Kinetics of Immobilized Enzymes

For industrial uses of enzymes, processes have to be technically and economically feasible. Immobilization of the enzyme, which is usually carried by entrapment within a porous solid matrix, has advantages over soluble enzymes (Pereira et al, 2001 Tramper et al 2001). This includes ease of reuse and enhancement of both enzyme activity and selectivity (D Souza, 2001 Gianfreda and Scarfl, 1991). Immobilized enzymes have applications in various industries and in different reactor configurations. Among these reactors, packed bed reactors have shown promise in many processes. This is widely used with an immobilized enzyme system due to long retention times and ease of operation (Abu-Reesh, 1997). 

On the other hand, the use of immobilized enzymes in continuous operation may have a negative effect on reaction kinetics. This can be either by conformational (structural), steric, or microenvironmental change (Kalthod and Uckenstein, 1982). The main shortcoming lies in mass transfer limitations that affect the activity of the enzyme, especially if immobilized on the internal surface of a porous support. 

---

Considerable progress has been made within the last decade in elucidating the effects of the microenvironment (such as electric charge, dielectric constant and lipophilic or hydrophilic nature) and of external and internal diffusion on the kinetics of immobilized enzymes (7). Taking these factors into consideration, quantitative expressions have been derived for the kinetic behavior of relatively simple enzyme systems. In all of these derivations the immobilized enzymes were treated as simple heterogeneous catalysts. 

Further progress in understanding the mechanism of action and kinetics of immobilized enzymes will require the study, both theoretical and experimental, of more complex enzyme systems. Of particular interest will be the study of the highly specific interactions between an enzyme embedded in a native membrane and the adjacent high or low molecular weight compounds. 

Although the kinetics of immobilized enzymes is especially relevant to industrial process chemistry, there is growing recognition that enzymes in crowded solutions may likewise experience diffusional control of the catalytic properties. 

D. L. Regan, M. D. Lilly, and P. Dunnill, Influence of intraparticle diffusional limitation on the observed kinetics of immobilized enzymes and on catalyst design, Biotechnol. Bioeng. 1974, 16, 1081-1093. 

Kinetics of Immobilized Enzymes. Another major factor in the performance of immobilized enzymes is the effect of the matrix on mass transport of substrates and products. Hindered access to the active site of an immobilized enzyme can affect the kinetic parameters in several ways. The effective concentration of substrates and products is also affected by the chemistry of the matrix especially with regard to the respective partition coefficients between the bulk solution and the matrix. In order to understand the effects of immobilization upon the rate of an enzyme-catalyzed reaction one must first consider the relationship between the velocity of an enzyme-catalyzed reaction and the... 

The choice of an appropriate reactor for applications of immobilized enzymes as well as for soluble enzymes depends on the kinetics of the reaction. Kinetics of immobilized enzymes are not only a function of enzyme activity but also of substrate transport to the enzyme, which is affected by the matrix used for immobilization. For a description of immobilized enzyme kinetics the reader is referred to the comprehensive literature in this field[35 40, 138 14H. Additionally, the use of immobilized enzymes is treated in Chap. 6 of this book. 

To study the kinetics of immobilized enzymes a recirculation reactor may be used. This reactor allows one to perform kinetic measurements with defined external mass transfer effects, reached by establishing a high flow rate near the catalyst, minimizing mass transfer resistance. The reactor behaves as a differential gradientless reactor allowing initial-rate kinetic measurements to be made. 

The attachment of catalytlcally active sites to materials which may be easily recovered from a reaction mixture has been the sine qua non of most useful examples of catalysis outside of enzymology, and this latter area has begun to follow suit (see for example, the six Enzvme Engineering Conferences,as well as several summary texts ). In a pleasantly exhaustive review of the kinetics of immobilized enzyme systems, Goldstein several years ago assigned "the effects of Immobilization on the kinetic behavior of an enzyme" to four situations ... 

Fig. 4.8 Kinetics of immobilized enzyme under external diffusional...

In recent years there has been much interest in immobilized enzymes, where the enzyme has been attached to a solid support. This has the advantage in technical work of allowing an enzyme preparation to be used many times. The study of the kinetics of immobilized enzymes is also valuable in leading to an understanding of how enzymes behave in living systems, where they are often immobilized. Kinetic studies have shown that in such systems the rate is sometimes diffusion controlled (Section VII). 

Immobilized enzyme reactors are increasingly popular due to their advantages over conventional catalysts. For efficient reactor design and performance prediction, quantitative knowledge of reaction kinetics and the factors affecting them is required. In this chapter, enzyme catalytic mechanisms are described and the kinetic models developed from these mechanisms are discussed. The chapter also discusses the kinetics of immobilized enzymes and their related mass transfer effects. Diffusion restrictions are described with a particular focus on packed bed reactors. The chapter concludes with a brief discussion of immobilized enzyme reactor design and scale-up. 

Semetz, M. Puchinger, H. Couwenbergs, C. Ostwald, M. A new method for the evaluation of reaction kinetics of immobilized enzymes... 

The response of the immobilized enzyme electrode can be made independent of the enzyme concentration by using a large excess of enzyme at the electrode surface. The electrode response is limited by the mass transport of the substrate. Using an excess of enzyme often results in longer electrode lifetimes, increased linear range, reduced susceptibiUty to pH, temperature, and interfering species (58,59). At low enzyme concentrations the electrode response is governed by the kinetics of the enzyme reaction. 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

In the design and operation of various bioreactors, a practical knowledge of physical transfer processes - that is, mass and heat transfer, as described in the relevant previous chapters - are often also required in addition to knowledge of the kinetics of biochemical reactions and of cell kinetics. Some basic concepts on the effects of diffusion inside the particles of catalysts, or of immobilized enzymes or cells, is provided in the following section. 

Enzyme thermistors have also found applications in more research-related topics, such as the direct estimation of the intrinsic kinetics of immobilized bio-catalysts [64]. Here, the enzyme thermistor offered a rapid and direct method for the determination of kinetic constants (K , Km and Vm) for immobilized enzymes. For the system being investigated, saccharose and immobilized invertase, the results obtained with the enzyme thermistor and with an independent differential reactor system were in very good correlation, within a flow-rate range of 1 to 1.5 ml/min. 

In addition to the analytical applications, there was sporadic work on the employment of flow calorimetry for the investigation of enzyme kinetics [23,24]. In 1985 Owusu et al. [25] published the first report on the use of flow microcalorimetry for the study of immobilized enzyme kinetics approaching... 

The properties of immobilized enzyme preparations are governed by the properties of both the enzyme and the carrier material. The specific interaction between the latter provides an immobilized enzyme with distinct chemical, biochemical, mechanical and kinetic properties (Fig. 1). 

Fig. 6. Kinetics of immobilization of glutaryl-7-ACA-acylase on epoxy-activated polymethacrylate. The Gl-7-ACA-acylase was incubated with the epoxy-activated carrier. At definite times aliquots were taken from the reaction suspension. Supernatant and carrier-fixed enzyme were separated by centrifugation. The carrier-fixed enzyme was washed with water to remove non-covalently linked enzyme. The activities of the immobilized enzyme and supernatant were determined (5 mM potassium phosphate buffer pH 8,37°C, 2% glutaryl-7-amino cepha-losporanic acid, pH-stat 8.0). Simultaneously, an aliquot of carrier-fixed enzyme was boiled in sodium dodecylsulfate (SDS)/glycine buffer and the supernatant was subjected to SDS-polyacrylamide electrophoresis (see insert from left to right lane 1 Carrier-fixed enzyme, 2 h lane 2 Carrier-fixed enzyme, 4 h lane 3 Carrier-fixed enzyme, 6 h lane 4 Carrier-fixed enzyme, 21 h lane 5 Carrier-fixed enzyme, 69 h lane 6 Dialyzed enzyme lane 7 Supernatant, 2 h lane 8 Supernatant, 21 h lane 9 Supernatant, 69 h lane 10 Molecular weight calibration markers)...

The peroxidase activity of catalase in solution and in immobilized state on both types of soot was studied on the oxidation of phenol. The kinetics of the enzyme reaction was monitored spectrophotometrically by the decrease in the concentration of the substrate at Xma. = 270 nm. 

By comparison with feedback methods, generation collection offers greater sensitivity to low activities of immobilized enzyme. An estimate of the minimum catalytic rate, kCM, of the immobilized enzyme which can still be detected and quantified can be made on the basis of the analytical sensitivity of the tip collector. If c is the detection limit of the tip, then enzyme kinetic data can be obtained if... 

The other important phenomenon that, in addition to the mass transfer, occurs when enzymes become heterogeneous catalysts, is the partitioning of substrates, products, inhibitors, metal and hydrogen ions between a bulk solution and a carrier. An elegant and simple theory describing the effect of microenvironment inside the particles of immobilized enzymes on their kinetics, has been developed by the group... 

In many cases it is not possible or desirable to register the reaction catalyzed by an enzyme. It may be that the product is difficult to detect or that the sensitivity in the analysis one needs to apply is not high enough. Then the use of one or more additional enzymes is quite common. The strategy has been worked out for soluble enzymes, and, in the flow systems, the enzymes are either immobilized separately, or coimmobilized. The latter approach has certain advantages in the sense that a better kinetic performance can be observed in a coimmobilized enzyme sequence as compared to when the enzymes are immobilized separately [52]. However, since most assays are based on the use of an excess of immobilized enzymes, no dramatic differences are observed. 

The kinetic aspects of immobilized enzymes are rather complicated. A typical situation is when the enzyme is immobilized within some polymeric material, which may be cut into slices and immersed in a suitably buffered solution of the substrate. This is the type of situation that occurs in a biological system, an example being a muscle (in which the enzyme myosin is immobilized) surrounded by a solution of the substrate ATP. For reaction to occur, the substrate has to diffuse through the polymeric material in order to reach the enzyme. Reaction then occurs and the products must diffuse out into the free solution. Since diffusion in polymeric materia occurs more slowly than in water, there is now a greater possibility of diffusion control (see p. 403) the overall rate of reaction may depend to some extent on the rates with which these diffusion processes occur. 

---