When enzymes are to be used in organic media, noncovalent immobilization methods are often used because enzymes in general are insoluble in those media. Usually, immobilization increases the observed catalytic activity drastically as compared with nonimmobilized enzyme powder. The main reason is that immobilization spreads the enzyme on a relatively large area, which facilitates the mass transfer of substrates and products. The morphology of the support material is thus of crucial importance. In many applications, a monolayer of enzyme on the support provides catalysts with good activity. In general, the enzyme is first present in an aqueous solution and water is then removed using a drying procedure. Simple and effective methods for the adsorption of lipase on porous polypropylene and deposition of a-chymotrypsin on Celite® are presented in this chapter.
Key Words: Adsorption; a-chymotrypsin; Celite; deposition; lipase; porous polymer. 1. Introduction
As a result of intense research over the past two decades, it is now generally realized that many enzymes express catalytic activity in organic media, and many useful applications in synthesis have therefore been presented (1). In this chapter "organic media" is defined as media consisting predominantly of organic solvents or other organic substances. Solvent free media, in which the substrates to be converted function as solvents as well, constitute an important example. The organic media contain small amounts of water, which the enzymes need in order to express catalytic activity. Ionic liquids and supercritical fluids have also been used as media for enzymatic reactions. In these applications, they share several characteristics with organic media, mainly because of the low water content in all these media. Immobilization of enzymes for use in ionic liquids and supercritical fluids is treated separately in Chapters 23 and 24.
Proteins are soluble in very few organic solvents (2). This means that in the majority of the organic media the enzymes are insoluble. It is therefore unnecessary to immobilize the enzymes in order to make it easier to separate them from the reaction mixture after the reaction. In many small-scale reactions, enzyme powders have been used as catalysts. However, immobilization is usually beneficial in order to improve the properties of the enzyme preparation. It is therefore quite common to immobilize enzymes by adsorption or deposition on porous supports before using them in organic media. The support material can be chosen so that it has suitable properties to be used in the intended reactor. In mechanically stirred reactors the materials should be resistant to shear forces, whereas in packed bed reactors the compressibility is more important because of its influence on the pressure drop of the reactor. In general, immobilization of the enzyme on a suitable support increases the observed catalytic activity as a result of effective dispersion of the enzyme on the surface of the support (3).
Enzyme immobilization methods for organic media are generally quite simple. An aqueous solution of the enzyme is mixed with the support, and the water is removed so that the enzyme is deposited on the surface of the support. The pH value of this solution is important. It has been said the enzyme "remembers" the pH of the last aqueous solution in which it was dissolved (4). The ionization state of the enzyme will remain essentially unchanged—unless acids or bases are present in the reactant solution—once the water has been removed and the enzyme preparation is put into an organic medium . Usually the aqueous solution used for enzyme immobilization is thus buffered to a pH value close to the pH optimum of the enzyme.
In some cases, enzymes adsorb spontaneously on the support. For example, it is possible to adsorb lipases efficiently on hydrophobic supports, such as porous polypropene. In this case, it is practical to use a rather large volume of aqueous solution in contact with the support. Lipases and other hydrophobic substances adsorb on the support whereas other substances remain in the solution. After complete adsorption, the immobilized enzyme is recovered by filtration. In this method, polar impurities in the enzyme powder used to prepare the solution are efficiently removed and thus immobilization simultaneously constitutes an enzyme purification step.
Not all enzymes are suitable for immobilization by adsorption. Some enzymes are adsorbed too strongly and thereby lose activity whereas others are not adsorbed efficiently enough. For the latter case, "deposition" constitutes a useful alternative method. Here, the enzyme is dissolved in a smaller volume of aqueous buffer, which is mixed with the support and followed by drying of the complete mixture. In this case, everything present in the solution is deposited on the support. This procedure has been found useful for a wide range of enzymes on a wide range of supports. Celite® is a typical support used in the deposition method.
The removal of water is a critical step in the immobilization processes for organic media. Most commonly, this is achieved by drying or freeze drying, but in some cases these procedures cause partial inactivation of the enzyme. Removal of water by treatment with water-miscible solvents such as propanol has been presented as a method to prepare highly active immobilized enzyme preparations for organic media (5).
Whatever immobilization method is used, the specific activity of an enzyme that has been immobilized on a support material and used in an organic medium is influenced by the nature of the support and by the enzyme loading (amount of enzyme per amount of support material). At low-enzyme loadings, low-specific activities are often observed as a result of inactivation of the enzyme (6). Presumably, an interaction between the enzyme and the support that is too strong is the main reason, and the chemical nature of the support is thus of key importance. In addition, the morphology of the support has a large influence (7). The specific surface area is a key parameter and it should be considered in relation to the enzyme loading. It has been observed that maximal specific activity is often obtained when the enzyme forms a monolayer on the surface of the support. At enzyme loadings too low to form a monolayer, the specific activity can often be increased by adding another protein (e.g., albumin) or another polymer (e.g., polyethylene glycol [PEG]) to protect the enzyme from inactivation (6). Under other conditions, mass transfer limitations can reduce the observed specific activity of the immobilized enzyme. This is common at high-enzyme loading and/or large particle size. Finally, the pore size of the support is of vital importance. The pores should be large enough to permit penetration by the enzyme during the immobilization procedure and diffusion of substrates and products during the reaction. In a lipase-catalyzed reaction, it was observed that maximal specific activity was obtained at a mean pore diameter of about 100 nm (8).
Simple protocols for adsorption of enzymes on hydrophobic supports and deposition of enzymes on Celite are described in Subheading 3. A third method of importance uses adsorption on ion exchange materials. Quite efficient adsorption is achieved when the ion exchanger and the enzyme are oppositely charged. Finally, it should be mentioned that covalent attachment of enzymes on supports is sometimes used in organic media, particularly when the reaction mixtures contain surfactants or other substances that can cause solubilization of the enzyme in the medium. Many of the methods described for covalent immobilization for aqueous media can be used in organic media as well.
2.1.Adsorption of Lipase on Porous Polypropene
1. Porous polypropene, Accurel MP1000 (Membrana GmbH, Obernburg, Germany; see Note 1).
2. 4.5-mL Glass vials with caps.
5. Lipase powder. Lipases immobilized using this procedure include those from
Aspergillus niger, Candida rugosa, Penicillium roquefortii, Pseudomonas fluorescence, Rhizopus oryzae, and Thermomyces lanuginosus (9).
6. End-over-end mixer.
7. Sintered-glass filter funnel, Buchner flask, and connection to suction pump.
8. Vacuum desiccator.
9. Vacuum pump (to give a pressure of 20 mbar or lower).
2.2. Deposition of a-Chymotrypsin on Celite
4. Deionized water.
5. Nitric acid.
6. Overhead stirrer.
8. Vacuum desiccator.
9. Vacuum pump (to give a pressure of 20 mbar or lower). 3. Methods
This procedure has been used for the immobilization of several different lipases (9). The immobilized preparations obtained are useful for a wide range of reaction including ester synthesis (3), racemate resolution (10), and lipid conversions (11).
1. Dissolve the lipase in 20 mM phosphate buffer, pH 6.0 (see Note 5). A suitable concentration is 0.1 to 1.0 mg lipase protein/mL.
2. Mix 50 mg Accurel MP1000 with 150 ||L of 95% ethanol in a 4.5 mL capped vial.
3. Fill the glass vial completely with enzyme solution.
4. Incubate the vial on an end-over-end mixer at room temperature for at least 3 h (see Note 6).
5. Separate the immobilized preparation from the remaining solution by filtration. Wash the immobilized preparation briefly with the immobilization buffer (see Note 7).
6. Dry the immobilized preparation at reduced pressure in a vacuum desiccator overnight (see Note 8).
This procedure is a slightly modified version of a previously published method used to prepare Celite-immobilized a-chymotrypsin (12). Virtually the same method has been used for the immobilization of several other enzymes, including other proteases (13), lipases (14), oxidoreductases (15), and lyases (16). It is often very useful when trying a new enzyme in organic media.
1. Wash 50 g of Celite twice with 500 mL 95% ethanol each followed by 1500 mL deionized water each time for 15 times. After each washing, the liquid is decanted thus removing fines.
3. Add 7 g of the washed Celite to a round flask containing 50 mL concentrated nitric acid. Stir the mixture with an overhead stirrer at 90°C for 4 h. Avoid grinding the particles (see Note 9).
4. Wash the Celite ten times with 100 mL deinonized water.
6. Dissolve 180 mg a-chymotrypsin in 6 mL 50 mMTris-HCl buffer, pH 7.8 (see Note 10).
7. Add the a-chymotrypsin solution to 6 g of the acid washed Celite. Mix thoroughly (see Note 11).
8. Spread out the preparation on the bottom of a wide beaker, which is put in a vacuum desiccator. Dry under vacuum overnight (see Note 8)
1. Different types of porous hydrophobic materials are useful for enzyme adsorption. Porous polypropene has been used extensively. The activity of the immobilized enzyme varies somewhat depending on the characteristics of the support. The porous polypropene Accurel EP-100 from Akzo has provided immobilized preparations with high activities in many studies, but this material is no longer available. However, the new material Accurel MP1000 available from Membrana GmbH gives similar results.
2. In order to get maximal catalytic activity, the buffer used should have a pH value close to the pH optimum of the enzyme. The buffer mentioned here, 20 mM Na-phosphate buffer, pH 6.0, has been used successfully for a wide range of lipases.
3. Celite of different types can be used. Depending on the chacteristics, differences in catalytic activity are obtained. Celite intended for gas chromatography from BDH (available from VWR) with mesh size 30 to 80 or 80 to 120 has been widely used, but there are many other useful types commercially available.
4. The buffer should be chosen to fit the enzyme to be immobilized (see Note 2). Both the type of buffer and its concentration will have an influence on the catalytic activity of the immobilized preparation.
5. If the lipase powder contains substances which are insoluble in water, those can be removed by centrifugation or filtration.
6. The adsorption process can be followed by measuring protein content and enzyme activity in the solution. Lipase adsorption is usually complete after 3 h. Often adsorption has been carried out overnight with similar results.
7. A small amount of aqueous buffer will remain in the immobilized preparation after filtration. During drying, the buffer salts will be deposited on the support surface. Because the buffer salts can influence the activity of the immobilized preparation, it is advisable to standardize the immobilization procedure to get reproducable results.
8. The drying process can be monitored gravimetrically or by water analysis.
9. Different types of Celite vary concerning the need for acid washing. By using the procedure described, immobilized preparations with high and reproducible catalytic activity have been obtained.
10. Make the solution just before using it. Prolonged incubation can lead to autolysis and reduced catalytic activity.
11. Here, 1 mL of aqueous solution is used with 1 g of Celite. If it is difficult to achieve uniform wetting of the Celite with these proportions, the volume of the aqueous solution can be increased (by dilution with water).
1. Koskinen ,A. M. P. and Klibanov, A. M. (1996) Enzymatic Reactions in Organic Media. Chapman & Hall, London, UK.
2. Chin, J. T., Wheeler, S. L., and Klibanov, A. M. (1994) On protein solubility in organic solvents. Biotechnol. Bioeng. 44, 140-145.
3. Persson, M., Mladenoska, I,. Wehtje, E.,and Adlercreutz, P. (2002) Preparation of lipases for use in organic solvents. Enzyme Microb. Technol. 31, 833-841.
4. Zaks, A. and Klibanov, A. M. (1985) Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. USA 82, 3192-3196.
5. Moore, B. D., Partridge, J., and Halling, P. J. (2001) Very high activity biocatalysts for low-water systems: Propanol-rinsed enzyme preparations. In: Enzymes in Nonaqueous Solvents (Vulfson, E. N., Halling, P. J., and Holland, H. L., eds.) Humana Press, Totowa, NJ, pp. 97-104.
6. Wehtje, E., Adlercreutz, P., and Mattiasson, B. (1993) Improved activity retention of enzymes deposited on solid supports. Biotechnol. Bioeng. 41, 171-178.
7. Barros, R. J., Wehtje, E., Garcia, F. A. P., and Adlercreutz, P. (1998) Physical characterization of porous materials and correlation with the activity of immobilized enzyme in organic medium. Biocatal. Biotransform. 16, 67-85.
8. Bosley, J. A. and Clayton, J. C. (1994) Blueprint for a lipase support: Use of hydrophobic controlled-pore glasses as model systems. Biotechnol. Bioeng. 43, 934-938.
9. Gitlesen, T., Bauer, M., and Adlercreutz, P. (1997) Adsorption of lipase on polypropylene powder. Biochim. Biophys. Acta 1345, 188-196.
10. Persson, M. , Costes, D., Wehtje, E., and Adlercreutz, P. (2002) Effects of solvent, water activity and temperature on enzyme enantioselectivity. Enzyme Microb. Technol. 30, 916-923.
11. Adlercreutz, P. (1994) Enzyme-catalyzed lipid modification. Biotechnol. Genet. Eng. Rev. 12, 231-254.
12. Björup, P., Wehtje, E., and Adlercreutz P (1996) Effects of acetonitrile-water mixtures on a-chymotrypsin catalyzed dipeptide synthesis. Biocatal. Biotransform. 13, 189-200.
13. Clapes, P., Torres, J.-L., and Adlercreutz, P. (1995) Enzymatic peptide synthesis in low water content systems: preparative synthesis of [Leu] - and [Met]-enkepha-lin derivatives. Bioorg. Med. Chem. 3, 245-255.
14. Triantafyllou, A. O., Wehtje, E., Adlercreutz, P., and Mattiasson, B. (1995) Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media. Biotechnol. Bioeng. 45, 406-414.
15. Andersson, M., Samra, B. K., Holmberg, H., and Adlercreutz, P. (1999) Use of Celite-immobilised chloroperoxidase in predominantly organic media. Biocatal. Biotransform. 17, 293-303.
16. Costes, D., Wehtje, E., and Adlercreutz, P. (1999) Hydroxynitrile lyase catalysed synthesis of cyanohydrins in organic solvents. Parameters influencing activity and enantiospecificity. Enzyme Microb. Technol. 25, 384-391.
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