IMMOBILIZED ENZYMES

by M. K. Goel, 1994

Outline of Preparation Techniques

Methods used for the immobilization of enzymes fall into four main categories:
  1. Physical adsorption onto an inert carrier,
  2. Inclusion in the lattices of a polymerized gel,
  3. Cross-linking of the protein with a bifunctional reagent, and
  4. Covalent binding to a reactive insoluble support.

Adsorption

Physical adsorption of an enzyme onto a solid is prabably the simplest way of preparing immobilized enzymes. The method relies on non-specific physical interaction between the enzyme protein and the surface of the matrix, brought about by mixing a concentrated solution of enzyme with the solid.
A major advantage of adsorption as a general method of insolubilizing enzymes is that usually no reagents and only a minimum of activation steps are required. As a result, adsorption is cheap, easily carried out, and tends to be less disruptive to the enzymic protein than chemical means of attachment, the binding being mainly by hydrogen bonds, multiple salt linkages, and Van der Waal's forces. In this respect, the method bears the greatest similarity to the situation found in biological membranes in vivo and has been used to model such systems.
Because of the weak bonds involved, desorption of the protein resulting from changes in temperature, pH, ionic strength or even the mere presence of substrate, is often observed. Another disadvantage is non-specific further adsorption of other proteins or other substances as the immobilized enzyme is used. This may alter the properties of the immobilized enzyme or, if the substance adsorbed is a substrate for the enzyme, the rate will probably decrease depending on the surface mobility of enzyme and substrate.
Adsorption of the enzyme may be necessary to facilitate the covalent reactions described later. Stabilization of enzymes temporarily adsorbed onto a matrix has been achieved by cross-linking the protein in a chemical reactiion subsequent to its physical adsorption.

Occlusion

Confining enzymes within the lattices of polymerized gels is another method for immobilization. This allows the free diffusion of low molecular weight substrates and reaction products. The usual method is to polymerize the hydrophilic matrix in an aqueous solution of the enzyme and break up the polymeric mass to the desired particle size.
As there is no bond formation between the enzyme and the polymer matrix, occlusion provides a generally applicable method that, in theory, involoves no disruption of the protein molecules. However, free radicals generated on the course of the polymerization may affect the activity of entrapped enzymes. Another disadvantage is that only low molecular weight substrates can diffuse rapidly to the enzyme, thus making the method unsuitable for enzymes that act on macromolecular substrates, such as ribonuclease, trypsin, dextranase, etc.
The broad distribution in pore size of synthetic gels of the polyacrylamide type inevitably results in leakage of the entrapped enzyme, even after prolonged washing. This may be overcome by cross-linking the entrapped protein with glutaraldehyde. Alternatively, ultrafiltration membranes of well-defined pore size may be used to occlude the enzyme.

Cross-Linking

Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix.. Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support, resulting in relatively low enzymic activity. Generally, cross-linking is best used in conjunction with one of the other methods. Preventing leakage from polyacrylamide gels has already been mentioned, but it is used much more widely as a means of stablilizing adsorbed enzymes.
Since the enzyme is covalently linked to the support matrix, very little desorption is likely using this method. Marshall (1973), for example, reported that carbamyl phosphokinase cross-linked to alkylamine glass with glutaraldehyde lost only 16% of its activity after continuous use in a column at room temperature for fourteen days.

Covalent Binding

The most intensely studied of the insolubilization techniques is the formation of covalent bonds between the enzyme and the support matrix. When trying to select the type of reaction by which a given protein should be insolubilized, the choice is limited by the fact that the binding reaction must be performed under conditions that do not cause loss of enzymic activity, and the active site of the enzyme must be unaffected by the reagents used.
The functional groups of proteins suitable for covalent binding under mild conditions include (i) the alpha amino groups of the chain and the epsilon amino groups of lysine and arginine, (ii) the alpha carboxyl group of the chain end and the beta and gamma carboxyl groups of aspartic and glutamic acids, (iii) the phenol ring of tyrosine, (iv) the thiol group of cysteine, (v) the hydroxyl groups of serine and threonine, (vi) the imidazile group of histidine, and (vii) the indole group of tryptophan.
A small number of reactions have been designed to couple with functional groups on the protein other than the amino and phenolic residues. Aminoethyl cellulose has been coupled to the carboxylic acid residues of enzymic protein in the presence of carbodiimide, and thiol residues of a protein have been oxidatively coupled to the thiol groups of a cross-linked copolymer of acrylamide and N-acryloyl-cystein.
It is possible in some cases to increase the number of reactive residues of an enzyme in order to increase the yield of insolubilized enzyme and to provide alternative reaction sites to those essential for enzymic activity. As with cross-linking, covalent bonding should provide stable, insolubilized enzyme derivatives that do not leach enzyme into the surrounding solution. The wide variety of binding reactions, and insoluble carriers with functional groups capable of covalent coupling, or being activated to give such groups, makes this a generally applicable method of insolubilization, even if very little is known about the protein structure or active site of the enzyme to be coupled.

Choice of Immobilization Method

When immobilizing an enzyme on a surface it is most important to choose a method of attachment aimed at reactive groups outside the active catalytic and binding site of that enzyme. Considerable knowledge of active sites of particular enzymes will enable methods to be chosen that would avoid reaction with the essential groups therein. Alternatively, these active sites can be protected during attachment as long as the protective groups can be removed without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate of the enzyme or a competitive inhibitor; this also contributes towards retention of tertiary structure of the enzyme.
The surface on which the enzyme is immobilized has several vital roles to play such as retaining of tertiary structre in the enzyme by hydrogen bonding or the formation of electron transition complexes. Retention of tertiary structure may also be a vital factor in maximizingthermal stability in the immobilized state. In this respect it is wise to follow closely the new findings in the chemical nature of soluble thermostable enzymes. The microenvironment of surface and the immobilized enzyme has an anionic or cationic nature of the surface that can cause a displacement in the optimum pH of the enzyme of up to 2 pH units. This may be accompanied by a general broadening of the pH region in which the enzyme can work effectively.
Immobilization by cross-linking the protein enzyme in order to insolubilize it or merely immobilize it in desired location has many possibilities and is relatively cheap. Several aldehydes and other cross-linking agents are now available for this purpose. Extension of this approach to a process where an enzyme is an integral component of a copolymer could permit designing for reversible polymerization.

Choice of Enzyme Reactor

In enzyme reactor, the highest specific activity, in terms of weight of enzyme and support employed, is desirable. It is considered an added bonus if the support is employed to fulfil another function. The most important of these functions is separation since, if this occurs simultaniously with reaction, unfavorable equilibria may be displaced. One approach is to use a molecular sieve as the support and, in packed reactors to pulse the reactor bed alternatively by passage of substrate solution and water, so that bands of unused substrate and product are progressing down the column. It so happens that these enzymes for which this would be useful are also those which in some cases benefit in having the enzyme immobilized on a porous support.
For an industrial reactor it is preferable to use supports that are non-biodegradable such as glass, silica, Celite, Bentonite, alumina, or titanium oxide, if possible. Even the linkages between enzyme and support can be non-biodegradable, as with the titanium procedure. As far as the biodegradability of the enzyme, in some cases, when in active use, enzymes will partially protect themselves, notably all those that effect oxidation with production of hydrogen peroxide as a by product.
Many types of reactors have been proposed: packed beds with downward flow, suspended particles in a fluid ben with upward flow of substrate, a simple stirred reactor, tubular reactors, membrane reactors, and many others. In some of these the physical nature of the surface becomes a major problem. Thus some supports that form excellent packed beds fail to do so when coated with enzyme and particles which ideally self-suspend in a fluid bed then aggregate during use requiring more power to pump through substrate allied to a lowered catalytic activity from a decreased surface area. Many problems were encountered with porous glass supports until it was realized that the enzyme was not simply losing activity but the glass itself could dissolve. This has now been overcome by the zirconium treatment of the glass surface. Additives to enzymes intended to preserve enzyme activity are also rarely fully disclosed. This often causes confusion as to the weight of protein in the enzyme product and can even interfere with the coupling procedure or decrease its efficiency.

Properties of Immobilized Enzymes

It is important to understand the changes in physical and chemical properties which an enzyme would be expected to undergo upon insolubilization if the best use is to be made of the various insolubilization techniques available. Changes have been observed in the stability of enzymes and in their kinetic properties because of the microenvironment imposed upon them by the supporting matrix and by the products of their own action.

Stability

The stability of the enzymes might be expected to either increase or decrease on insolubilization, depending on whether the carrier provides a microenvironment capable of denaturing the enzymic protein or of stabilizing it. Inactivation due to autodigestion of proteolytic enzymes should be reduced by isolating the enzyme molecules from mutual attack by immobilizing them on a matrix. It has been found that enzymes coupled to inorganic carriers were generally more stable than those attached to organic polymers when stored at 4 or 23 ° centigrade. Stability to denaturing agents may also be changed upon insolubilization.

Kinetic Properties

Changes in activity of enzymes due to the actual process of insolubilization have not been studied very much. There is usually a decrease in specific activity of an enzyme upon insolubilization, and this can be attributed to denaturation of the enzymic protein caused by the coupling process. Once an enzyme has been insolubilized, however, it finds itself in a microenvironment that may be drastically different from that existing in free solution. The new microenvironment may be a result of the physical and chemical character of the support matrix alone, or it may result from interactions of the matrix with substrates or products involved in the enzymatic reaction.
The Michaelis constant has been found to decrease by more than one order of magnitude when substrate of opposite charge to the carrier matrix was used. Again, this only happened at low ionic strengths, and when neutral substrates were used. The electrostatic potential was calculated by insertion of the Maxwell-Bottzmann distribution into the Michaelis-Menton equation using the changes in Michaelis constant, and good agreement was obtained with the value for the electrostatic potential calculated from the pH-activity shifts.
The diffusion of substrate from the bulk solution to the micro-environment of an immobilized enzyme can limit the rate of the enzyme reaction. The rate at which substrate passes over the insoluble particle affects the thickness of the diffusion film, which in turn determines the concentration of substrate in the vicinity of the enzyme and hence the rate of reaction.
The effect of the molecular weight of the substrate can also be large. Diffusion of large molecules will obviously be limited by steric interactions with the matrix, and this is reflected in the fact that the relative activity of bound enymes towards high molecular weight substrates has been generally found to be lower than towards low molecular weight substrates. This, however, may be an advantage in some cases, since the immobilized enzymes may be protected from attack by large inhibitor molecules.