Enzyme preparation solid enzymes

By gentle warming, dissolve 5 g. of salicin in 100 ml. of water contained in a 250 ml. conical flask. Add about 20 ml. of the emulsin solution as prepared above or o 2-0 3 solid enzyme prepara-... 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Catalytic processes frequently require more than a single chemical function, and these bifunctional or polyfunctional materials innst be prepared in away to assure effective communication among the various constitnents. For example, naphtha reforming requires both an acidic function for isomerization and alkylation and a hydrogenation function for aromati-zation and saturation. The acidic function is often a promoted porous metal oxide (e.g., alumina) with a noble metal (e.g., platinum) deposited on its surface to provide the hydrogenation sites. To avoid separation problems, it is not unusual to attach homogeneous catalysts and even enzymes to solid surfaces for use in flow reactors. Although this technique works well in some environmental catalytic systems, such attachment sometimes modifies the catalytic specifici-... 

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). 

Several enzymes like lipases, esterases, and dehydrogenases have been active in hydrophobic environments. Thermodynamic water activity is a good predictor of the optimal hydration conditions for catalytic activity [51]. Enzyme preparation can be equilibrated at a specific water activity before the reaction [52]. When water concentration is very low, enzyme is suspended in the solid state in the water-immiscible organic solvent [46]. Enzymes are easily recovered after the reaction by the method of filtration. 

In another study, the carrier protein was replaced by an enzyme compatible solid-phase resin (PEGA), and enzyme-catalyzed cyclization was used to probe substrate specificity. This study demonstrated also that oxo-esters are tolerated as substrates for TE domains, and then-preparation in library format served as an excellent tool for substrate specificity studies, as well as for preparation of cyclized peptides. Figure 13.11 shows how the TycA TE showed selectivity for only residues 1 and 9 (colored in red), and changes at all other residues were tolerated [42]. Hydrogen bonding interactions are shown in green. Several compounds made from this series were shown to demonstrate improved therapeutic indices (with respect to hemolysis) while retaining antimicrobial activity. 

There are at least three reasons for attempting to prepare solid-phase catalysts that resemble enzymes. Synthetic procedures would generally be simplified. Catalytic groups are fixed on the support so that they cannot interact with one another, for example, thiols cannot deactivate by forming disulfides and metal ions cannot deactivate by forming binuclear structures. Finally, if the successful catalyst is eventually made, it will almost certainly be used in heterogeneous systems. 

Flitsch, S.L, Lahja, S., Turner, N.J. Solid phase preparation and enzymic and non-enzymic bond cleavage of sugars and glycopeptides, PCT Int. Appl. 1997, patent EP 9605535, CAN 127 81736. 

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

For practical purposes it is often beneficial to use a heterogeneous system with the enzyme as a solid preparation which easily can be separated from the product in the liquid phase. Solid enzyme preparatiorrs can conveniently be used in packed bed and stirred tank reactors. As in other cases with heterogeneous catalysis, mass trarrsfer limitations can reduce the overall reaction rate, but usually this is no major problem. 

Solubilised enzyme preparations are well suited for many ftmdamental studies, for example spectroscopic investigations reqtriring transparent solutiorrs. When the solubilised preparatiorrs are used as catalysts it is an advantage that mass trarrsfer limitations are normally absent, but product isolation and ertzyme recovery are usirally more difficult than with solid enzyme preparations. Methods used to separate the enzyme from the product solution include precipitation of the enzyme, and the use of membranes which retain the enzyme but not the product. 

If a solid enzyme preparation is used, mass transfer may hmit the reaction rate. 

In a liquid/liquid biphasic system (Figure 9.1a), the enzyme is in the aqueous phase, whereas the hydrophobic compounds are in the organic phase. In pure organic solvent (Figure 9.1b) a solid enzyme preparation is suspended in the solvent, making it a liquid/solid biphasic system. In a micellar system, the enzyme is entrapped in a hydrated reverse micelle within a homogeneous organic solvent... 

Immobilization. The fixing property of PEIs has previously been discussed. Another application of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on solid supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus facilitating preparative work. This technique has been applied to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

For immobilized lipase preparations, a more complex mechanism is expected to occur since esterification efficiency is also highly dependent on the hydration state of the enzyme preparation, which can be greatly modified by the nature of the substrate and the support (1,4)- In the case of butyl butyrate synthesis, analysis of substrate polarity measured as partition coefficient (Table 1) showed a higher value for butanol than for butyric acid, favoring butanol migration to the solid phase (immobilized lipase). Thus, there should be more alcohol than acid at the active site of the immobilized lipase, requiring an excess of acid in the reaction medium to provide equimolar amounts of reactants and satisfactory yields (7). 

Sacrificial sampling was used to assess the activity of sol-gel-immo-bilized CPO over a 3-h period. H202 was provided via the in situ reaction with GOx. Experiments were conducted in by preparing solutions of sol-gel enzyme preparations in assay buffer in a centrifugal filter unit. About 10 mg of sol-gel enzyme was used for each assay. The purpose of the filter was to allow easy separation of the solid-phase enzyme for measurement... 

The enzyme mixture of 20 ml containing immobilized recombinant penicillin G amidase as the enzyme, 10% hydroxyethyl ester of 4-hydroxy-D-phenylglycine, 4% cefprozil (amine source), and 8% enzyme (immobilized recombinant penicillin G amidase, equivalent to 32 IU/ml of enzyme) was made up without buffer. The above prepared ester solution (6.9 ml) was mixed with water (2 ml) and adjusted to pH 7.5 with 10 N NH4OH. Then the amine source (0.8 g) was added to it and the pH adjusted to 7.5 with 1 N NH4OH and the volume to 18.4 ml. Then the mixture was cooled to 5-15°C and solid enzyme (1.6 g 640 IU) was added to it. The pH was not maintained at 7.5 and fell about 0.6 units during the reaction. The reaction mixture was analyzed by HPLC on a C18 Reverse Phase column. The mobile phase was 10% acetonitrile/0.3% H3P04. The isomers of cefprozil appeared at 2.9 minutes (cis) and at 5.1 minutes (trans). The final product was obtained with a maximum yield of 92-96%. The whole experiment was completed in 25-50 min. 

Interesterification of blends of milk fat and palm kernel olein by a mycelium-bound lipase from Rhizomucor miehei or a commercially immobilized enzyme preparation resulted in a lower slip melting point and solid fat content. An interesterified product made from a 70 30 mixture of palm kernel olein and anhydrous milk fat was considered to be suitable for use in ice cream (Liew et al., 2001). 

Bohenin occurs as a white to light tan, waxy solid. It is a triglyceride containing behenic acid at the 1- and 3-positions and oleic acid at the 2-position. Behenic acid is a saturated fatty acid that occurs naturally in peanuts, most seed fats, animal milk fat, and marine oils. It is produced by the interesterification of triolein and ethyl behenate in the presence of a suitable lipase enzyme preparation. It melts at approximately 52°. It is insoluble in water soluble in hexane, in chloroform, and in acetone and slightly soluble in hot ethanol. 

Most commercial products are a mixture essentially of four individual enzymes pectin esterases, polygalacturonases, pectin lyases and pectate lyases. Depending on the intended use, the enzyme preparations have different contents of the individual pectolytic enzymes. The enzyme preparations are commercially available in liquid or solid form they originate mainly from mould cultures. 

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