Immobilization enzymes catalyzing

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-... 

Dezotti, M. Innocentini-Mei, L.H. Duran, N. Silica immobilized enzyme catalyzed removal of chlorolignins from eucalyptus Kraft effluent. J. Biotechnol. 1995, 43, 161-167. 

The results obtained can be interpreted as shown in Fig. 5C. The immobilized enzyme catalyzed the hydrolysis of urea into ammonia and carbon dioxide ... 

Siddique MH, St Pierre CC, Biswas N et al (1993) Immobilized enzyme catalyzed removal of 4-chlorophenol from aqueous solution. Water Res 27 883-890... 

This is useful when the overall performance of an immobilized enzyme catalyzed process has to be competitive with other technologies, such as preparation of the desired product by fermentation or by whole cell biotransformations. 

We are presuming that the intrinsic reaction kinetics of the immobilized enzyme catalyzed reaction is of the Michaelis-Menten type, therefore eq. (9.231) takes the form... 

Important progress has been made on the use of immobilized enzyme catalyzed polycondensation reactions to prepare a wide range of polyesters (2,83). In this book, Hunsen et al (20) describe how an inunobilized catalyst from Humilica insolens has excellent activity for polycondensation reactions between diols and diacids. The natural role of cutinases is hydrolysis of the outer cutin polyester layer on plant leaves. Cutin is made of long chain... 

Al-Muftah, A. E., and I. M. Abn-Reesh. 2005. Effects of Internal Mass Transfer and Prodnct Inhibition on a Simnlated Immobilized Enzyme-Catalyzed Reactor for Lactose Hydrolysis. Biochemical Engineering Journal 23 139-153. 

Enzyme and microbial kinetics involve the study of reaction rates and the variables that affect these rates. It is a topic, that is critical for the analysis of enzyme and microbial reacting systems. The rate of a biochemical reaction can be described in many different ways. The most commonly used definition is similar to that employed for traditional reactors. It involves the time change in the amount of one of the components participating in the reaction or of one of the products of the reaction this rate is also based on some arbitrary factor related to the system size or geometry, such as volume, mass or interfacial area. In the case of immobilized enzyme catalyzed reactions, it is common to express the rate per unit mass or per unit volume of the catalyst. 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

Impractical and Theoretical Considerations The operation of an enzyme electrode is illustrated in Figure 6-1. The immobilized enzyme layer is chosen to catalyze a reaction, which generates or consumes a detectable species ... 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

For desymmetrization of diesters 3 via their hydrolysis in water, pig Hver esterase [12], o -chymotrypsin [12, 13a], and Candida antarctica Hpase (CAL-B) [14] were successfully used. However, further studies showed that respective anhydrides 5 can be used as substrates for enzyme-catalyzed desymmetrization in organic solvents [15]. The desired monoesters 4 were obtained in high yield in this way, using immobilized enzymes Novozym 435 or Chirazyme L-2 (Scheme 5.3). After the reaction, enzymes were filtered off, organic solvents were evaporated, and the crude products were crystalHzed. This was a much simpler experimental procedure in which control of the reaction progress was not necessary, and aU problems associated with extraction of products from aqueous phase and their further purification were omitted [15]. 

An intriguing influence of a cosolvent immiscible with water on the enantioselec-tivity of the enzyme-catalyzed hydrolysis was observed. It was proven that enzyme enantioselectivity is directly correlated with the cosolvent hydrophobicity. In the best example, for ethyl ether as cosolvent, the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7% ee. This finding may be of great practical importance, particularly in industrial processes [24], since it will enable better optimization of enzyme-catalyzed processes. It is clear that, in future, immobilized enzymes, as heterogeneous catalysts, wiU be widely used in most industrial transformations, especially in the preparation of pharmaceuticals [25]. 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Other solutions to dealing with interferences in the detection of H O have included the use of a copperfll) diethyldithiocarbamate precolumn to oxidize the sample before it reaches the immobilized enzyme, as well as the use of a palladium/gold sputtered electrode which catalyzes the oxidation of hydrogen peroxide In addition, peroxidase has been used to catalyze the reaction between hydrogen peroxide and iodide ferrocyanide and organo-fluorine compounds Am-... 

In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context. 

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