e-mail: nsw@umd.edu
Of the three gels, polyacrylamide is the most widely used matrix for entrapping enzymes. It has the advantage that it is non-ionic. The consequence is that the properties of the enzymes are only minimally modified in the presence of the gel matrix. At the same time, the diffusion of the charged substrate and products is not affected, neither. However, dimethylaminopropionitrile, the polymerization initiator, is highly toxic and must be handled with great care. The requirement to purge the monomer solution with nitrogen is also troublesome, although not totally crippling.
Calcium alginate is just as widely used as polyacrylamide. Unlike polyacrylamide gels, gelation of calcium alginate does not depend on the formation of more permanent covalent bonds between polymer chains. Rather, polymer molecules are cross-linked by calcium ions. Because of this, calcium alginate beads can be formed in extremely mild conditions, which ensure that enzyme activity yields of over 80% can be routinely achieved. However, just as easily as calcium ions can be exchanged for sodium ions, they can also be displaced by other ions. This property can both be advantageous and disadvantageous. If needed, enzymes or microbial cells can be easily recovered by dissolving the gel in a sodium solution. On the other hand, proper caution must be exercised to ensure that the substrate solution does not contain high concentrations of those ions that can disintegrate the gel.
The main attraction of using gelatin as the immobilization media is that the gel formation process requires only simple equipment and that the reagents are relatively inexpensive and nontoxic. The retention of enzymatic activities for immobilization with a gelatin gel is typically 25-50% of the original free enzyme. Gelatin gel has the advantage that the mass transfer resistance is relatively low compared to other entrapment methods, but the rate of enzyme loss due to leakage is high.
In summary, the efficiency of an immobilization process can be measured by the following criteria. Most important of all, a high percentage of the enzymes must be initially retained in gel matrices. Secondly, the enzyme activity must be preserved. And thirdly, the enzymes must be physically restrained from diffusing back into the substrate solution at a later time. It is quite difficult to create a fine and uniform mesh so as to prevent the entrapped enzymes from leaking out of the matrices. On the other hand, highly cross-linked matrices can result in higher mass transfer resistances for both the substrate and the product.
Bacterial alpha-amylase will be used in this experiment to demonstrate and compare the effectiveness of various entrapment techniques. The class will be divided into groups of three. Before attempting the experiment, identify the major steps and devise a work plan among the group members. Each member should be explicitly assigned an equal portion of the responsibilities. For example, one member may be responsible for making all three types of gels, and other members may be assigned the tasks of measuring the enzyme leakage and immobilized enzyme activities. Alternatively, each member may be responsible for remain responsible throughout all phases of the study associated only one type of gel. It is the responsibility of the entire group to make sure that each member is carrying out the work according to the plan, whatever the plan is. Work closely together and help each other if needed to coordinate the smooth execution of the plan. Share the data at the end.
Numerous methods exist for enzyme immobilization, sometimes referred to as enzyme insolubilization. The overwhelming majority of the methods can be classified into four main categories: matrix entrapment, microencapsulation, adsorption, and covalent binding. Of these methods, matrix entrapment is the focus of this experiment.
Many entrapment methods are used today, and all are based on the physical occlusion of enzyme molecules within a "caged" gel structure such that the diffusion of enzyme molecules to the surrounding medium is severely limited, if not rendered totally impossible. What creates the "wires" of the cage is the cross-linking of polymers. A highly cross-linked gel has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages. The degree of cross-linking depends on the condition at which polymerization is carried out. Because there is a statistical variation in the mesh size, some of the enzyme molecules gradually diffuse toward the outer shell of the gel and eventually leak into the surrounding medium. Thus, even in the absence of loss in the intrinsic enzyme activity, there is a need to replenish continually the lost enzymes to compensate for the loss of apparent activity. In addition, because an immobilized enzyme preparation is used for a prolonged period of operation, there is also a gradual, but noticeable, decline in the intrinsic enzyme activity even for the best method. Eventually, the entire immobilized enzyme packing must be replaced.
Besides the leakage of enzymes, another problem associated with the entrapment method of immobilization is the mass transfer resistance to substrates, products, and inhibitors. Because the average diameter of a typical bead of enzyme impregnated gel is much larger compared to the average diffusion length, substrate cannot diffuse deep into the gel matrix, as in any other conventional non-biological immobilized catalysts. At the same time, the diffusional resistance encountered by the product molecules can sometimes cause the product to accumulate near the center of the gel to an undesirable high level, leading to product inhibition for some enzymes. Thus, ideally the network of cross-linking should be coarse enough so that the passage of substrate and product molecules in and out of a gel bead is as unhindered as possible. For this reason, entrapment is not suitable for special cases where the substrate has a large molecular weight such that it cannot easily move freely in the gel matrix.
Unlike the adsorption and covalent bonding methods, most polymerization reactions that cause cross-linking and gel formation in entrapment methods do not directly involve the formation of bonds between the support material and the enzyme molecules. There are reports that these bonds change the conformation of the enzyme protein and modify the enzyme properties. Since the enzyme molecules do not themselves participate in the polymerization reaction in the entrapment methods, the same entrapment techniques can be successfully applied to a wide range of enzymes with only minor modifications between different enzymes.
e-mail: nsw@umd.edu