Biomedical Application of Enzymes

ABSTRACT

Reports on chemical immobilization of proteins and enzymes first appeared in the 1960s. Since then, immobilized proteins and enzymes have been widely used in the processing of variety of products and increasingly used in the field of medicine. Here, we present a review of recent developments in immobilized enzyme use in medicine. Generally speaking, the use of immobilized enzyme in medicine can be divided into two major categories: biosensors and bioreactors. A brief overview of the evolution of the biosensor and bioreactor technology, of currently existing applications of immobilized enzymes, of problems that researchers encountered, and of possible future developments will be presented.

INTRODUCTION Reports on chemical immobilization of proteins and enzymes first appeared ~30 years ago. Since then, immobilized proteins have been widely used for the processing of a variety of products spanning industries from food to environmental control. In addition to their use in processing, immobilized proteins and enzymes have also been found useful in many bioanalytical and biomedical applications. These applications include the use of immobilized antibodies or antigens in bioaffinity chromatography, of immobilized receptors or legends in the study of their interactions, and of immobilized cells in biosensors. Although there have been a large number of articles discussing immobilization techniques of proteins and enzymes as well as their applications in certain fields,13 a review on the applications of immobilized proteins and enzymes in medicine and particularly during the past decade (i.e., 1990s) is still lacking. Presently, immobilized proteins/enzymes are used routinely in the medical field, such as in the diagnosis and treatment of various diseases. For example, immobilized antibodies, receptors, or enzymes are used in biosensors and ELISA for the detection of various bioactive substances in the diagnosis of disease states; encapsulated enzymes are also used in bioreactors for the removal of waste metabolites and correction of inborn metabolic deficiency. Moreover, the use of artificial cells as well as the development of controlled release drug delivery systems to release encapsulated enzymes or proteins may also be considered a form of immobilized enzyme use.
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However, this article will concentrate only on the review of current applications of traditionally defined immobilized enzymes in medicine; specifically in the areas of biosensors and bioreactors.

IMMOBILIZED ENZYME USED IN BIOSENSORS Enzyme-based electrodes represent a major application of immobilized enzymes in medicine. The high specificity and reactivity of an enzyme towards its substrate are properties being exploited in biosensor technology. Biosensors possess advantages such as reliability, sensitivity, accuracy, ease of handling, and low-cost compared with conventional detection methods. These characteristics, in combination with the unique properties of an enzyme already mentioned, render an enzyme based biosensor ideal for biomedical applications.

Enzyme Immobilization An enzyme-based electrochemical sensor is formulated by immobilizing a thin layer of enzyme( s) on the surface of the membrane of an electrode. The analyze to be monitored diffuses into the enzyme layer where the catalytic eaction occurs, either consuming a substrate or generating a product that can be detected electrochemically. The electro active species produced are monitored either potentiometrically or amperometrically, and the electrochemical signal can be correlated to the concentration of the analyze to be measured. The immobilization method may affect the activity of the immobilized enzyme, and thus it can contribute significantly to the sensitivity of the biosensor. Immobilization methods that are currently being used include absorption, cross-linking, and selfassembly, whereas materials into which enzymes are incorporated include carbon paste, conducting or nonconducting polymers, and different types of gels. The ultimate aim of these efforts is to develop an inexpensive and easy-to-prepare method that can yield a stable matrix possessing the maximum retention of the biological activity of the immobilized enzyme. Generally speaking, physical entrapment of enzymes in polymeric membranes is one of the most advantageous methods because it is rapid and simple, the retained enzyme activity is usually quite high, plus no chemical reaction that may lead to the inactivation of the enzyme is required. The major limitation of physical entrapment is, however, that unlike chemical immobilization where all
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types of substrates, whether they are large or small, can all reach the immobilized enzyme for interaction, the physically entrapped enzyme is prohibited from interaction with large substrates that are unable to diffuse into the matrix. Therefore, for assaying a large substrate, chemically immobilized enzyme should be used. In search of suitable matrices for enzyme immobilization, there has been a growing interest in attaching the enzymes to the solid electrolyte Nafion. The Nafion membrane possesses a high surface adhesion property and a low swelling capability in aqueous media. Being a negatively charged polyelectrolyte matrix, Nafion exhibits a reduced ermeability to negatively charged substances, therefore improving the sensor selectivity and virtually eliminating the interference of reductants such as ascorbate and acetaminophen. On the other hand, a Nafion film possesses a high ability to entrap positively charged redox-active compounds that can be used as mediators in bioelectrocatalytic reactions.

Sensitivity and Selectivity Amperometric biosensors based on oxidase enzymes that generate hydrogen peroxide are the most widely used biosensors. Glucose determination utilizing the enzyme glucose oxidase (GOD) is an example of such an mperometric biosensor, and the principle of such glucose electrodes is based on the following reactions: Immobilized layer: glucose + O2 ->gluconic acid + H2O2 Electrode: H2O2->O2 + 2H+ + 2eObviously, this type of sensor is based on either oxygen depletion (cathodic) or the electrochemical oxidation of H2O2 (anodic). However, direct oxidation of H2O2 requires a highly positive overpotential, and hence this type of sensor suffers from interferences due to the presence of easily oxidizable species in the biological fluids. Although the cathodic measurement of molecular oxygen concentration is perfectly suitable for glucose monitoring in solution, these devices also suffer potential difficulties when operating in vivo or in undiluted biological samples where the response is limited by physiological amounts of oxygen available. To solve the oxygen deficit problem, attempts have been made in glucose detection to use an additional catalase in recycling O2 from H2O2,45 or to develop approaches that allow O2 to be delivered externally to the electrode surface, thereby producing an excess level of O2 (compared with that of glucose) at the electrode surface. This excess amount of O2
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will allow for better regulation of the reaction environment and, hence, oxygen (instead of glucose) does not become the limiting reagent in the reaction.

IMMOBILIZED ENZYMES USED IN BIOREACTORS The therapeutic application of immobilized enzyme in a bioreactor started as early as the 1960s. Since then, significant efforts have been made to apply this technology to the correction of inborn errors of metabolism, cancer treatment, blood detoxification, and removal of waste metabolites. Generally speaking, the enzyme(s) can be either immobilized on a solid support or encapsulated in solgel or an artificial cell (red blood cell or liposome) to construct the bioreactor. Although there are some reports on the immobilization of enzymes including phospholipase A2 and heparinase in bioreactors for blood detoxification, most efforts have been centered on the application of encapsulated enzymes for disease treatment. Compared with immobilized enzyme, enzymes entrapped in an artificial cell not only convert substrates that diffuse into the cell, but also possess low immunogenicity (due to the ability of artificial cells to mask their immune determinants) and prolonged blood clearance time. Initially, only a few enzymes, such as asparaginase and urease, had been used. Also, encapsulation of only single enzymes in the artificial cell had been attempted. At present, however, multiple enzymes are being encapsulated in one artificial cell, and such a multienzyme system has been used for the correction of metabolic imbalances.

Red Blood Cell as A Carrier for Enzymes As a new therapeutic approach, human and animal red blood cells (RBCs) have been used as the carrier vehicle for a number of exogenous enzyme drugs intended to be disseminated in an organism. Because of their biocompatible nature, these cells will elicit little or virtually no immune response, thereby providing advantages of protecting the activity of encapsulated substances from rapid clearance and of avoiding toxic side effects. In addition, RBCs can be readily obtained, and a large quantity of the drug can be entrapped into a rather small volume of the cell using certain well-defined methods. Techniques for induction of exogenous substances into RBCs include endocytosis, passive diffusion,electroporation,84 and hypotonic dialysis isotonic resealing etc. Among them, hypotonic dialysis/isotonic resealing is currently the most widely used technique because it is
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simple to use, it provides an efficient encapsulation of a large amount of the protein, it is adaptable for scale-up purposes, and it provides a carrier cell possessing the same in vivo halflife as that of the normal RBC. Electroporation is another simple and efficient method for enzyme encapsulation. If the intensity of the electric field and the exposure time are maintained below certain critical values, the membrane rupture process required can be controlled to be completely reversible. Compared with hypotonic dialysis, electroporation provides several advantages, such as the preparation of the cell suspension can be achieved in an isotonic solution and the procedure can be manipulated easily and carried out routinely.84 In addition to the loading techniques, the sources of RBCs can also affect the capacity for drug loading. Among different animal species from which the RBCs are obtained and treated with the hypotonic dialysis technique, the rat and rabbit have been reported to be the species that could present problems for enzyme encapsulation because these RBCs yield relatively low drug-loading capacity compared with human erythrocytes.

CONCLUSION This paper presents a brief review of recent (i.e., during the past decade) developments and medical applications of immobilized enzymes, particularly in the areas of biosensors and bioreactors. Although the progress in therapeutic use of immobilized enzymes is slow and somewhat staggered, because of the complexity of the human body system to be applied, the future prospect for application of immobilized enzymes in biosensing is promising. This promising future is particularly true because of the current advancement in technologies in microprocessing and microelectronic devices. Such technologies would allow the biosensors to be miniaturized during manufacturing, integrated with signal processing steps on a chip, and arrayed for more complicated substrate analysis. In addition, the progress in optical transducing devices provides another promising avenue that can be incorporated in biosensor development.