Enzymes loading

For excess substrate concentrations, based on the Michaelis-Menten assumption, the enzyme concentration increases as the rate of the reaction increases. This is due to more enzyme molecules (and thus more active sites) being available to catalyze the reaction. Therefore, more enzyme-substrate complexes are formed. However, in real situations a point is reached where every substrate molecule gets bound to the enzyme, and thus further increases in enzyme concentration do not increase the reaction rate. This point is usually referred to as substrate saturation. The Michaelis-Menten model does not take this into consideration. 

Since enzymes are proteins, any environmental factor that affects protein structure may chauge enzyme activity. Denaturation is the distraction of the secondary and tertiary structures of the enzyme. This can be reversible or irreversible. Denaturation is said to be reversible when the enzyme regains its native structure after removing the enlistment stick, whereas in irreversible denaturation, regaining its structure is not possible. Such structural changes are the result of bond disruption and changes in the ionization of the active sites. 

Supercritical Fluids Technology in Lipase Catalyzed Processes 

The pH can also affect ionization of the substrate and rednce its concentration. The effect on ionized snbstrate is shown in Equation 4.29  

Equations 4.30 and 4.31 represent the rate equations for the two cases, respectively, where in both cases, Michael s constant, K f, is affected. 

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Erythrocyte Entrapment of Enzymes. Erythrocytes have been used as carriers for therapeutic enzymes in the treatment of inborn errors (249). Exogenous enzymes encapsulated in erythrocytes may be useful both for dehvery of a given enzyme to the site of its intended function and for the degradation of pathologically elevated, diffusible substances in the plasma. In the use of this approach, it is important to determine that the enzyme is completely internalized without adsorption to the erythrocyte membrane. Since exposed protein on the erythrocyte surface may ehcit an immune response following repeated sensitization with enzyme loaded erythrocytes, an immunologic assessment of each potential system in animal models is required prior to human trials (250). 

Enzymatic hydrolysis is affected by numerous process factors including pH, temperature, reaction time, and enzyme loading. These factors often interact with one another. In the current study the main and interactive effects of four factors (reaction time, enzyme to... 

Multiple-enzyme conversion of CO2 to formate, then formaldehyde, then methanol by FateDH, FaldDH, and ADH Enzymes loaded in CaC03 followed by LbL deposition and dissolution of core, then encapsulation into a gel bead... 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

Capsules with high enzyme loading and activity prepared by templating BMS spheres can also be used as biomimetic reactors [89]. For example, PGA/ PLL capsules with pre-loaded urease (prepared via a BMS sacrificial template) are capable of catalyzing the hydrolysis of urea and have been shown to induce the exclusive formation of CaC03 particles inside the capsules [89]. 

Stone et al. use this method to simultaneously synthesize the silica and entrap the butyrylcholinesterase which retains all its activity after the process of encapsulation, a high enzyme loading (90 %) is reached and the stability is increased [168]. The method has been further developed to simultaneously entrap catalase and horseradish peroxidase with inorganic magnetic nanopartides [169] which will fadlitate the separation [170,171]. 

In ion-selective FETs (ISFETs), an ion-selective membrane replaces the gate electrode. When an enzyme-loaded gel is combined with the membrane, the device can be used to measure substrates which enzymically generate charged species. 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

An alternate method to produce templated electrodes is the use of chemical reduction of the monomer in the presence of a track-etched or alumina membrane. Parthasarathy et al. [46] have produced enzyme-loaded nanotubules by a combination of both electrochemical and chemical deposition. Initially, the alumina membrane was sealed at one end with a thick Au film (Figure 1.9a), after which the membrane was placed into a mixture of pyrrole and Et4NBF4. The pyrrole was then electropolymerized to form a small plug of polypyrrole at the closed end of the alumina membrane (Figure 1.9b). Subsequently, the membrane was placed into a... 

Figure 1.9 Schematic diagram for the method used to produce and enzyme load microcapsule arrays. (Reprinted with permission from Ref [46]. 1994 Nature Publishing Group.)...

Conversely, controlled immobilization of enzymes at surfaces to enable high-rate direct electron transfer would eliminate the need for the mediator component and possibly lead to enhanced stability. Novel surface chemistries are required that allow protein immobilization with controlled orientation, such that a majority of active centers are within electrontunneling distance of the surface. Additionally, spreading of enzymes on the surfaces must be minimized to prevent deactivation due to irreversible changes in secondary structure. Finally, structures of controlled nanoporosity must be developed to achieve such surface immobilization at high volumetric enzyme loadings. 

Both digester systems exhibit extremely low levels of detectable cellulase activities (exoglucanase, endoglucanase, and -glucosidase) when compared to industrial saccharifying processes (See Table III) in which the hydrolysis of cellulose in the feedstock is optimized with respect to enzyme loading. Therefore, the data indicate the level of improvement that may be made to attain maximum rates for cellulose hydrolysis in the anaerobic reactor system. 

The use of a monolithic stirred reactor for carrying out enzyme-catalyzed reactions is presented. Enzyme-loaded monoliths were employed as stirrer blades. The ceramic monoliths were functionalized with conventional carrier materials carbon, chitosan, and polyethylenimine (PEI). The different nature of the carriers with respect to porosity and surface chemistry allows tuning of the support for different enzymes and for use under specific conditions. The model reactions performed in this study demonstrate the benefits of tuning the carrier material to both enzyme and reaction conditions. This is a must to successfully intensify biocatalytic processes. The results show that the monolithic stirrer reactor can be effectively employed in both mass transfer limited and kinetically limited regimes. 

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