Solid enzymes

The clear filtrate is very active and may be used directly for the following experiments. Alternatively the solid enzyme may be isolated as follows. To the iiltrate add an equal volume of alcohol to precipitate the enzyme filter off the... 

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-... 

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). 

Quality improvement, 27 171-178 history of, 27 171 impact of, 27 179 Quality, in maintenance, 75 479 Quality management, 27 173-178 Quality manual, 27 165 Quality-of-life indicators, 24 176 Quality, of solid enzyme formulations, 70 273... 

The most straightforward way of using sohd enzymes in organic media is to suspend the solid enzyme directly in the solvent. If one wants to get quick results from a bioconversion and does not want to optimise the efficiency of the enzyme, this method is the obvious first choice. There are many example in the hterature where enzymes have been used successfully in synthesis just as powders directly from the enzyme manufacturer. Sometimes there is a need to dissolve the enzyme powder and re-lyophilise it from a buffer with a more suitable composition, see the section 9.6 pH control in non-conventional media . 

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... 

Granulation of Enzymes. Although the trend is to market industrial enzymes as liquid products, solid enzyme is needed. Examples of this are enzymes for solid detergents, animal feed, and flour improvement. 

Instead of assuming a solid enzyme, in which active center the substrate molecule is bent (the concept of substrate strain), the idea was developed (Koshland 1958,1966) that enzymes can embrace the substrate molecule flexibly in the active center and effect reaction by the formation of specific interactions, the so-called induced jit . This picture is especially appropriate with allosterically activated enzymes or in situations in which part of the enzyme molecule has to turn or move over longer distances to effect catalysis (hinge movement), as for instance with most NAD(P)(H)-dependent enzymes (Stillman, 1999). 

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. 

Gunnlaugsdottir et al. (151) studied the phase behavior of the reaction system cod liver oil + ethanol + immobilized lipase in SCCO2 using a sapphire view cell. They concluded that at 9-24 MPa and 313 K, the reaction system was comprised of three phases solid (enzyme)-liquid-vapor throughout the reaction (151). 

Because of the long-term instability of proteins in aqueous solution, enzyme producers and formulators have attempted to produce stable solid enzyme formulations since enzymes were first used in detergents. Initially, commercially produced protease-containing detergents contained spray dried enzymes. As discussed earlier, proteases have the ability to digest themselves via autolysis and are often incompatible with surfactants. These problems are easily overcome by storing the... 

It is well known that the water content of the reaction medium (i.e., the solvent and solid enzyme-containing phase) has a strong impact on nonaqueous enzymology. Moreover, for a given reaction, enzyme preparation, and medium composition, there is an optimal water content for maximizing the enzyme activity, or the initial rate of reaction. The optimal value is a strong function of the presence and concentration of substrates, and properties of the solid phase. Moreover, the enzyme, immobilization matrix, and continuous phase all compete for adsorption/retention of water molecules. Polar solvents are known to strip away water molecules from solid-phase enzymes. ... 

For operation of continuous bioreactors, the solvent type employed has a significant effect on the water retention during esterification. One role of the flowing solvent is to remove the generated water. However, when the solvent is lipophilic, snch as hexane, its water capacity is too small to remove the water at a rate eqnal to water prodnction hence, water accumulates in the solid enzyme phase and leads to irreversible inactivation. The inclusion of a polar co-solvent, or the use of a solvent of intermediate polarity, can improve water removal. The optimal concentration of polar co-solvent is predetermined by equating water removal rate of the co-solvent mixtnre s (water solnbility multiplied by the flow rate) with the water generation rate. Water accnmnlation also occurs when the polarity of the fluid phase decreases... 

After some orientating experiments employing the newly selected liquid enzyme preparation a simple extraction procedure comprising three extractions at pH 8.5 and two at pH 2 could be developed. On comparison of the solid enzyme preparation Optimase M 440 with Solvay Protease L 660, it turned out that the removal of... 

As mentioned in Section 5.3.2.3.2, three pilot batches from 120 to 150 kg of (R,S)-2 had to be carried out according to not yet optimized conditions without a phase separator and using the solid enzyme preparation [38]. Nevertheless, the experiments were successful and a total of 160 kg of (S)-3 in high chemical and optical purity were produced. 

Owing to experience previously gained concerning work-up with the solid enzyme formulations [22] only the liquid enzyme preparations of Novo and Solvay, Alkalase 2.5 L and Protease L 660, respectively, were used in the following studies. Both preparations exhibited nearly the same specific activity in the present reaction. An enzyme concentration of <5% (v/w) with respect to 9 (s/e> 20 [24]) was used in the subsequent experiments of this section. 

Solid enzymes have been used as fixed bed catalysts with vaporized substances.79 As an example, allyl alcohol has been oxidized to acrolein with aldehyde dehydrogenase (9.3). 

One of the expected benefits from using enzymes in supercritical fluids (SCFs) is that mass transfer resistance between the reaction mixture and the active sites in the solid enzyme should be greatly reduced if the reactants and products are dissolved in an SCF instead of running the reaction in a liquid phase. It is expected that the high diffusivity and low viscosity of SCFs will accelerate mass-transfer controlled reactions. 

Two types of mass- transfer can be distinguished for catalysis with heterogeneous catalyst particles. External mass transfer refers to molecular transport between the bulk reaction mixture and the surface of the enzyme particle through a boundary layer. Internal mass transfer is the molecular transport inside the solid enzyme phase. Internal mass transfer occurs within the pores of the catalyst particle to and from the particle surface. Figure 4.9-4 illustrates the definitions of external and internal mass transfer. 

Such solid enzyme preparations are conventionally produced by means of freeze drying or spray drying. As freezedrying process is unsuitable for large-scale productions, spray drying is used as the most fitted process for the industrial mass production of solid enzyme preparations. Especially in the case of solid enzyme preparations to be used in detergents, spray-drying process is most frequently used. 

An addition of 0.5% lactose in a Bacillus alkaline protease spray-drying process is enough to improve the recovery of active enzyme after drying and thermal treatment at 90°C (Table 48.5). In the same way, the exhaust air temperature could be increased by 10°C reaching severe drying conditions without significant loss of protease activity (Table 48.6). The solid enzyme preparation obtained in this process was also excellent in resistance to mechanical pressure [16]. 

N. Yamada and Y. Shoga, Solid enzyme preparation and process for producing the same, EP Patent 0,501,375 A1 (1992). 

Preparation 4r-l Candida strains may be cultivated in a nutritive medium containing assimilable carbon and nitrogen sources, essential mineral matter, trace elements and the like under aerobic conditions, and the medium may be constructed in a conventional manner. After the cultivation, insoluble substances are removed by filtration or centrifuging to prepare a concentrated solution of liquid enzyme, and a culture solution may be subsequently evaporated or concentrated by reverse osmosis. The concentrated solution may be precipitated in a solvent capable of being mixed with salts or water, for example ethanol, or may be dried in a conventional spray manner to prepare a solid enzyme preparation. 

The properties of supported enzyme preparations are governed by the properties of both the enzyme and the carrier material. The interaction between the two provides an immobilized enzyme with specific chemical, biochemical, mechanical and kinetic properties. The support (carrier) can be a synthetic organic polymer, a biopolymer or an inorganic solid. Enzyme-immobilized polymer membranes are prepared by methods similar to those for the immobilized enzyme, which are summarized in Fig. 22.7 (a) molecular recognition and physical adsorption of biocatalyst on a support membrane, (b) cross-linking between enzymes on (a), (c) covalent binding between the biocatalyst and the membrane, (d) ion complex formation between the biocatalyst and the membrane, (e) entrapment of the biocatalyst in a polymer gel membrane, (f) entrapment and adsorption of biocatalyst in the membrane, (g) entrapment and covalent binding between the biocatalyst and the membrane, (h) entrapment and ion complex formation between the biocatalyst and the membrane, (i) entrapment of the biocatalyst in a pore of an UF membrane, (j) entrapment of the biocatalyst in a hollow-fiber membrane, (k) entrapment of biocatalyst in microcapsule, and (1) entrapment of the biocatalyst in a liposome. 

The addition of enzyme-stabilizing agents may improve the stability of the solid enzyme preparation significantly. 

Some of the methods for the removal of water have inherent disadvantages and are therefore not trivial. Evaporation of water from the reaction mixture can only be efficient if the alcohol and acid reactants have a low volatility (high boiling point). On the other hand, recovery of (solid) enzymes from organic solvents in the presence of solid inorganic water-scavengers such as salts or molecular sieves may be troublesome. 

Solid enzymes can be introduced in organic solvents under three main forms crude solid, immobilized, or crystals. 

Crude Solid. The simplest way to use enzymes in organic solvents is to suspend a precipitate or a lyophilisate. The enzyme does not need to be of high purity, but some care should be taken during the preparation. In aqueous solution, the enzyme has an optimal pH, dictated by the ionization state under which the amino acids involved in the catalysis must be to allow activity. The solid enzyme must be in the same ionization state when used in organic solvents (15). For this purpose, it is important to precipitate the enzyme or lyophilize it from a solution buffered at this pH. This applies to the other forms of solid enzyme preparations. The other important point is the drying of the preparation. It has been observed that the secondary structure of proteins can be affected by lyophilization (16). This can be avoided by the use of lyoprotectants such as sorbitol (17) or salts such as KCl (18). 

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