Immobilized enzymes production

Other immobilized enzyme product successes (where annual production of immobilized enzymes has surpassed 1 ton/year) comprise Aminoacylase (Amano), hydantoinase (Smith Kline Beecham), lactase (Valio), lipase (Novo Nordisk), penicillin G acylase (Gist-brocades, Smith Kline Beecham, Rohm) and penicillin V acylase (Novo Nordisk/Gist-brocades) (Poulsen, 1984). 

Active immobilized enzyme production of 216 D-gluconic acid from o-glucose... 

Active immobilized enzyme production of D-glucose from starch Active immobilized enzyme... 

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). 

Fig. 6. Diagram of an immobilized enzyme electrode, where S is the substrate and P is the enzyme-bound substrate product.

Multienzyme Electrodes. Coupling the reactions of two or more immobilized enzymes increases the number of analytes that can be measured. An electro-inactive component can be converted by an enzyme to a substrate that is subsequentiy converted by a second enzyme to form a detectable end product (57). For example, a maltose [69-79-4] sensor uses the enzymes glucoamylase and glucose oxidase, which convert... 

In the early years of the chemical industry, use of biological agents centered on fermentation (qv) techniques for the production of food products, eg, vinegar (qv), cheeses (see Milk and milk products), beer (qv), and of simple organic compounds such as acetone (qv), ethanol (qv), and the butyl alcohols (qv). By the middle of the twentieth century, most simple organic chemicals were produced synthetically. Fermentation was used for food products and for more complex substances such as pharmaceuticals (qv) (see also Antibiotics). Moreover, supports were developed to immobilize enzymes for use in industrial processes such as the hydrolysis of starch (qv) (see Enzyme applications). 

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. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

Membrane reactors, where the enzyme is adsorbed or kept in solution on one side of an ultrafHtration membrane, provides a form of immobilized enzyme and the possibiHty of product separation. 

When selecting a suitable feed symp, the main criteria are optimization of enzyme productivity and minimization of the formation of by-products. Typical feed symp specifications are shown in Table 5. Higher symp concentration and higher viscosity results in a reduced isomerization rate due to diffusion resistance in the pores of the immobilized enzyme. A deaeration step is desirable to remove dissolved oxygen that would otherwise iacrease the formation of by-products. The pH is adjusted to the optimum level for the productivity of the enzyme. 

During operation, the immobilized enzyme loses activity. Most commercial enzymes show decay as a function of time (Eig. 12). The glucose isomerase ia a reactor is usually replaced after three half-Hves, ie, when the activity has dropped to around 12.5% of the initial value. The most stable commercial glucose isomerases have half-Hves of around 200 days ia practical use. To maintain the same fmctose content ia the finished symp, the feed-flow rate is adjusted according to the actual activity of the enzyme. With only one isomerization reactor ia operation, the result would be excessive variations ia the rate of symp production. To avoid this, several reactors at different stages ia the cycle of enzyme decay are operated ia combiaation. 

Immobilized system the air circulates over a film of microorganisms that grows on a solid surface. In an immobilized bioreactor, particulate biocatalysts for enzyme production and conversion of penicillin to 6-aminopenicillanic acid are used. 

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]. 

Since many years, pectolytic enzymes have been widely used in industrial beverage processing to improve either the quality and the yields in fruit juice extraction or the characteristics of the final product [1,2]. To this purpose, complex enzymatic mixtures, containing several pectolytic enzymes and often also cellulose, hemicellulose and ligninolytic activities, are usually employed in the free form. The interactions among enzymes, substrates and other components of fruit juice make the system very difficult to be investigated and only few publications are devoted to the study of enzymatic pools [3-5], An effective alternative way to carry out the depectinisation process is represented by the use of immobilized enzymes. This approach allows for a facile and efficient enzymatic reaction control to be achieved. In fact, it is possible to avoid or at least to reduce the level of extraneous substances originating from the raw pectinases in the final product. In addition, continuous processes can be set up. 

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

Biocatalytic membrane electrodes have an ISE or a gas sensing electrode in contact with a thin layer of biocatalytic material, which can be an immobilized enzyme, bacterial particles or a tissue slice, as shown in Fig. 3 The biocatalyst converts substrate (the analyte) into product, which is measured by the electrode. Electrodes of this type are often referred to as biosensors . 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

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-... 

Unlike in the previous example, here the catalysts are not reported specifically to deactivate one another. Rather, the immobilization of the lipase in these silica elastomer spheres allows access to reaction conditions that are otherwise unavailable, namely higher temperatures, as the lipase is no longer deactivated and is able to undergo multiple reaction cycles, resulting in a much higher enzyme productivity, in terms of mass of product per unit mass of protein. The activity of the lipase is also observed to have increased, possibly not only from its ability to access higher temperatures when immobilized, but also due to the increased stability of the active... 

For application of protein-immobilized porous materials to sensor fields, use of an electroactive substance as the framework material is important. DeLouise and Miller demonstrated the immobilization of glutathione-S-transferase in electrochemically etched porous silicon films [134], which are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. Not limited to this case, practical applications of nanohybrids from biomolecules and mesoporous materials have been paid much attention. Examples of the application of such hybrids are summarized in a later section of this chapter. 

---