Commercial Applications of Enzymes

Transfer of methyl groups 2.2. Transfer of glycosyl groups 

Reaction CH2CH2OH + NAD — CH3CHO + NADH + H Systematic Name alcohol NAD oxidoreductase (1.1.1.1.) Trivial Name alcohol dehydrogenase 

Because an enzyme is a protein whose function depends on the precise sequence of amino acids and the protein s complicated tertiary structure, large-scale chemical synthesis of enzymes is impractical if not impossible. Enzymes are usually made by microorganisms grown in a pure culture or obtained directly from plants and animals. The enzymes produced commercially can be classified into three major categories (Crueger and Crueger, 1984)  

Industrial enzymes, such as amylases, proteases, glucose isomerase, lipase, catalases, and penicillin acylases 

Analytical enzymes, such as glucose oxidase, galactose oxidase, alcohol dehydrogenase, hexokinase, muramidase, and cholesterol oxidase 

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Another large successful commercial application of enzymes is in the amino acid industry. Amino acids for food and feed fortification, nutritional supplements, or as feedstock for downstream products can be made by fermentation processes, from protein hydrolysates or by chemical synthesis. While chemical synthesis is cheaper for a number of amino acids,, it often produces a racemic mixture. The racemic mixture is successfully resolved on a commercial scale by acylating the amino acids, then using an aminoacylase to remove the acyl group from the L-amino acid and separating the free L-amino acid from the still acylated-D-amino acid. Ajinamoto and other companies, especially in Japan, make large amounts of amino acids by this process. 

Enzymatic reactions in nonaqueous solvents have generated a great deal of interest, fueled in part by the commercial application of enzymes as catalysts in specialty synthesis. The increasing demand for enantiopure pharmaceuticals has accelerated the study of enzymatic reactions in organic solvents containing... 

Enzymes when hosted in reverse micelles can catalyze reactions that are not favored in aqueous media. Products of high-added value can be thus produced in these media. The potential technical and commercial applications of enzyme-containing microemulsions as microreactors are mainly linked to their unique physicochemical properties. The potential biotechnological applications of microemulsions with immobilized biocatalysts such as enzymes are described in Chapter 12 by Kunz and coworkers and in Chapter 13 by Xenakis and coworkers. 

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

Enzymes have been proposed as a means of subtractive shrink-resist treatment. Their use has been discussed already in section 10.4-2. There are difficulties, however, in the commercially successful application of enzymes to wool at present. 

Many applications of enzymes exist today in many, veiy different industries. In this chapter, first a short description is given of the various industries where enzymes are used as processing aids or processed into final products. Further in this chapter examples from the detergent, feed, textile and food industiy are worked out in detail, highlighting technical, commercial and social aspects to reckon with when developing and applying enzymes for these purposes. 

As commercial interest grows in this area and more cost-effective microbial enzymes become available, it is inevitable that the application of enzymes in feed will further expand. In areas where feed enzymes are already widely applied, they have been acclaimed as the most important development in mono-gastric feeding this decade. 

The commercial availability of enzymes or whole cell biocatalysts for a desired biotransformation is freqnently a limiting factor for commercial application of biocatalysts. Enzymes that are cheaply available are typically used in detergents, processing of food, feed and textiles, as well as in waste management applications. Most of these are hydrolytic enzymes, bnt also isomerases (e.g. glucose isomerase) and oxidorednctases are used on indnstrial scale (Table 5.1). 

Analytes are also used to specify the application. Glucose enzyme sensor is an enzyme biosensor measuring the glucose. Characteristics and commercial varieties of enzyme electrodes, especially using glucose oxidase, have been extensively reviewed by Kuan and Guilbault (17). 

Control of Juice Bitterness. A number of advances have been reported in this field since it was last reviewed (3). A commercial application of the cellulose acetate adsorption technique for the removal of limonin from citrus juices was undertaken (49). New sorbent gel forms of cellulose esters for adsorption of limonin were developed (50). Knowledge was gained that limonoids are biosynthesized in citrus leaves and translocated to the fruit (12) and that specific bioregulators can inhibit accumulation of XIV in citrus leaves (15). Additional studies were carried out on the use of neodiosmin to suppress limonin and other types of bitterness (30,51). The influence of extractor and finisher pressures on the level of limonin and naringin in grapefruit juice was reported (34). Also, further studies were conducted on the microbial sources and properties of limonoate dehydrogenase (52), the enzyme that converts XIV to XV and can be used to prevent limonin from forming in freshly expressed citrus juices (53). 

Researchers at Degussa AG focused on an alternative means towards commercial application of the Julia-Colonna epoxidation [41]. Successful development was based on design of a continuous process in a chemzyme membrane reactor (CMR reactor). In this the epoxide and unconverted chalcone and oxidation reagent pass through the membrane whereas the polymer-enlarged organocatalyst is retained in the reactor by means of a nanofiltration membrane. The equipment used for this type of continuous epoxidation reaction is shown in Scheme 14.5 [41]. The chemzyme membrane reactor is based on the same continuous process concept as the efficient enzyme membrane reactor, which is already used for enzymatic a-amino acid resolution on an industrial scale at a production level of hundreds of tons per year [42]. 

In current research, oxidoreductases are second in the number of applications of enzymes in organic synthesis. The number of commercially available biocatalysts of this class has increased tremendously during the last few years and various screening kits for oxidation and reduction are sold. Many oxidoreductases are rather easy to handle, though, in contrast to hydrolases, they are dependent on cofactors [22]. 

The first large-scale commercial application of cross-linked enzyme crystals was the use of glucose isomerase CLCs to produce high-fructose com syrup. While this is not a pharmaceutical or a biotechnological application, it is included here because it serves to demonstrate the economic viability of the technology in a very cost-sensitive business. In this application the CLCs were attached to the surface of a polystyrene-cellulose-titanium oxide composite carrier in a ratio of 9 1 carrier enzyme. The catalyst had a half-life of 150 days at 57°C, and 12-18 tons of dry sugar product could be produced per kilogram of enzyme [37],... 

This book gathers and analyzes information of both basic and applied aspects of heme peroxidases. Peroxidases are oxidoreductases that catalyze the oxidation of a wide range of molecules, using peroxide as electron acceptor. Although they have been proposed for applications in several fields (see for example [8, 9]) there are few industrial processes that utilize peroxidases. The commercial applications of these enzymes are reduced to diagnosis and research [10]. Unfortunately, the... 

More recently, covalent chemical modification has been used as a powerful tool to enhance the functionality and stability of enzymes, for example, the covalent link of flavin to papain turned a protease into an oxido-reductase [107]. The use of this methodology was rekindled as a result of the explosion in the interest in commercial and synthetic applications of enzymes [108]. As a consequence, enzymes with new properties such as stability at extreme pH conditions, temperature, or solubility in organic solvents are being generated. 

While the application of enzymes and proline as catalysts for the (commercial) formation of carbon-carbon bonds is relatively new, transition metal catalysts are well established for the industrial synthesis of carbon-carbon bonds. Although in themselves not always perfectly green, transition metal catalysts often allow the replacement of multi-step and stoichiometric reaction sequences with one single catalytic step. Thus, the overall amount of waste generated and energy used is reduced drastically [61-64]. 

Collagen membranes also bind a variety of enzymes (141). The binding procedure is particularly mild because the enzyme never comes in contact with the chemical resents, avoiding all risks of denaturation. Such membranes, however are too thick and too fragile, especially at 37 °C, to be recommended for in vivo applications of enzyme electrodes (142). Several commercial preactivated membranes are available that provide simple and fast procedures for immobilizing membranes (90-92, 143). The stability of the enzymatic membranes were excellent More than 400 cissays were performed within 50 days. 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

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