Conversion rate, immobilized enzyme

The measurable activity reflects the biocatalytic efficiency of an immobilized enzyme. In homogeneous solution the initial rate of substrate conversion rises linearly with enzyme concentration. The reaction rate is influenced by substrate diffusion only at extremely large degrees of conversion. With immobilized enzymes the measured reaction rate depends not only on the substrate concentration and the kinetic constants Km and vmax but also on so-called immobilization effects. These effects are due to the following alterations of the enzyme by the immobilization process (Kobayashi and Laidler, 1974). 

A significant advantage of immobilized enzymes is the total absence of catalytic activity in the product. Moreover, the degree of substrate-to-product conversion can be controlled during processing, eg, by adjusting the flow rate through a packed-bed column reactor of immobilized enzyme. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

The rate equation of a reaction with an immobilized enzyme is r = 5tjC/(0.05+C). The inlet concentration to a three-stage CSTR is 1.2 and 80% conversion is required. Find the required residence time per stage when the effectiveness is 60% or 100%. 

It can be seen from Eq. (5) that the maximum possible concentration on the surface, c, influences significantly the transport rate. This parameter is a function of the available surface area as well as of the density of the reactive sites. Because of that, the matrix structure plays a very important role in such adsorp-tion/desorption processes. In the case of biological reactions, where the chemical conversion is performed by immobilized enzymes, the immobilization also plays an important role in order to achieve an optimal enzyme density on the reactive surface. 

Immobilized enzymes used in conjunction with ion-selective electrodes provide very convenient methods of analysis. The immobilized enzyme may be held in a gel or membrane around the electrode and the substance to be measured diffuses into the enzyme gel. Its conversion to the product alters the ionic equilibrium across the ion-selective membrane (Figure 8.23). It is important that the enzyme layer is thin, to minimize any problems caused by slow diffusion rates through the layer. 

When immobilized enzymes are employed in a continuous reactor, many of these limitations are avoided. Moreover, in this case the output signal is recorded at the reactor outlet, and this procedure therefore cannot affect the processes taking place in the reactor, and the signals obtained can be used in another system as actual concentrations without conversion. Yet, in this configuration the cofactor enters the reactor in the feed stream, which requires large amounts of cofactor, especially when a high flow rate is employed. 

Conversely, controlled immobilization of enzymes at surfaces to enable high-rate direct electron transfer would eliminate the need for the mediator component and possibly lead to enhanced stability. Novel surface chemistries are required that allow protein immobilization with controlled orientation, such that a majority of active centers are within electrontunneling distance of the surface. Additionally, spreading of enzymes on the surfaces must be minimized to prevent deactivation due to irreversible changes in secondary structure. Finally, structures of controlled nanoporosity must be developed to achieve such surface immobilization at high volumetric enzyme loadings. 

Figu re 3.3 Conceptual process model for application of a coupled tyrosinase-laccase reaction converting tyrosol. Immobilized enzymes are first characterized with respect to substrate conversion rates, using tyrosol and hydroxytyrosol as substrates for tyrosinase and laccase, respectively. One hundred percent conversion can be achieved in Reactor 1 by use of sufficient tyrosinase... 

In case of reversible immobilization, minimum amount of the enzyme can be added, whenever required, to obtain optimum conversion rates. This results in considerable economy in cases where costly enzymes are required. 

Highly efficient enzyme membrane reactors can be also produced by immobilizing enzymes in membranes or in hollow fibers. For example, enzymes can be confined in the porous support matrix of an asymmetric capillary membrane, while substrate-containing solution flows through the fiber lumen. The dense skin layer at the lumen wall should be impermeable to the enzyme molecules. The latter diffuse through the inner wall of the fiber to the enzyme into the spongy part, where the conversion takes place. Applied transmembrane pressure and axial flow rate are parameters that contribute to control of the reactor performance. 

Experiments with enzymes in SCFs revealed very early that scCOa could strip the essential water from the enzyme. Randolph et al. found that damp immobilized enzyme lost its activity when exposed to bone-dry carbon dioxide. The activity was quickly regained when they injected a small amount of water into the system [12]. Dumont also reported results from an immobilized lipase/C02 system where the conversion rate decreased when the enzyme was in contact with a dry COa/substrate flow. Reaction rates were restored completely when they directed the CO2 flow through a water saturator [13]. 

Chirazyme L2-C2 (CAL-B) proved to be a very useful enzyme for the development of an acylation process for the large-scale production of vitamin A (retinol, 91) at Roche (Scheme 27) [90,91]. In the plant process of vitamin A, intermediate 88 is partially acylated and then subjected to acid-catalyzed dehydration and isomerization to yield the vitamin A ester 90 via acetate 89. Contrary to the chemical acylation, an enzymatic approach allowed for a highly selective monoacylation of 88, and Chirazyme L2-C2 showed a very high conversion rate at 30% (w/w) substrate concentration. A first continuous process on the laboratory scale was set up with a 15 ml fixed-bed reactor containing 5.0-8.0 g of immobilized biocatalyst 4.9 kg of 89 was synthesized within 100 days in 99% yield and with 97% selectivity for the primary hydroxyl group. The laboratory process was implemented in a miniplant (120 g of biocatalyst), which could convert 1.4 kg of 88 into 1.6 kg 89 per day. After 74 days the conversion efficiency was still 99.4%. Further development of this transformation led to a modified process, which uses Thermomyces lanuginosus lipase immobilized on Accurel MPlOOl for the continuous production of 89 [92]. 

The efficiency factor for an immobilized enzyme. In general the conversion rate of an immobilized enzyme is lower than that of an equal amount of the free en me. This decreased activity is caused by diffusional limitations to the rate at which the subtra-te is transported to the site of reaction in the immobilized enzyme particles. In chemical engineering the subject of the interplay between diffusional limitations and chemical kinetics in heterogeneous catalysis has been extensively studied. The state of the art on this subject is described by Satterfield (). 

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