Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Kinetics of Enzyme-Catalyzed Reactions
Enzyme Inactivation
Enzyme Stabilization Methods

In addition to searching for more stable natural forms, there are several Methods for increasing the stability of already known Enzymes. These methods can be divided into three main groups:

a) adding stabilizing agents to the medium in which the enzyme is stored or the enzymatic reaction is conducted;

b) chemical Modification of the soluble protein;

c) immobilization of the protein On the surface or within the bulk of an insoluble solid carrier or matrix. In our brief review, we will focus primarily on the first two stabilization methods, while immobilization and its effect on protein stability will be discussed in the next chapter.

Substances that increase protein stability include substrates, organic Solvents, and salts. Since the Active Site of an enzyme is often also the most labile part of its molecule, the presence of a substrate can stabilize the enzyme by locking a portion of the protein molecule in the form of an enzyme-substrate complex. On the other hand, cases of enzyme destabilization by their respective substrates are also known. Solvents such as polyhydric alcohols, which stabilize certain enzymes, may increase the stability of intramolecular Hydrogen Bonds within the protein.

At low salt concentrations (<0.1 M), several cations, such as Ca²⁺, Zn²⁺, Mn²⁺, Fe²⁺, Mo²⁺, and Cu²⁺, specifically interact with special enzymes called metalloenzymes. Some of these cations are Cofactors, and their presence stabilizes the enzyme. The Ca²⁺ cation is involved in stabilizing the Tertiary Structure of several Proteins. By forming ionic bonds with two different amino acid residues, Ca²⁺ ions can function as a stabilizing bridge, similar to Disulfide Bonds.

Class="center">Table 3.11. Examples of enzyme stabilization methods

Examples of the effects of various substances on enzyme stability are given in Table 3.11, and more detailed information can be found in review [4]. It should be emphasized that The Effect of a given substance or factor On the Stability of a particular enzyme does not always mean that this substance or factor will act in the same way on Other Enzymes. On the other hand, the stabilizing effect is often manifested only under a specific combination of solution composition and Temperature; for example, The addition of small amounts of certain salts stabilizes enzymes, whereas higher concentrations of the same salts usually cause their Denaturation. This rule also applies to organic solvents.

To increase enzyme stability, Chemical modification of proteins, an important tool in biochemical research, has also been used with relative success. In one variant of this method, the side chains of Certain amino acid residues are modified, for example, by acylation, reductive alkylation, or condensation of the amino groups of the native protein with the side chains of polyamino acids.

Another variant of chemical Protein Stabilization methods is based on The Use of bifunctional Reagents, such as glutaraldehyde. Such reagents form cross-links between the amino groups of the protein, thereby, first, hindering the access of proteases to the protein and, second, potentially locking the active conformation of the protein. Diimides, which link amine and carboxyl groups via cross-linking amide bonds, can also be used for chemical stabilization. We will consider these reagents and the corresponding reactions in more detail when studying their application for Enzyme Immobilization.