has also been studied. In these systems, both the enzyme and its substrate (also protein) are likely to be associated with the mineral surface. In early studies, Ensminger and Gieseking [30] found that smectite reduced the hydrolysis of protein by pepsin, whereas kaolinite had minimal effects. The proteolysis of denatured lysozyme adsorbed on kaolinite by chymotrypsin was approximately two-thirds of the rate obtained in homogeneous solution [31]. Subsequent work by Estermann et al. [32] showed that lysozyme adsorbed on smectite was even more resistant to proteolysis than lysozyme adsorbed on kaolinite. The higher degree of protective effect of smectite was attributed to lower accessibility of interlayer protein, leading to greater difficulty in forming the clay-substrate-enzyme complex [5]....