Properties of immobilized biocatalysts

Immobilization by chemical cross-linking without the addition of an inert carrier or matrix can provide the means to stabilize and reuse a biocatalyst without dilution of volumetric activity. A major deficiency in all of the aforementioned immobilization methods is that a substantial amount of a catalytically inert carrier or matrix is used to bind or contain the biocatalyst. In many cases, the amount of carrier is two orders of magnitude higher than the protein catalyst. Unfortunately, direct cross-linking of the enzyme, followed by precipitation of an amorphous solid often results in low activity and poor mechanically properties and so this method is not often used. Recently, however, cross-linked enzyme crystals have been reported to give many of the desirable properties of immobilized enzymes without the need for a support material (Sect. 6.4.1). 

As with most heterogeneous catalysts, it is often difficult to characterize immobilized enzymes at a molecular level. Most immobilized preparations are often complex mixtures with a distribution of chemically modified protein species. The gross catalytic properties observed are a composite of those of a range of differentially modified individual proteins, often irregularly distributed within the sample. Mass transfer limitations and microenvironment effects further complicate characterization. 

---

Stefuca V, Gemeiner P, Kurillova L, Danielsson B, Bales V (1990) Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system. Enzyme Microb Technol 12 830 - 835 Stefuca V, Welwardova A, Gemeiner P, Jakubova A (1994) Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst. Biotech-nolTech 8 497-502... 

In the following section examples are given that illustrate the application of the principles introduced above to the study of the properties of immobilized biocatalysts. 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

Experience shows that flow microcalorimetry is a universal technique that is suitable for the investigation of the catalytic properties of immobilized biocatalysts. This review has summarized all basic examples of its application, but has not exhausted all of their potential possibilities. As an example, the steady-state measurement of a bi-substrate enzyme reaction with a co-immobilized glucose oxidase-catalase system was reported [26]. However, there is no report on the evaluation of kinetic properties of partial enzymes in co-immobilized systems. Even the measurement of the overall heat produced in such systems does not provide direct information about partial reactions. We believe that new approaches to analyze these systems based on mathematical modeling can be developed. 

The complexity of the physical and catalytic properties of immobilized biocatalysts and the difficulty in comparison of effectiveness based on literature descriptions has led to the publication of guidelines for the characterization of immobilized bio-catalysts1351. The authors suggest that description of parameters listed in Table 6-4 should be the minimum required for characterization of an immobilized preparation. 

Gemeiner et al. (1993) presented a similar method for the direct determination of catalytic properties of immobilized cells. Cephalosporin C transforming Trigonopsis variabilis were immobilized by three different methods, filled into a column and set into the ET. After thermal equilibration, Cephalosporin C solutions (0.1-50 mmol/1) were continously pumped through the ET until steady-state heat production was obtained. Again, the ET was shown to be suitable for a rapid and simple estimation of the kinetic properties of immobilized cells. Microkinetic factors such as mass transfer were taken into account (Stefuca et al. 1994). Thus, ET measurements allow us to obtain intrinsic data, even from immobilized cells. Moreover, the data can be applied to optimize biocatalyst design and bioreactor models (Gemeiner et al. 1996). 

Another type of stability of immobilized biocatalysts is the retention of activity after periodic use in batch processes, as has been reported previously for penicillin acylase entrapped in polyacrylamide gel [40]. This option can be used to advantage for rapid monitoring of biocatalyst activity under conditions of industrial application. Apart from the measurement of activity as an indication of the necessity to replace the biocatalyst, the periodic analysis of the variation of kinetic properties permits greater insight into deviation from the optimal parameters. 

Flow microcalorimetry, which makes many rapid and accurate measurements of the activity of immobilized biocatalysts, provides a tool for researchers that can be used to discriminate between different preparatives of immobilized biocatalysts. Table 3 shows previous experiments where the characterization of kinetic properties by flow micro calorimetry was used to compare different techniques of purified enzyme immobilization [27, 30, 31, 35] as well as the immobilization of enzymes fixed in cells [28,29,40]. More details can be found in our recent review article [41]. 

Several kinds of states in which enzymes may be used for various reactions in aqueous-organic biphasic systems have been developed in previous work (Table 2). In biphasic media, the biocatalyst is easily recovered after the reaction then it is not always necessary to be immobilized. Nevertheless, the immobilization can confer important properties, such as improved stability of biocatalyst. Furthermore, protection of the biocatalyst against a damaging turbulent environment can also play a role. 

A remaining crucial technological milestone to pass for an implanted device remains the stability of the biocatalytic fuel cell, which should be expressed in months or years rather than days or weeks. Recent reports on the use of BOD biocatalytic electrodes in serum have, for example, highlighted instabilities associated with the presence of 02, urate or metal ions [99, 100], and enzyme deactivation in its oxidized state [101]. Strategies to be considered include the use of new biocatalysts with improved thermal properties, or stability towards interferences and inhibitors, the use of nanostructured electrode surfaces and chemical coupling of films to such surfaces, to improve film stability, and the design of redox mediator libraries tailored towards both mediation and immobilization. 

Because the monoliths allow total convection of the mobile phase through their pores, the overall mass transfer is dramatically accelerated compared to conventional porous structures. Based on the morphology and porous properties of the molded monoliths, which allow fast flow of substrate solutions, it can be safely anticipated that they would also provide outstanding supports for immobilization of biocatalysts, thus extending the original concept of monolithic materials to the area of catalysis. 

The recent literature in bioelectrochemical technology, covering primarily the electrochemical aspects of enzyme immobilization and mediation, includes few reports describing engineering aspects of enzymatic biofuel cells or related devices. Current engineering efforts address issues of catalytic rate and stability by seeking improved kinetic and thermodynamic properties in modified enzymes or synthesized enzyme mimics. Equally important is the development of materials and electrode structures that fully maximize the reaction rates of known biocatalysts within a stable environment. Ultimately, the performance of biocatalysts can be assessed only by their implementation in practical devices. 

In Figure 10.1 the time course of thermodynamically and kinetically controlled processes catalysed by biocatalysts are compared. The product yield at the maximum or end point is influenced by pH, temperature, ionic strength, and the solubility of the product. In the kinetically controlled process (but not in the thermodynamically controlled process) the maximum yield also depends on the properties of the enzyme (see next sections). In both processes the enzyme properties determine the time required to reach the desired end point. The conditions under which maximum product yields are obtained do not generally coincide with the conditions where the enzyme has its optimal kinetic properties or stability. The primary objective is to obtain maximum yields. For this aim it is not sufficient to know the kinetic properties of the enzyme as functions of various parameters. It is also necessary to know how the thermodynamically or the kinetically controlled maximum is influenced by pH, temperature and ionic strength, and how this may be influenced by the immobilization of the biocatalysts on different supports. 

Petri A, Gambicorti T, Salvadori P (2004) Covalent Immobilization of Chloroperoxidase on Silica Gel and Properties of the Immobilized Biocatalyst. J Mol Catal B Enzym 27 103... 

From the previous paragraph it can be discerned that the half-life of a biocatalyst is determined not only by the deactivation properties of the free enzyme but also by the mass-transfer properties of an immobilisate. Figure 19.7 reveals the factor by which the dependence on temperature increases operating stability in the case of immobilized enzyme. 

The biocatalyst a-chymotrypsin s ability to hydrolyze 20 is inhibited in the presence of copolymer 19a loaded with 0.2 mol% of the triphenyl carbinol units. 47b Photoirradiation of 19a results in heterolytic bond cleavage and the formation of the cationic copolymer 19b. In this polymer structure, the biocatalyzed hydrolysis of 20 is activated (V = 1.0 pM min-1). The polymer-induced photostimulated activation and deactivation of a-chymotrypsin in the different membrane environments correlates with the permeability and transport properties of the substrate 20 through the different structures of the polymer membranes.1471 Flow dialysis experiments showed that the polymer states 17a, 18a, and 19a are nonpermeable to 20, and hence the biocata-lytic functions of the immobilized enzyme are blocked. The polymer structures 17b,... 

The choice of reactor configuration depends on the properties of the reaction system. For example, bioconversions for which the homogeneous catalyst distribution is particularly important are optimally performed in a reactor with the biocatalyst compartmentalized by the membrane in the reaction vessel. The membrane is used to retain large components, such as the enzyme and the substrate while allowing small molecules (e.g., the reaction product) to pass through. For more labile molecules, immobilization may increase the thermal, pH and storage stability of biocatalysts. 

Aoun S, Chebli C, Baboulene M (1998) Noncovalent immobilization of chloroperoxidase onto talc catalytic properties of a new biocatalyst. Enzyme Microb Technol 23 380-385... 

The different properties of ILs, with regard to their polarity, hydrophobicity, and solvent miscibility behavior through combination with different anions, are the reason for the different biocatalyst activities. Good to excellent activity of CALB was observed with a decrease in polarity and hydrophobicity and a viscosity increase of the ILs. In [bmim][PF6] a conversion of (R)-l-phenylethanol into the ester of 48.9% and an ee of 95.6% were achieved after 5h and 100% of (R)-l-phenylethanol was converted into the enantiopure (R)-l-phenylethyl acetate after a 1-day reaction. Immobilized CALB exhibited excellent stability, activity, and selectivity towards the (R)-enantiomer of 1-phenylethanol in [bmim][PF6]. In some research bis(trifluoromethylsulfonyl)imide-based ILs have been regarded as very suitable media for biocatalysis [39, 46, 50]. On the contrary, in the present work, lower suitability of the same IL was demonstrated. Since immobilized CALB catalyzed both hydrolytic and transesterification reactions, its enantioselectivity for long reaction times was lower. 

Membrane reactors using biological catalysts can be used in enantioselective processes. Methodologies for the preparation of emulsions (sub-micron) of oil in water have been developed and such emulsions have been used for kinetic resolutions in heterogeneous reactions catalyzed by enantioselective enzyme (Figure 43.4). A catalytic reactor containing membrane immobilized lipase has been realized. In this reactor, the substrate has been fed as emulsion [18]. The distribution of the water organic interface at the level of the immobUized enzyme has remarkably improved the property of transport, kinetic, and selectivity of the immobilized biocatalyst. 

---