Solid biocatalyst

FIG. 1 Reaction systems containing organic solvent, and corresponding theoretical concentration profiles for water insoluble substrate. solid biocatalyst organic phase aqueous phase or biocatalyst dissolved in aqueous phase. 

One of the most extensively applied techniques to retain and reuse the enzyme in continuous operation is the immobilization onto a support. The outlet port is provided with a nylon membrane to retain the solid biocatalyst inside the... 

Visual observations of the phase behavior at different reaction conditions are presented in Figure 8.2. The reaction was carried out in a liquid reaction bulk in the contact with the solid biocatalyst at 9 MPa. At a pressure close to 11 MPa at the chosen temperature and composition conditions the supercritical phase was attained for the whole reaction bulk. 

Figu re 8.2 Visual vapour-liquid phase equilibrium observations of the reaction bulk liquid reaction bulk in contact with the solid biocatalyst up to 6MPa (left panel) liquid reaction bulk in contact with the solid biocatalyst up to 9MPa (middle panel) supercritical reaction bulk in contact with the solid catalyst upon 15 MPa (right panel). 

The biocatalytic process takes place in stirred tank reactors with sieves in the bottom and a working volume of several cubic meters (Fig. 7). The first biocatalytic step is a three-phase reaction with a solid biocatalyst, immobilized DAO on a spherical carrier, the liquid Ceph C solution and oxygen or air as the gaseous phase. The second step is a two-phase reaction with immobilized GA on a solid carrier and the glutaryl-7-ACA in solution. 

In aqueous media, the most usual state of the enzyme molecules is dissolved in the reaction medium. In this case, the previous treatment of the enzyme has little effect on catalytic activity, provided irreversible inactivation is avoided. In contrast, in low-water media, the enzyme molecules are usually present in solid particles. The way the solid biocatalyst is prepared will clearly affect the state of the enzyme molecules, and hence their catalytic properties. Furthermore, as hydration of the enzyme molecules is reduced, it is clear that conformational changes can become much slower. As a result the previous history of the enzyme has important effects, not just the final conditions. In other words, there may be pronounced hysteresis effects. 

Often the easiest way to set the initial water activity of components of the reaction mixture is by pre-equilibration with a saturated salt solution. The relative humidity or water activity is fixed above a saturated solution of a given salt at a known temperature. As water equilibrates in or out of the solution, solid salt will tend to dissolve or crystallize to maintain saturation and hence the fixed water activity in the headspace. Any other material placed in contact with the headspace will eventually equilibrate to the same water activity. The reaction mixture component can simply be placed inside a closed vessel together with the salt solution, such that water can transfer between the two via the vapour phase. Wide-mouth screw cap jars are convenient, with salt solution over the base and an open vial containing the sample (Fig. 8-3). The rate of equilibration depends on the surface areas exposed and the amount of water that must be transferred. Typically 1-2 days is sufficient for either solid biocatalyst preparations or liquid phases based on relatively non-polar organic solvents. The rate of equilibration may be checked by weighing or Karl Fischer analysis respectively. 

A decision must be made about the sequence and timing in which components are combined to make the final reaction mixture. The choices made can have large effects on the final hydration conditions and biocatalyst behavior. It is usually best initially to prepare as separate phases (i) a non-aqueous solution or mixture of the reactants and (ii) the solid biocatalyst preparation (lyophilized powder, immobilised enzyme, cross-linked crystal etc). The best treatment to apply then depends on the objective of the experiment. 

Immobilized system the air circulates over a film of microorganisms that grows on a solid surface. In an immobilized bioreactor, particulate biocatalysts for enzyme production and conversion of penicillin to 6-aminopenicillanic acid are used. 

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. 

Catalytic transformations can be divided on the basis of the catalyst-type - homogeneous, heterogeneous or enzymatic - or the type of conversion. We have opted for a compromise a division based partly on type of conversion (reduction, oxidation and C-C bond formation, and partly on catalyst type (solid acids and bases, and biocatalysts). Finally, enantioselective catalysis is a recurring theme in fine chemicals manufacture, e.g. in the production of pharmaceutical intermediates, and a separate section is devoted to this topic. 

Separation of the aqueous, oil, and biocatalyst (solid/biomass paste) phases ... 

A fixed bed or slurry bioreactor incorporates the biocatalyst immobilized on a solid support in an aqueous solution, mineral nutrients and an assimilable source of carbon. 

The concept of zeolite action was tested in a particular reaction where the enzyme is exposed from the beginning to an acidic environment the esterification of geraniol with acetic acid catalyzed by Candida antarctica lipase B immobilized on zeolite NaA [219]. Lipases have been used for the hydrolysis of triglycerides and due to their ambivalent hydrophobic/hydrophilic properties they are effective biocatalysts for the hydrolysis of hydrophobic substrates [220]. When water-soluble lipases are used in organic media they have to be immobilized on solid supports in order to exhibit significant catalytic activity. 

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