CHARACTERIZATION OF ENZYME STABILITY

In many enzyme-related studies, an index of enzyme stability is required. Enzyme stability can be characterized kinetically or thermodynamically. 

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For the phenomenological kinetic characterization of enzyme stability, the discussion will be restricted to the case where losses in activity, or decreases in concentration of native enzyme, foUow a first-order decay pattern in time (Fig. 12.1a). This process can be modeled as... 

A common parameter used in the characterization of enzyme stability is the half-fife (0/2). As described in Chapter 1, the reaction half-life for a first-order reaction can be calculated from the rate constant ... 

A specialized parameter used by certain disciplines in the characterization of enzyme stability is the decimal reduction time, or D value. The decimal reduction time of a reaction is the time requited for one logio reduction in the concentration, or activity, of the reacting species (i.e., a 90% reduction in the concentration, or activity, of a reactant). Decimal reduction times can be determined from the slope of logio([N,]/[No]) versus time plots (Fig. 12.3). The modified first-order integrated rate equation has the following form ... 

The frequency factor A (time" ) is a parameter related to the total number of collisions that take place during a chemical reaction, Ea (kJ mol" ) the energy of activation, R (kJ mol" K" ) the universal gas constant, and T (K) the absolute temperature. From Eq. (12.17) we can deduce that for a constant value of A, a higher Ea translates into a lower k. As discussed previously, at a constant A, the higher the value of ko, the more thermostable the enzyme. Thus, the rate constant of denaturation, ko, and the energy of activation of denaturation, Ea, are useful parameters in the kinetic characterization of enzyme stability. 

For the thermodynamic characterization of enzyme stability, the denaturation process is also considered a one-step, reversible transition between the native and denatured states ... 

For the kinetic characterization of enzyme stability, enzyme solutions are incubated at a particular temperature and aliquots removed at the appropriate times. Enzyme activity in these samples is then measured at the enzyme s temperature optimum. This activity is usually determined immediately after the temperature treatment. These data will be used in the kinetic characterization of enzyme activity. 

The persistence of some enzymic activities in soils, and the possibility that active abiontic enzymes may be stabilized by combination with soil colloids, has led a minority of workers to attempt the characterization of enzymes in soil extracts and in other fractions. Elucidation of the mechanisms by which abiontic enzymes are stabilized in soil may be important in the wider context of understanding the processes which confer biological resistance on soil organic N. 

The stability of the enzyme-polymer complex and its dissociation upon the variation of pH depends on the structural and other physico-chemical properties of CP and enzyme molecule. Thus, a Biocarb-T heteroreticular biosorbent (Fig. 26) is characterized by a stability of its complex with ot-amylase (under the condition of its stabilization) in acid solutions and a complete dissociation of the complex during isolation of the active enzyme at pH 7-8. 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

Methylmalonyl-CoA mutase (MCM) catalyzes a radical-based transformation of methylmalonyl-CoA (MCA) to succinyl-CoA. The cofactor adenosylcobalamin (AdoCbl) serves as a radical reservoir that generates the S -deoxyadenosine radical (dAdo ) via homolysis of the Co—C5 bond [67], The mechanisms by which the enzyme stabilizes the homolysis products and achieve an observed 1012-fold rate acceleration are yet not fully understood. Co—C bond homolysis is directly kineti-cally coupled to the proceeding hydrogen atom transfer step and the products of the bond homolysis step have therefore not been experimentally characterized. 

The laccase molecule is a dimeric or tetrameric glycoprotein, which contains four copper atoms per monomer, distributed in three redox sites. More than 100 types of laccase have been characterized. These enzymes are glycoproteins with molecular weights of 50-130 kDa. Approximately 45% of the molecular weight of this enzyme in plants are carbohydrate portions, whereas fungal laccases contain less of a carbohydrate portion (10-30%). Some studies have suggested that the carbohydrate portion of the molecule ensures the conformational stability of the globule and protects it from proteolysis and inactivation by radicals (Morozova and others 2007). 

Thus, the use of subzero temperatures in cryosolvents has allowed a series of intermediates in the elastase reaction to be identified, characterized, and stabilized. Temporal resolution of the significant steps in this reaction was difficult to achieve by any other method. Analogous results with other serine proteases suggest that these results are general for this class of enzymes. 

The value and potential usefulness of a new enzyme depends on its properties and the extent to which it has been characterized. The initial characterization of an enzyme often involves the determination of its pH optimum, stability, gross physical properties, and substrates. The enzymes of L. edodes, typically show pH optima between 3.5 and 5.0, maximal activity at 50 to 60"C, little activity loss until over 70"C, and high relative specific activities (9,14). Below we will highlight some of the other characteristics determined for the major ligninase, p-(l,4)-D-xylanase, and a-(l,3)-L-arabinosidase purified from wood-grown cultures of L. edodes. 

A detailed scientific study on the properties of the five major isozymic forms of the lignin peroxidases produced in our pilot reactor has recently been published 12), Our purified enzyme in this study is composed of two isozymes having isoelectric points of 3.85 and 3.80 and molecular masses of 42 000. In this study we have characterized the enzyme s stability as an industrial product. 

Biological catalysts in the form of enzymes, cells, organelles, or synzymes that are tethered to a fixed bed, polymer, or other insoluble carrier or entrapped by a semi-impermeable membrane . Immobilization often confers added stability, permits reuse of the biocatalyst, and allows the development of flow reactors. The mode of immobilization may produce distinct populations of biocatalyst, each exhibiting different activities within the same sample. The study of immobilized enzymes can also provide insights into the chemical basis of enzyme latency, a well-known phenomenon characterized by the limited availability of active enzyme as a consequence of immobilization and/or encapsulization. 

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