Use of immobilized enzymes

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. 

Guilbault, G, Analytical Uses of Immobilized Enzymes, New York, Marcel Dekker 1984... 

In homogeneous catalytic systems we witnessed a new process for the production of acetic acid from methanol and carbon monoxide using a transition metal complex, thus displacing the earlier process employing ethylene as the starting material. The use of immobilized enzymes makes possible the commercial conversion of glucose into fructose. 

When immobilized glucose isomerase was introduced in the early seventies, it was believed, that other industrial applications of immobihzed enzymes would soon be found, but this turned out not to be trae. The main limitation to the use of immobilized enzymes is that the substrate has to be soluble and highly purified in order to avoid clogging of the enzyme bed. In the case of glucose isomerase the cost of purifying the syrup (carbon treatment, ion exchange) before the fixed bed isomerase reactors is substantially higher than the enzyme costs. 

G. G. Guilbault, in Analytical Uses of Immobilized Enzymes, Modern Monographs in Analytical Chemistry, Vol. 2, (G.G. Guibault, Ed.), Marcel Dekker, New York (1984). 

Immobilized Enzymes in Diagnostic Reagents. The use of immobilized. instead of soluble, enzymes for measurement of analytes lias received considerable attemion. especially lor clinical analyses. Use of immobilized enzymes offers the advantages of greater accuracy, stability, and convenience. Only a few meihods utilizing immobilized enzymes have become commercially available. 

Another approach to dealing with the nonelectrochemically active nature of most amino acids is to generate, in situ, chemical reactions at the electrode surfaces to produce electrochem-ically active products for detection. Related to this concept, is the online use of immobilized enzymes (142) to react with amino acids. A by-product of this reaction is hydrogen peroxide, which is then quantified by amperometric detection. 

Polarimetry takes advantage of the optical activity of carbohydrates. The high selectivity of this procedure makes it especially suitable in the case of complex food extracts, where other components would interfere with ultraviolet or refractive index detection. However, a major disadvantage is its lower sensitivity. The use of immobilized enzymes (51) with detection by fluorescence or electrochemistry has also been applied in fermentation juices (52) and other particular cases. 

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

Gal-(1— 3)-D-GalNAc. By using 0.7 U of STB, as a soluble preparation readily obtained from 300 g of porcine liver, the sialylation of /J-D-Gal-(1 — 3)-d-G1cNAc was performed on a one-mmol scale and sialylated trisaccharide 65 was obtained in 21 % isolated yield. In this respect, the purification of reaction mixtures is still troublesome, especially because of the presence of Triton X-100 from our experience, the use of immobilized enzymes, eliminating the need for detergent, greatly facilitates the purification procedure. 

Carbohydrates are used increasingly for the synthesis of antibiotics and their analogues. This subject was discussed by J. G. Moffat and S. Umezawa in London (1978) and by C.-H. Wong in Hamburg (2000). Enzymes came into more extensive use S. David discussed the use of immobilized enzymes in preparative carbohydrate chemistry (Ithaca 1986) and C.-H. Wong further developed this subject in Paris (1992). The mechanism of the action of enzymes was discussed by S. G. Withers in his Whistler Award lecture in Cairns (2002). 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Fan and Zhang determined acetylcholine and choline in rat brain tissue by a fluorescence immunoassay method, making use of immobilized enzymes and chemiluminescence detection [50]. Tissue was homogenized with a 10 fold volume of 0.6 M HC104, the homogenates were kept on ice for 30 min, and then centrifuged at 2000 G for 20 min. The pellets were... 

It can be expected that the use of immobilized enzymes and cells in the food, pharmaceutical, and chemical industnes will continue to expand in the future... 

Descriptions of the current commercial use of immobilized enzyme technology have appeared over the past several years (3,4,29,30) consequently, this area will be summarized briefly to allow more time for discussion of developing technology. 

One of the most important recent developments in the use of enzymes for industrial food processing is the fixing of enzymes on water-insoluble inert supports. The fixed enzymes retain their activity and can be easily added to or removed from the reaction mixture. The use of immobilized enzymes permits continuous processing and greatly increased use of the enzyme. Various possible methods of immobilizing enzymes have been listed by Weetall (1975) and Hultin (1983). A schematic representation of the... 

Many examples of the use of immobilized enzymes in food processing have been reported. One of the most important of these is the use of immobilized glucose isomerase obtained from Streptomyces for the production of high-fructose com syrup (Mermel-stein 1975). In this process, the enzyme is bound to an insoluble carrier such as diethyl amino ethyl cellulose or a slurry of the fixed... 

Examples of the use of immobilized enzymes in food processing and analysis have been listed by Olson and Richardson (1974) and Hultin (1983). L-aspartic acid and L-malic acid are produced by using enzymes contained in whole microorganisms that are immobilized in a polyacrylamide gel. The enzyme aspartase from Escherichia coli is used for the production of aspartic acid. Fumarase from Brevibacterium ammoni-agenes is used for L-malic acid production. 

Hultin, H.O. 1983. Current and potential uses of immobilized enzymes. Food Technol. 37, no. 10 66-82, 176. 

In another advance in the use of immobilized enzymes in analysis, glucose oxidase was combined with an oxygen electrode to give an enzyme electrode for determining glucose concentration (15, 16). The... 

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