Enzyme support system

Plasma-polymerised HMDSO film was used to produce a biocompatible surface and an enzyme support system [85]. The adsorption of urease onto a well-defined solid support, petroleum-based activated charcoal, has been achieved to provide the enzymatic hydrolysis of urea. The adsorption of urease, and the activity and stability of the enzyme on the support were studied and optimised, improving its availability for clinical applications. 

The characteristics of a support material are of great importance to the measured enzyme activity [79, 101]. Hydrophobic carriers have a low ability to attract water, thus leaving more available for the enzyme, hence Wehtje et al. [102, 103] have shown that celite is a suitable carrier for the PaHnl to yield an immobilized form of the enzyme. In contrast, controlled pore glass (CPG) and Sephadex G25 were found to be less well suited to enzyme support as, using these systems, cyanohydrin synthesis was significantly reduced (over 30%). Sephadex also promoted the spontaneous addition of HCN to benzaldehyde [102]. A series of batch experiments showed that if the solvent (diisopropyl ether) surrounding the immobilised PaHnl contained insufficient water (i. e. less than 2 %), it would be extracted from the enzyme preparation and consequently enzyme activity was lost [102]. These results were the basis for the production... 

Artificial liver support systems aim at the extracorporeal removal of water soluble and protein-bound toxins (albumin being the preferential binding protein) associated with hepatic failure. Albumin contains reversible binding sites for substances such as fatty acids, hormones, enzymes, dyes, trace metals and drugs [26] and therefore helps elimination by kidneys of substances that are toxic in the unbound state. It should be noticed that the range of substances to be removed is broad and not completely identified. Clinical studies showed that the critical issue of the clinical syndrome in liver failure is the accumulation of toxins not cleared by the failing liver. Based on this hypothesis, the removal of lipophilic, albumin-bound substances, such as bilirubin, bile adds, metabolites of aromatic amino acids, medium-chain fatty acids, and cytokines, should be benefidal to the dinical course of a patient in liver failure. 

Many enzymes are stable and catalyze reactions in supercritical fluids, just as they do in other non- or microaqueous environments (7). Enzyme stability and activity may depend on the enzyme species, supercritical fluid, water content of the enzyme/support/reaction mixture, decompression rates, exposure times, and pressure and temperature of the reaction system. 

In fermentation reactors, cell growth is promoted or maintained to produce metabolite, biomass, transformed substrate, or purified solvent. Systems based on macro-organism cultures are usually referred as tissue cultures. Those based on dispersed non-tissue forming cultures of micro-organisms are loosely referred as microbial reactors. In enzyme reactors, substrate transformation is promoted without the life-support system of whole cells. Frequently, these reactors employ immobilized enzymes, where an enzyme is supported on inert solids so that it can be reused in the process. Virtually all bioreactors of technological importance deal with a heterogeneous system involving more than two phases. 

The development of enzyme-catalyzed processes in organic solvents makes it possible to perform enzymatic analysis in organic solvents. Earlier work involved the addition of moderate amounts of solvents to improve substrate solubility, but the new trend is to operate in almost water-free conditions. The selection of reaction parameters is important. Thus, it is necessary to optimize the solvent (118,119) as well as the enzyme support (120). The polarity of the solvent is also important the more polar the solvent, the less stable the enzyme (119). Thus, extremely hydrophobic solvents are useful, provided the substrates and products are soluble. The choice of support is governed by its tendency to attract minute amounts of water present in the system. The supports are characterized with regard to their aquaphilicity There is an inverse correlation between aquaphilidty and catalytic activity of the adsorbed enzyme (121). 

In applying this system, a biotinylated binding molecule (e.g., antigen, primary or secondary antibody) is allowed to interact with a target system (cells, microtiter plates, and so on). By uang an appropriate avidin-conju-gated probe (e.g., fluorescent or electron-dense marker, solid support, enzyme), the system can be used for a variety of different applications. 

Water affects the reaction rate through its effect on reaction kinetics and protein hydration, which is required for optimal enzyme conformation and activity. Enzymes need a small amount of water to maintain their activity however, increasing the water content can decrease the reaction rate as a result of hydrophilic hin-drance/barrier to the hydrophobic substrate, or because of denaturation of the enzyme (189). These opposite effects result in an optimum water content for each enzyme. In SCFs, both the water content of the enzyme support and water solubilized in the supercritical phase determine the enzyme activity. Water content of the enzyme support is, in turn, determined by the distribution/partition of water between the enzyme and solvent, which can be estimated from water adsorption isotherms (141, 152). The solubility of water in the supercritical phase, operating conditions, and composition of the system (i.e., ethanol content) can affect the water distribution and, hence, determine the total amount of water that needs to be introduced into the system to attain the optimum water content of the support. The optimum water content of the enzyme is not affected by the reaction media, as demonstrated by Marty et al. (152), for esterification reaction using immobilized lipase in n-hexane and SCC02- Enzyme activity in different solvents should, thus, be compared at similar water content of the enzyme support. 

A distribution coefficient is used widely in various areas involving two-phase systems [43,44] to describe behaviour of immobilized enzymes, electrode systems, different kinds of chromatographic separation and, in particular, makes it possible to correlate analytically parameters describing equilibria on a surface with parameters of column and thin-layer chromatography, whose success is determined mostly by extensive use of pristine and modified silicas as adsorbents and supports. 

Other Friedel-Crafts catalysts are also being developed these include clay-supported metal halides [32] and mesoporous silica supported systems [33], Even enzymes can be used to perform Friedel-Crafts reactions [34]. 

Enzymes are not catalytically active if water is completely absent. The often cited explanation is that at least a monolayer of water per enzyme molecule is necessary to keep the enzyme active [40]. Apparently, the essential noncova-lently bound water maintains the enzyme s native protein structure. In an enzymatic reaction under supercritical conditions, the water partitions between the enzyme, the enzyme support and the reaction mixture. In an essentially non-aqueous system, the existing water partitions preferrably to the solvent with increasing hydrophilicity. If there is little water in the system and if the solvent is relatively hydrophilic, the solvent may strip the essential water from the enzyme, making it inactive. When Zaks and Klibanov first noted that enzymes were more active in hydrophobic solvents than in hydrophilic organic solvents. 

The water content has probably the strongest effect in reaction rates of all process parameters of the supercritical reaction system. The water concentrations in the fluid phase and in the enzyme support phase depend on the adsorption isotherms between the fluid and solid phases. The form of the adsorption isotherm depends on the fluid, the enzyme support material, and on the substrates in the reaction mixture. Water partitioning data for SCFs and enzyme supports are available from several papers. 

For example, Yoon et al. report that the water adsorption isotherm for an immobilized lipase (Lipozyme IM) and pure scCOa follows eq (4.9-1) where w is the water concentration in the enzyme support (wt%), C is the water concentration in CO2 (mM) and Co is the water solubility in CO2 (mM) under system pressure and temperature [41], Marty et al. report water adsorption isotherms (33 °C, 40 °C, 50 °C) between a microporous anionic resin support and ethanol containing SCCO2 at three pressures (110, 130 and 170 bar) [9]. Water partitioning data for an anionic resin support and pure SCCO2 are also available [42,43]. 

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