Enzyme preparation immobilized enzymes

Enzyme preparation. The enzyme preparation Lipozyme IM, which is a Rhizomucor miehei lipase, immobilized on a macroporous anion exchange resin, was kindly donated from NOVO Nordisk AS (Copenhagen, Denmark). 

Fig. 3 Thermostability of acetylated P-glucosidase from Novozym 188 preparation in free and immobilized forms. I Free enzyme, 2 immobilized enzyme, t 70 °C, pH 5.0...

The method of preparation of the lipase is important and can greatly affect its reactivity. The pH of the aqueous solution the enzyme was last in as well as the method by which water was removed can both influence the enzyme activity. Immobilized enzymes can aid recovery and reuse of the biocatalyst In addition, there are a range of different enzyme supports available. 

Adsorption is the simplest and fastest way to prepare immobilized enzymes. Adsorption can roughly be divided into two classes physical adsorption and chemical adsorption. Physical adsorption is weak and occurs mainly via van der Waals interactions. Chemical adsorption is stronger and involves the formation of covalent bonds. 

In these systems, solid enzyme preparations (e.g. lyophilized or immobilized on a support) are suspended in an organic solvent in the presence of enough aqueous buffers to ensure catalytic activity. Although the amount of water added to the solvent (as a rule of thumb <5% v/v) may exceed its solubility in that solvent, a visible discrete aqueous phase is not apparent because part of it is adsorbed by the enzyme. Therefore, the two phases involved in an organic solvent system are a liquid (bulk organic solvent and reagents dissolved in it) and a solid (hydrated enzyme particles). 

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]. 

It is not only the activity that can be altered by incorporation of noncoded amino acids. Introduction of structures possessing certain chemical functions leads to the possibility of highly regioselective modification of enzymes. For example, selective enzymatic modification of cystein residues with compounds containing azide groups has led to the preparation of enzymes that could be selectively immobilized using click chemistry methods [99]. 

A commercial pectinase, immobilised on appropriately functionalised y-alumina spheres, was loaded in a packed bed reactor and employed to depolymerise the pectin contained in a model solution and in the apple juice. The activity of the immobilized enzyme was tested in several batch reactions and compared with the one of the free enzyme. A successful apple juice depectinisation was obtained using the pectinase immobilised system. In addition, an endopolygalacturonase from Kluyveromyces marxianus, previously purified in a single-step process with coreshell microspheres specifically prepared, was immobilised on the same active support and the efficiency of the resulting catalyst was tested. 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

The porosity of hybrid nanocomposites provides access of the substrates to immobilized enzyme and their proper functioning. It is attributable to the absence of volume shrinkage of synthesized materials after their preparation. Although the compacting does not occur as in the common two-stage processes (Figure 3.7), enzyme macromolecules are held inside the silica matrix and not easily washed out of it. 

Materials required AChE preparations, commercial acetylcholinesterase, horse (blood) serum preparation, human (blood) serum preparation, or matrix biotests with these immobilized enzyme (see section 15.2.1)... 

Finlay, T.H., Troll, V., Levy, M., Johnson, A.J., and Hodgins, L.T. (1978) New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine. Anal. Biochem. 87, 77-90. 

L. Korecka, Z. Bilkova, M. Holeapek, J. Kralovsky, M. Benes, J. Lenfeld, N. Mine, R. Cecal, J.-L. Viovy, and M. Przybylski, Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments. Journal of Chromatography, B Analytical Technologies in the Biomedical and Life Sciences 808, 15-24 (2004). 

Precision was generally excellent, and sensitivities were 100-1000-fold higher than with spectrophotometric methods. The immobilized-enzymes preparations were stable for several months and each reactor could be used for 600-800 analyses [229],... 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Effective and simple immobilization of enzymes can be obtained by the cross-linking of enzyme aggregates, so-called CLEAs [55]. In this way, essentially any enzyme, including crude preparations, can be transformed into a heterogeneous type of material, insoluble in both water and organic solvents, that is stable and recyclable with high retention of the enzyme s original activity [56], These enzyme preparations are, therefore, of special value for both bio-bio and bio-chemo cascade processes. 

Enzymes are valuable analytical tools and offer sensitive and specific methods of quantitation for many substances. The increasing availability of highly purified enzyme preparations, both in solution and in immobilized forms, permits the development of a wide range of methods. 

There are many instruments designed for either enzyme assays or substrate assays using enzymes. Information on the analytical capabilities of these instruments will be supplied by the manufacturers. This will often include protocols for specified assays using kits of commercially available pre-prepared reagents. These may be in liquid or dry form and may, for substrate assays, include immobilized enzymes. The facility to be able to develop additional automated methods on a particular instrument will depend upon its design and some instruments are dedicated solely to specified analyses. 

It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. 

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