WO2008151822A1 - Thermally modified clay minerals as carrier materials for enzymes - Google Patents

Abstract

The invention relates to a solid enzyme complex, comprising at least one enzyme that is immobilized on a carrier obtained by heating a clay mineral to such a temperature and for such a duration that the carrier has a swellability in water of less than 15 ml/2 g. The invention further relates to a method for the production of the solid enzyme complex and the use thereof in enzymatically catalyzed reactions.

Description

 THERMALLY MODIFIED SOUND MINERALS AS SUPPORT MATERIALS FOR ENZYMES

The invention relates to a solid enzyme complex, a process for its preparation and the use of the solid enzyme complex.

Because of their high selectivity and relatively low energy requirements, enzymatically catalyzed reactions are becoming increasingly important, even for syntheses carried out on an industrial scale. However, if the production costs of the enzymes are high, it is necessary that they can be used repeatedly. They must therefore be cost-effectively separated from the reaction mixture. However, methods such as chromatography or ultrafiltration are generally too expensive for industrial processes. One therefore tries the enzymes in an appropriate manner to immobilize. There are essentially two methods available for this purpose.

In the first method, the enzymes are bound on a carrier. The binding can be done in different ways. Physical binding can be achieved by adsorption of the enzyme on the surface of the support. Binding occurs via hydrophobic interactions or by ionic forces, with charged groups of the enzyme interacting with oppositely charged groups on the surface of the support. The advantage of this method is its ease of execution and the relatively low influence on the activity of the enzyme. The disadvantage, however, is that the enzymes can be displaced relatively easily from the surface of the carrier again. An irreversible binding of the enzyme can be achieved by forming a covalent bond between the enzyme and the carrier. Here, however, must be taken into account that under certain circumstances, the activity of the enzyme is lowered, since the enzyme may for example be fixed on the surface, that the active center is no longer accessible. Finally, the stability of the enzyme / carrier complex can be further increased by the enzymes are crosslinked by at least bifunctional molecules. This results in larger aggregates which have a lower solubility. However, the control of immobilization is very difficult in this method. Furthermore, a significant deactivation of the enzyme usually has to be accepted, since its conformation is greatly altered or the active center is no longer freely accessible.

In the second method, the enzyme is entrapped in a spherical or tubular matrix. The matrix must be permeable to the educts and products of the catalyzed reaction, but not to the enzymes. For this technique, natural polymers, such as alginates, gelatin or agar, or even syn- thetische polymers, such as polyacrylamide or Polyvmylalkohol used. In this method, the enzyme is protected from being inactivated by the solvent in reactions in organic media. However, it must be accepted as a disadvantage that the matrix can act as a diffusion barrier for the educts and products of the enzyme-catalized reaction.

Phyllosilicates have already been proposed as carriers for the immobilization of enzymes. Thus, DE 2 154 672 describes a process for preparing a solid enzyme complex, wherein an enzyme in an aqueous buffer solution is contacted with a support m which has been previously modified with a coupling agent. The carrier used is preferably Bentomt, which has been treated with cyanuric chloride as the coupling agent. The immobilization of the enzyme is very rapid, ie within a few minutes, and is carried out at temperatures of less than 40 ⁰ C, preferably at about 0 ⁰ C, so that the thermal load of the enzyme remains low.

US Pat. No. 6,180,378 describes solid complexes in which an enzyme is immobilized on a sheet silicate. For immobilization, the phyllosilicate is first delaminated in water. For this purpose, the ions of the layered silicate can also initially be exchanged for sodium or alkylammonium ions. To suspend the delaminated sheet silicate, the enzyme and a crosslinking agent, such as tetraethyl orthosilicate, are then added. As the reaction progresses, larger aggregates form, with the enzyme trapped in interlayers of the layered silicate. The solid can be washed neutral with a suitable buffer and then dried. When drying, care must be taken that the conditions are chosen to be sufficiently mild so that the structure of the layered silicate is not destroyed and the enzyme is not denatured. When dried under reduced pressure, the activity of the enzyme remains largely intact. - A -

while during freeze-drying a clear deactivation occurs. In the solids macropores form which allows Dif ^¬ fusion of the substrate to the active centers of the enzyme molecules.

In US 5,279,948, an immobilized enzyme on a polymer is described which led, for example, in columns ver ^¬ contact. The polymer used is either a homopolymer of 1-aminoethylene or a copolymer of 1-aminoethylene and N-vinylformamide. The polymer is added to an aqueous solution of the enzyme and then a crosslinking agent is added which can react with amino groups. Suitable crosslinking agents are, for example, glutaraldehyde, diisocyanates and polyazetidine. A precipitate is obtained, which is separated from the liquid medium and dewatered, so that a paste-like mass is obtained, which can be processed into particles of a desired size. However, the preparation of such an immobilized in a solid matrix enzyme is relatively expensive.

HW Morgan and CT. Corke, Can. J. Microbiol. Vol. 22, 1976, - s. 684-693, describe the adsorption of glucose oxidase on different clays. For the investigations, montmorillonite and kaolinite with a particle size of less than 2 μm were used. The tone was implemented in a sodium acetate buffer at 25 ⁰ C with glucose oxidase. The maximum amount of enzyme adsorbed was observed at a pH which was below the isoelectric point of the enzyme. At the beginning of adsorption, the adsorbed enzyme showed significantly lower activity than the free enzyme. As the amount of adsorbed enzyme increased, however, its activity also increased and approached that of the free enzyme.

MS Carrasco, JC Rad and S. Gonzales-Carcedo, Bioresource Technology 51 (1995), 175-181 report the immobilisation tion of alkaline phosphatase to Natnumsepiolit. The best conditions for immobilization were found in a phosphate buffer (pH 7.2) at a temperature of 4 ° C, an enzyme concentration of 0.25 mg / ml and an enzyme / clay ratio of 10 ml / g. The immobilized phosphatase can be used to improve the bioavailability of phosphorus compounds in soil. For the preparation of the immobilized enzyme, sepiolite having a particle size of <2 mm was first converted into the sodium form by repeatedly slurrying the sepiolite in a sodium chloride solution. For the immobilization, the enzyme is reacted in a phosphate buffer (pH 7) at 4 ^° C. with the sepiolite present in the sodium form and the solid is separated by centrifuging. The separated pellet is washed with buffer, sodium chloride being added in the second washing step to separate weakly bound enzyme from the clay. Subsequently, the enzyme activity is determined.

T. Tietjen and R. G. Wetzel, Aquatic Ecology, 37, 331-339, 2003, examined the influence of immobilization of enzymes on clay minerals on their activity. For this purpose, various enzymes were adsorbed on montmorillonite as well as on a clay from a natural source, whereby a reduction in the enzyme activity was found. When the enzyme complexes were exposed to sunlight, a reduction in activity was observed, but not significantly different from the reduction in activity caused solely by the immobilization. For the immobilization, a suspension of the clay particles, which had a particle size of less than 2 μm, was mixed with the respective enzyme.

The immobilization of cellulase on silicate clay minerals is reported by AAS Smegani, G. Emtiazi, and H. Shariatmdari, J. Colloid Interface Sei. 290, 2005, 39 - 44. As a sound mine For example, illite, kaolinite, montmorillonite, palygorskite and an undefined soil sample were used. The adsorbed amount of enzyme could be increased by coating the minerals with aluminum hydroxide. After adsorption of the enzyme, the crystal structure of the clay minerals does not show any widening of the layer distance, so that, according to the authors, no enzyme is incorporated into the intermediate layers of the crystal structure.

Y. Yesiloglu, Process Biochemistry, 40, 2005, 2155-2159 reports on the adsorptive immobilization of the enzyme lipase from Candida rugosa on bentonite. Experiments on the temperature stability of the enzyme show that the half-life of the supported lipase in comparison to the unsupported enzyme at 50 ⁰ C increased from 17 to 45 minutes.

K. Buchholz and I. Mikhael, Zuckerind. 114, 1989, 558-561 describe the isolation of the enzyme tyrosinase from sugar beets by adsorption on bentonite. The enzymatic oxidation of tyrosine and dopa was then investigated using the immobilized enzyme.

In the cited references, the enzymes are immobilized on clay minerals, in particular bentonite, which are present in an aqueous buffer solution in colloidal form. However, such a finely divided suspension is not suitable for industrial applications because the fine particles are difficult to separate from the reaction medium after the end of the reaction. Furthermore, it is not possible to package such fine clay particles in the form of a column that can be used in a continuous flow process.

In US 5,342,768 a particulate is immobilized

Lipase, which consists essentially of a thermostable

Lipase consists of a microorganism from the group of

Species of the genus Humicola, Candida antarctica and Rhizomucor miehei was isolated and adsorbed on particles of a macroporous carrier. The carrier consists of at least 65% silica gel or silicates, wherein at least 90% of the particles have a particle size between 100 and 1000 microns and wherein at least 80% of the pores of the particles have a diameter which is 12 to 45 times the diameter of a Lipasemole - kuls corresponds, and wherein the water content of the particulate immobilized lipase is between 1 and 20%. For adsorption, the particulate carrier is placed in an aqueous solution of the lipase. The loaded carrier particles are separated, optionally washed and dried to the specified water content.

C. Buttersack, K. Novikow, A. Schaper and K. Buchholz (Zuckerind.1199 (1994) No. 4, pp. 284-291) report the use of layered silicates to separate enzymes from the filtered juice of homogenized sugar beets. By adsorption on sodium bentonite and subsequent desorption with a pH buffer enzymes could be separated in a simple manner in a pure form. The work shows the good suitability of layered minerals for the separation or enrichment of proteins or enzymes. The handling of the layered minerals is, however, complicated, as they lead to a strong increase in the viscosity of the sludge in water and the layer minerals disintegrate into very small particles. It is therefore very difficult to carry out the accumulation of proteins in such a way that the layered mineral is packed in a column, through which the liquid containing the protein is then passed. Without assistance, the column clogged relatively quickly, as the layer mineral swells strongly. However, to allow separation of the enzymes in a column, the authors suggest that sodium bentonite be poured into a calcium alginate gel. The hydrogenated homogenate is pumped at pH = 5.0 across the column packed with beads of immobilized bentonite. After the non-adsorbed - -

Fraction has escaped from the column, the enzyme is eluted with a buffer solution adjusted to pH = 8. For the large-scale separation of proteins, the use of sodium bentome, which is fixed in a calcium-algmatite as described by the authors, is unsuitable because of the high costs involved.

The invention had the object of providing a stored enzyme available, which can be produced easily and inexpensively, can be used in industrial processes and has a high activity and a long half-life in the application.

This object is achieved with a solid enzyme complex having the features of patent claim 1. Advantageous developments of erfmdungsgemaßen solid enzyme complex are the subject of the dependent claims.

The suitability of phyllosilicates as carrier material for the immobilization of enzymes is already known. In order to avoid the disadvantages of this carrier material, its high swellability or the delamination into fine particles, a heat-treated clay mineral is used in the solid enzyme complex according to the invention. The heat treatment ensures that the swelling ability of the clay is significantly reduced or completely suppressed. At the same time, however, the good binding properties of the phyllosilicates for enzymes are retained. The heat-treated clay mineral can readily be agglomerated to form larger stable particles. It is therefore possible to easily separate the solid enzyme complex after completion of the reaction, for example, by allowing filtration or settling of the solid enzyme complex and decanting the liquid phase. On the other hand, the reactions can also be carried out in the flow, if the erfmdungsgemaße solid enzyme complex is provided as a column or in a cartridge. The erfmdungsgemaße solid enzyme - -

Complex can be produced very simply and inexpensively as well as in large quantities. The carrier is obtained by heating the clay mineral used as starting material until the desired properties are achieved. On the heat-treated clay material can then be immobilized by conventional methods, the enzyme of interest.

According to the invention, therefore, a solid enzyme complex is made available, comprising at least one enzyme which is immobilized on a carrier which has been obtained by heating a clay mineral to a temperature and for a duration that the carrier has a swelling capacity in water of less than 15 ml / 2 g.

The carrier obtained by heating a clay mineral has a swelling capacity which substantially corresponds to the sediment volume. Even with prolonged standing in water or an aqueous reaction medium, the carrier does not swell and also shows no delamination, so that, for example, it does not cause an increase in the viscosity of the reaction medium. The erfmdungsgemaße solid enzyme complex can therefore be easily added in large quantities to a liquid, especially aqueous medium, without thereby, for example, the viscosity of the suspension increases dramatically. Furthermore, the solid enzyme complex according to the invention can be separated again from the liquid medium without difficulty by conventional methods, such as filtration or centrifugation. The enzymes remain predominantly adsorbed on the carrier, so that the carrier can optionally be used in a further reaction or batch.

On the carrier obtained by heat treatment of a clay mineral, enzymes can be bound in such amounts that the solid enzyme complex has an activity which is interesting for industrial applications. By itself, according to prevailing opinion, phyllosilicates assume that in the case of complete The loss of the swelling capacity of the clay material greatly reduces the internal surface area available for the adsorption of enzymes because the layers are chemically bound together. It is therefore expected that the adsorption capacity of the clay mineral will be drastically reduced by a heat treatment. Surprisingly, it has now been found that the swelling capacity of clay minerals, in particular smectic phyllosilicates, such as bentonites, can be destroyed by a temperature treatment Bmdevermo- gen for enzymes is maintained.

The properties of the carrier can be controlled by the temperature treatment.

High ion exchange capacity generally correlates with good Bmdekapazitat for enzymes. In the carrier prepared from a clay mineral by heat treatment, the cation exchange capacity is preferably at least 5 meq / 100 g, preferably at least 10 meq / 100 g, more preferably at least 15 meq / 100g. The carrier preferably has the highest possible ion exchange capacity. Preferably, the carrier has a cation exchange capacity of at least 40 meq / 100 g, more preferably at least 60 meq / 100 g. Most preferably, the cation exchange capacity is in the range of 65 to 130 meq / 100g. The ion exchange capacity of the carrier prepared by a heat treatment from a clay mineral is an important criterion for distinguishing, for example, the carriers of clay materials contained in the solid enzyme complex of the present invention, such as those obtained by extracting with hot acid from natural clay minerals. The latter show little or no ability for ion exchange. High-digestion bleaching earths that no longer swell typically exhibit a cation exchange capacity in the range of less than 30 meq / 100 g. Due to the heat treatment, the inner surface of Tonπii- nerals is significantly reduced. Preferably, the carrier has a specific surface area of less than 300 m ² / g, particularly preferably less than 200 m ² / g, more preferably less than 120 m ² / g. In order to achieve a sufficient absorption capacity for enzymes, however, the specific surface must not be lowered too far, so the clay mineral will not be "burnt to death". Preferably, the specific surface of the carrier is at least 30 m ² / g, particularly preferably at least 60 m ^z / g.

The carrier contained in the inventive solid enzyme complex has almost no swelling capacity in water more. The swelling volume in this case corresponds to the sediment volume. Even with prolonged storage in water, no or almost no increase in sediment volume is observed. After storage in water for three days, the carrier preferably has a sediment volume of less than 15 ml / 2 g, preferably less than 10 ml / 2 g, particularly preferably less than 8 ml / 2 g.

The inventive solid enzyme complex can be provided as a fine powder or else in the form of large-sized moldings. Since the carrier has no or only a very low swelling capacity in water, for example, granules of any size can be prepared, on which then the enzyme is immobilized. In order to achieve sufficient stability for large-sized molded articles, so that the solid enzyme complex does not decompose, for example, in an aqueous reaction medium, the thermal treatment is preferably carried out only after shaping.

As such there are no limitations with respect to the enzyme which is provided in the form of the solid enzyme complex according to the invention. In principle, all enzymes can be incorporated in the solid enzyme complex according to the invention, which are suitable for the Durchfuhrung enzyme-catalyzed reactions are known. The enzyme is preferably selected from the group which is formed from lipases, oxidoreductases, such as glucose oxidase or pyruvate dehydrogenase, transferases, such as hexokmase or glycogenophosphorylase, hydrolases, such as amylases, chymotrypsm, peptidases or esterases, isomerases, such as glucose isomerase, ligases such as carboxylases, lyases such as aldolase or catalase. The enzyme is particularly preferably a lipase, in particular from the enzyme class of the hydrolases.

Lipases are enzymes that cleave lipids, such as triglycerides or diglycerides, to free fatty acids and glycine. Lipases (Tn acylglycerm hydrolases) catalyze the hydrolysis as well as the synthesis of esters of glycerm and long-chain fatty acids. Other lipase-catalyzed reactions include the transesterification of lipids and the esterification of primary, secondary and tertiary alcohols. The industrial use of these enzymes is very versatile. Addition to detergents is the largest field of application for lipases. Approximately 1000 tons of enzyme are added to detergents every year. In the cosmetics industry, lipases are used to make emulsifiers and flavorings. In the food industry lipases are used for the synthesis of glycerides and flavorings, as well as for the modification of fats. A further large field of application for lipases is the production of consumer and feminine chemicals. The use of the enzymes for stereoselective reactions is becoming increasingly important. Lipases are also used in the production of lubricants, hydraulic oils and biodiesel.

The enzyme can be bound to the carrier via a covalent bond or else via a non-covalent bond. For a covalent binding of the enzyme to the carrier coupling agents are used, as they are already known from the immobilization of enzymes or proteins. Such coupling agents are Molecules having at least two reactive groups, one group reacting with a hydroxy group provided on the surface of the support and the at least one further reactive group reacting with a corresponding group on the enzyme, such as a hydroxy group, an amino group or a thiol group, so that the enzyme is over the coupling agent molecule is fixed on the surface of the support by covalent bonds.

It is also possible that the enzyme is cross-linked by corresponding at least bifunctional molecules, so that the molecular weight of the enzyme is increased and thus its solubility in the reaction medium is deteriorated.

Preferably, the enzyme is bound to the carrier via a non-covalent bond. The carrier has a sufficiently high cation exchange capacity even after the thermal treatment, so that adsorption of the enzyme on the carrier can be carried out via ionic bonds. Likewise, an immobilization of the enzyme via hydrophobic interactions between enzyme and Trager take place. The noncovalent binding of the enzyme to the carrier lessens the structure of the enzyme so that the immobilization does not interfere excessively with the activity of the enzyme.

The amount of the enzyme immobilized on the carrier is preferably selected to be high enough to achieve a sufficiently high activity of the solid enzyme complex. Preferably, the proportion of the enzyme, based on the weight of the solid enzyme complex, between 3 and 35 wt .-%, particularly preferably 5 to 20 wt .-%, particularly preferably 8 to 15 wt .-%.

The enzyme contained in the solid enzyme complex is, as already explained, not subject to any particular restrictions. Preferably, the enzyme has a molecular weight in the range of 10 to 200 kDa. The erfmdungsgemaße solid enzyme complex can be produced in a simple and cost-effective manner, so that larger amounts of erfmdungsgemaßen solid enzyme complex are accessible. The invention therefore also relates to a process for the preparation of a solid enzyme complex as described above. The erfmdungsgemaße method comprises the following steps:

Providing a carrier obtained by heating a clay mineral to a temperature and for a period of time that the carrier has a swelling capacity in water of less than 15 ml / 2 g;

Providing an enzyme;

Contacting the enzyme and the carrier in a reaction medium, wherein the enzyme is immobilized on the carrier; and

if necessary, removal of the solid enzyme complex from the reaction medium.

In a first reaction step, the carrier is provided by subjecting a clay mineral to a thermal treatment so that it obtains a certain, very low swelling capacity. Since the clay mineral used as starting material is obtained from a natural source and therefore variations in the properties of the various Tonmmeralien may occur, it is preferred m the way that initially determined by screening the optimum temperature for the clay mineral, wherein the desired low swelling capacity of the carrier is set. Preference is given to ^¬ as low as possible selected temperature and optionally adjusted the duration of the temperature treatment. Are preferred for the heat treatment temperatures of at least 400 ⁰ C, preferably at least 450 ⁰ C, particularly preferably zumm- least 500 ⁰ C chosen. However, the clay mineral should also not experience too high a thermal load, otherwise the adsorption capacity will drop. Therefore, the temperatures are preferably less than 1000 ⁰ C, preferably lower than 900 ⁰ C, and particularly preferably chosen below 800 ° C. The duration of treatment depends on the amount of clay mineral used and the type of furnace. Rotary kilns are preferably used for the heat treatment, since they allow uniform heating of the clay mineral. The treatment time is preferably chosen as short as possible in order to avoid excessive thermal stress on the clay mineral. In an industrial implementation of the method treatment times of at least 10 minutes, preferably at least 20 minutes are selected. In order to avoid a thermal overload, the treatment time is preferably chosen shorter than 2 hours, particularly preferably shorter than 1 hour. The appropriate duration of treatment can be easily determined by one skilled in the art by periodically taking samples during the thermal treatment of the clay mineral and examining their swelling capacity. Further parameters, such as the ion exchange capacity, the specific surface or the pore volume, can also be used to assist in the monitoring of the thermal treatment.

As starting material for the production of the carrier can be used in any clay minerals, which are accessible from natural sources. Preference is given to using clay minerals which have a high ion exchange capacity and a high swelling capacity. Clay materials are preferably used which have an ion exchange capacity of at least 5 meq / 100 g, preferably at least 10 meq / 100 g, more preferably at least 20 meq / 100 g, especially preferably at least 40 meq / 100 g, and clay minerals are particularly preferably used. their cation exchange capacity is in the range of 50 to 130 meq / 100 g, more preferably in the range of 70 to 120 meq / 100 g. Furthermore, clay minerals which have a high swelling capacity are preferably used. Preferably, the clay minerals used as starting material have a swelling capacity in water after one hour of at least 5 ml / 2 g, preferably at least 8 ml / 2 g, particularly preferably at least 10 ml / 2 g. Preferably, the clay minerals used as Ausgangsmateπalien a swelling capacity in the range of 6 to 80 ml / 2 g, more preferably 9 to 40 ml / 2 g, particularly preferably 10 to 20 ml / 2 g. For example, sodium bentonites or calcium bentonites are suitable.

The clay minerals used for the preparation of the carrier preferably have neutral to weakly basic properties. A 2% by weight suspension of the clay mineral in distilled water preferably has a pH in the range of 6 to 11.

As starting materials for the production of the carrier smectitic sheet silicates are preferably used. These include, for example, charged clay minerals of the 2: 1 layer type with a negative charge of 0.2 to 0.6 per formula unit.

The cation exchange capacity of the clay minerals used as starting materials for the preparation of the carrier can be determined by exchange with ammonium chloride and subsequent nitrogen determination and is preferably in the above-described range of 10 to 130 meq / 100 g. Examples of suitable smectite clay minerals are bentonite, montmorillonite, beidellite, nontronite, hectorite and saponite. Another suitable smectite clay mineral is stevensite. Its structure is derived from talc, with the positions of the magnesium ions being replaced by vacancies.

By selecting the clay mineral, the proportion of charge interactions and hydrophobic interactions, with The selected enzyme is fixed on the surface of the carrier. For example, supports made from montmorillonite-containing clay minerals with high cation-exchange capacities of preferably more than 65 meq / 100 g, preferably more than 85 meq / 100 g, by thermal treatment are more suitable for adding the enzymes to be immobilized via charge interactions whereas carriers made, for example, from stevensites, kerolites or saponites are particularly suitable for the immobilization of enzymes which are bound to the carrier via hydrophobic interactions. Suitable saponites have, for example, a cation exchange capacity of preferably 5 to 60 meq / 100 g, and preferably a specific surface area of 100 to 170

If an enzyme is to be bound to the carrier preferably via hydrophobic interactions, preference is given to selecting clay minerals for the thermal treatment which have a cation exchange capacity of less than 80 meq / 100 g, preferably less than 75 meq / 100 g, particularly preferably in a range of 40 to 70 meq / 100 g. Preferably, these clay minerals have a high specific surface area. The clay minerals preferably have a specific surface area of more than 100 m ² / g, preferably more than 120 m ² / g, particularly preferably more than 150 m ² / g.

The clay minerals used for the preparation of the carrier may optionally be dried before the heat treatment, preferably to a moisture content of less than 20 wt .-%, in particular to a moisture content in the range of 5-15 wt .-%. Further, the raw clay material may be ground prior to the heat treatment, preferably to a mean grain size D ₅₀ of less than 3 mm, more preferably less than 1 mm, most preferably less than 0.2 mm. The clay minerals, in particular smectitic clay minerals, used as starting material for the preparation of the carrier may be in both the sodium form and in the calcium or magnesium form. Likewise, it is also possible to use mixed forms in which monovalent alkali metal ions and divalent alkaline earth metal ions are present as intermediate layer ions in the clay minerals. The proportion of monovalent or divalent cations can be adjusted before the heat treatment by reacting the clay mineral with a corresponding alkali metal salt, for example sodium carbonate or sodium bicarbonate. For this purpose, for example, a naturally moist calcium bentonite can be sprayed with a soda solution or kneaded with solid sodium carbonate. If the proportion of divalent alkaline earth metal ions to be increased, the clay mineral with a suitable Erdalkaliverbmdung, such as calcium chloride, can be implemented.

Preferably, the heat-treated Tonmmeral used in erfmdungsgemaßen method is prepared from a raw clay material having at least a proportion of divalent alkaline earth metal ions, preferably calcium ions on Zwischenschichtplatzen.

In the case of bentonites, the higher the binding capacity for proteins, the higher the proportion of alkali metal ions in the exchangeable ions arranged in the intermediate layers of the bentonite. The high binding capacity of sodium bentonites is attributed to the strong delamination that occurs when preparing a suspension in water. This greatly increases the surface area available for absorption. In the case of calcium bentonites, there is a stronger cohesion of the layers, as a result of which delamination only occurs to a lesser extent in the production of a moist suspension and thus also in comparison to sodium carbonate. bentonites lower surface is available for absorption.

The erfmdungsgemaßen process everα has shown that a higher absorption capacity can be achieved for enzymes, if it is assumed that a clay mineral, which has a proportion of alkaline earth metal on interlayer spaces.

In the case of the heat-treated clay material, the proportion of divalent cations in the ion exchange capacity is preferably at least 15%, particularly preferably at least 30%, particularly preferably at least 70%. The proportion of divalent cations, in particular calcium and magnesium, in the cation exchange capacity is preferably less than 95%, in particular less than 85%. It has been observed that a high proportion of exchangeable divalent cations can enhance the tendency of enzymes to bind to the heat-treated clay material. Without wishing to be bound by this theory, the inventors assume that the alkaline earth metals arranged in intermediate layers exert a support function during the thermal treatment of the clay mineral so that the layers of the clay mineral do not sink together as much as with pure natural bentonites. The proportion of divalent or monovalent cations can be adjusted accordingly before the heat treatment of the clay mineral. For example, this can be reacted with a suitable sodium salt, such as sodium carbonate, by spraying the clay mineral, for example with a soda solution, or by kneading it with solid sodium carbonate.

As already explained, the exchanger according to the thermal treatment preferably has a specific surface area of less than 300 m ² / g, more preferably less than 200 m ² / g, more preferably less than 120 rn ² / g. The surface of the carrier preferably amounts to at least 30 m ² / g, more preferably at least 35 m ² / g, particularly preferably at least 60 m ² / g. The spe- In addition to the swelling capacity already described, the cif- ic surface can therefore be used to monitor the thermal treatment of the clay mineral.

The carrier has almost no swelling capacity in water after the thermal treatment. The swelling volume in this case corresponds to the sediment volume. Even with prolonged storage in water, no or almost no increase in sediment volume is observed. Preferably, after storage in water for three days, the carrier has a swelling volume of less than 15 ml / 2 g, preferably less than 10 ml / 2 g, more preferably less than 8 ml / 2 g. The swelling volume may also take on lower values, for example values of less than 5 ml / 2 g

After the thermal treatment, the carrier is preferably no longer subjected to further activation. It can still be adjusted by physical process steps, such as grinding and sieving, a desired grain size. Preferably, the carrier is adjusted to a mean grain size D ₅₀ in the range of 10-200 μm. Granulation can also produce larger particles. The larger particles can be stabilized in their shape by a, possibly renewed, thermal treatment. Suitably, the particulate carrier, in particular the granules, is heated to a temperature in the range from 400 to 750 ^° C. for 2 minutes to 5 hours. Preferably, however, proceeding in such a way that the clay mineral is first granulated or molded and only then the thermal treatment is carried out, at the same time a stabilization of the granules or Formkorpers is achieved.

Preferably, the granulation of the clay mineral, which is preferably carried out before the thermal treatment, takes place in such a way that the clay mineral is introduced into a high-speed mixer and that an aqueous binder, such as For example, water or water glass is added in a short period of time in its entire amount. The granulation can be carried out both in a batch process and in a continuous process. For granulation in the batch process, a so-called egg mixer and for continuous granulation, for example, a continuously operating ploughshare mixer, as offered for example by the company Lodige, or a ring-layer mixer, such as a Lodige CB mixer, can be used.

In addition to the described granulation process, other shaping processes can also be used. For example, the dry powdered thermally treated clay mineral can be pressed under high pressure into molded articles which, if appropriate, can also be comminuted again to the desired size after shaping. Such a highly compacting shaping can be carried out, for example, in a tableting press, a disk press or a briquetting press. But it is also possible, for example, to moisten the dry powdered thermally treated clay mineral with water and to knead it into a plastic mass. This plastic mass can then be extruded, for example, into a strand which is comminuted to a granulate of the desired size. But it is also possible to break the plastic mass into smaller lumps and to dry them first. The dried lumps can then be broken and the granules of the desired size separated by sieving.

In addition to the carrier an enzyme is provided in erfmdungsgemaßen process, which is to be immobilized on the carrier. There are no restrictions on the choice of enzyme per se. Preferred enzymes have already been mentioned. The enzyme is preferably provided in the form of an aqueous solution. To stabilize the enzyme, the enzyme is preferably in a buffered solution provided. The pH of the buffer is selected depending on the enzyme to be immobilized and is preferably in the range of 3 to 9. The ideal range for the pH depends on the specific enzyme. For lipases, the pH of the buffer is preferably selected in the range of 3 to 5. In general, the buffer is suitably selected in such a way that its buffer effect is as large as possible at a pH which is approximately one unit below the isoelectric point of the enzyme in question. The enzyme then has a positive total charge and can be fixed by an ionic bond on the carrier. The concentration of the buffer is suitably adjusted in the range of 10 to 300 mmol / l, preferably 50 to 200 mmol / l. The concentration of the enzyme in the solution is preferably selected in the range of 1 to 10 mg / ml. In order to prevent premature deactivation of the enzyme, the immobilization is preferably carried out at a temperature in the range of 0 to 37 ° C, particularly preferably 4 to 10 ⁰ C. The enzyme and the carrier can be brought into contact in any desired manner. Thus, the carrier can be suspended in a solution of the enzyme. But it is also possible to spray the solution of the enzyme on the carrier while it is being moved, for example. The time required for the immobilization of the enzyme depends on the carrier used and the enzyme used. Preferably, the reaction time is chosen in the range of 10 minutes to 5 hours.

Finally, the reaction medium can still be separated from the solid enzyme complex. This can be done by conventional methods, for example by filtration or Zentπfugieren. The solid enzyme complex can then be washed to remove unbound enzyme. For example, the same buffer can be used for washing as has been used in the reaction of enzyme and carrier. Is relatively little solvent, especially water in the solid enzyme complex For example, because the enzyme has been sprayed on the carrier, the solvent can also be evaporated. For this example, the solvent can be distilled off under reduced pressure. Again, the temperature is chosen as low as possible in order to avoid premature deactivation of the enzyme.

In order to avoid denaturation of the enzyme, as well as to provide a suitable pH for the most efficient adsorption of the enzyme on the carrier, the carrier is preferably before the task of the enzyme to a pH of preferably 3.0 to 7.0 equilibrium. For this purpose, the carrier can be slurried, for example, in a suitable buffer medium.

Although the enzyme is immobilized by non-covalent bonds on the surface of the carrier. However, it is also possible to fix the enzyme via covalent bonds on the surface of the carrier. For this purpose, the carrier and the enzyme are reacted with a coupling agent which has at least two reactive groups, so that one of the groups with, for example, hydroxyl groups on the surface of the carrier and the other group with a suitable group can react on the enzyme, such as a hydroxyl, a mino- or a thiol group. A suitable coupling agent is for example Cyanurchloπd.

Particularly suitable as coupling agents are functionalized silanes, as are commonly used for the fixation of enzymes on to ^¬ organic Tragern. This binds first functionalized alkoxy or chlorosilanes on the carrier. As alkoxy groups, for example, methoxy or ethoxy groups are used. The attachment of the functionalized silane takes place for example via the formation of Si-O-Si bonds with elimination of alcohol or HCl. If one uses functionalized silanes, which carry functional groups, which can react directly with groups on the enzyme, so in a second - -

Step the enzyme to be bound directly to the carrier. A typical example of this are epoxysilane compounds, such as epoxyalkyltrialkoxysilanes. Here, the epoxy group of the silane after application to the support can react directly with free NHo groups of the enzyme.

In a second variant, the functionalized silane is first reacted after application on the support with an activating agent. With the activating agent, groups are introduced into the silane bound on the surface of the support which can react with a group on the enzyme, for example an amino group. Only after the activating agent has reacted with a group on the silane can the enzyme be attached in a further reaction step to a group of the activating agent. For example, the above-described carrier obtained by thermal treatment of a clay mineral can be reacted with an aminoalkoxysilane. For example, the ammoalkoxysilane reacts with a hydroxy group on the surface of the carrier. By fixing the aminoalkoxysilane amino groups are provided on the surface of the carrier. These amino groups can then be reacted with a bifunctional activating agent, for example a dialdehyde or a diisocyanate. One functional group reacts with the amino group provided on the surface of the support and the other functional group is available for reaction with a group on the enzyme. A suitable bifunctional aldehyde is, for example, glutaraldehyde.

Finally, the invention also relates to the use of the above-described solid enzyme complex for enzyme-catalyzed reactions. The reactions can be carried out per se under known conditions and in known devices. Thus, the reactions can be carried out in suspension, including the solid enzyme complex in a, preferably buffered, solution the substrate is slabbed, to which other conventional components such as coenzymes, ATP, etc. may be added. However, it is also possible to carry out the enzyme-catalyzed reaction continuously by packing the solid enzyme complex, for example in the form of a column, through which a solution of the substrate is then continuously passed.

The invention will be explained in more detail with reference to examples and with reference to the attached figures closer. Showing:

Fig. 1: an adsorption isotherm recorded for a lipase at pH 4 in a 50 mM acetate buffer of Bentomt I ₅₀₀ ;

FIG. 2: an adsorption isotherm recorded on Bentomt 3 ₅₀₀ for a lipase at pH 4 in a 50 mM acetate buffer; FIG. and

FIG. 3 shows an adsorption isotherm, which was recorded for a lipase at pH 4 in a 50 mM acetate buffer at saponite l ₆ oo.

The following analysis methods were used:

BET surface area / pore volume after BJH and BET:

The surface area and the pore volume were determined using a fully automatic nitrogen porosimeter from the company Mikromeritics, type ASAP 2010.

The sample is cooled in a high vacuum to the temperature of liquid nitrogen. Subsequently, nitrogen is continuously metered into the sample chamber. By detecting the adsorbed amount of gas as a function of pressure becomes more constant - -

Temperature determined an adsorption isotherm. After pressure equalization, the analysis gas is gradually removed and a desorption isotherm is recorded.

To determine the specific surface area and the porosity according to the BET theory, the data are evaluated in accordance with DIN 66131.

The pore volume is also determined from the measurement data using the BJH method (EP Barret, LGJoiner, PP Haienda, J. Am. Chem Soc 73 (1951, 373).) This procedure also takes into account effects of capillary condensation Pore volumes of certain pore sizes are determined by summation of incremental pore volumes obtained from the BJH adsorption isotherm analysis.The total pore volume according to the BJH method refers to pores with a diameter of 1.7 to 300 nm.

Particle size determination by means of dynamic light scattering (Maillvern)

The measurements are carried out with a device "Mastersizer" from Malvern Instruments Ltd., UK, according to the manufacturer's instructions. The measurements are carried out with the intended sample chamber ("dry powder feeder") in air and the values related to the sample volume are determined.

Water content:

The water content of the products at 105 ⁰ C is determined using the method DIN / ISO-787/2.

Elemental analysis:

This analysis is based on the total resolution of the clay materials or the corresponding product. After releasing the festival Substances, the individual components are analyzed and quantified using conventional specific analysis methods, such as ICP.

Ion exchange capacity:

For the determination of cation exchange the clay material to be tested over a period of 2 hours at 105 ⁰ C is dried. Thereafter, the dried clay material is reacted with an excess of aqueous 2N NH ₄ Cl solution for 1 hour under reflux. After a service life of 16 hours at room temperature, it is filtered, whereupon the filter cake is washed, dried and ground and the NH ₄ content in the clay material is determined by nitrogen determination (CHN analyzer "Vario EL III" from Elementar in Hanau) according to the manufacturer's instructions. The proportion and type of exchanged metal ions is determined in the filtrate by ICP spectroscopy.

Determination of sediment volume:

A graduated 100 ml measuring cylinder is filled with 100 ml of distilled water. 2 g of the substance to be measured are added slowly and in portions, each about 0.1 to 0.2 g, with a spatula on the surface of the water. After a drop of added portion, the next portion is added. After the 2 g of substance have been added and dropped to the bottom of the measuring cylinder, the cylinder is allowed to stand for one hour at room temperature. Then the height of the sediment volume in ml / 2 g is read off the graduation of the measuring cylinder. For the determination of the sediment volume after 3 tagiger storage in water of the sample preparation is sealed with Parafilm ^® and vibration-free for 3 days at room temperature, allowed to stand. Then the sediment volume is read off the graduation of the measuring cylinder. Determination of the montmonellite content via methylene blue adsorption

The methylene blue value is a measure of the inner surface of the

Clay materials.

a) Preparation of a Tetranatnumdiphosphat solution

5.41 g of tetrasodium diphosphate are weighed to the nearest 0,001 g into a 1000 ml volumetric flask and placed under debris up to the calibration mark with dist. Water filled up.

b) Preparation of a 0.5% methylene blue solution

In a 2000 ml beaker, 125 g of methylene blue in about 1500 ml dest. Water dissolved. The solution is decanted off and distilled to 25 l with dist. Water filled up.

0.5 g wet test bag with known internal surface are weighed to the nearest 0.001 g in an Erlen πyy piston. Add 50 ml of tetrasodium diphosphate solution and heat the mixture to boiling for 5 minutes. After cooling to room temperature, 10 ml of 0.5 molar H ₂ SO _{4 are} added and 80 to 95% of the expected final consumption of methylene blue solution is added. With the glass rod, a drop of the suspension is taken and placed on a filter paper. It forms a blue-black spot with a colorless yard. It is then added in portions of 1 ml more Methylenblaulosung and repeated the dab sample. The addition takes place until the farm turns slightly light blue, so the added Methylenblaumenge is no longer absorbed by the Testbentomt. c) Examination of audio material

The test of the clay wxrd was carried out in the same way as for the test bentonite. From the used amount of Methylenblaulosung can calculate the inner surface of the clay material.

381 mg of methylene blue / g of clay correspond to a content of 100% montmorillonite according to this method.

Determination of Trockensiebruckstandes

About 50 g of the air-dry clay material to be examined are weighed out on a sieve of the corresponding mesh size. The sieve is connected to a vacuum cleaner, which sucks all parts, which are finer than the sieve through the sieve through a suction slot circulating under the sieve bottom. The strainer is covered with a plastic lid and the vacuum cleaner is switched on. After 5 minutes, the vacuum cleaner is switched off and determines the amount of remaining on the screen coarser portions by differential weighing.

Determination of wet sieve pressure

First, a 5% suspension is prepared by stirring an appropriate amount of the clay material to be examined at about 930 rpm for about 5 minutes in water. The suspension is stirred for a further 15 minutes at about 1865 rpm and the suspension is then poured through a sieve of the desired mesh size. The residue is washed with tap water until the wash water runs clear. The screen with the residue is then placed in an ultrasonic bath for 5 minutes to remove residual females. The remaining residue is briefly washed with tap water and the sonication is repeated, if necessary, until during the sonication - -

no more fines pass to the water. The sieve is then dried to constant weight. For weighing, the residue left on the sieve is transferred to a weighed porcelain dish.

Determination of the pH of a bentomtprobe

2 g of the sample are dispersed in 98 ml of distilled water. Thereafter, the pH is determined with a calibrated glass electrode.

Determination of protein concentration according to the Lowry method

The protein quantification according to the modified Lowry protocol is based on the reaction of proteins with copper sulfate and copper tartrate in alkaline solution, which leads to the formation of a four-toothed copper-protein complex.

In the next step, reduction takes place by addition of the follin-Ciocalteau-phenol reagent (containing phosphomolybdate-phosphotungstic acid), whereby an intense blue coloration occurs. The concentration of the resulting reaction product is determined photometrically at 750 nm.

Execution :

The determination was carried out with reagents which were obtained ready for use from Sigma-Aldrich GmbH, Taufkirchen, DE.

100 μl of the protease / enzyme solution are pipetted into a cavity of a 96-well plate. 100 μl Lowry reagent is pipetted in and the mixture is homogenized. After 20 minutes, 50 μl of Folm-Ciocalteu reagent are added and the mixture is rehomogenized. 30 minutes after addition of Folm-Ciocalteu reagent the absorbance is measured against a blank value at 750 nm in the plate photometer.

Determination of protein concentration according to the BCÄ method

Bicinchoninic acid (BCA) is used as the detection system in the BCA method. Initially, complex formation of protein with Cu ²⁺ ions in alkaline solution occurs. The Cu ²⁺ ions of the complex are reduced to Cu ⁺ ions, which can be detected by complex formation with BCA by absorption measurement at 562 nm.

Execution :

The determination was carried out with working reagents obtained from Pierce, Illinois, US ready for use.

25 μl of the enzyme / proteme solution are pipetted into a cavity of a 96-well plate. 200 μl of working reagent are pipetted in and the mixture is homogenized. After incubation for 30 minutes at 37 ^° C., the absorbance at 550 nm is measured in the plate photometer.

Standard curve for both protein determinations:

Standard 1 0.125 mg / ml

Standard 2 0.25 mg / ml

Standard 3 0.5 mg / ml

Standard 4 1.0 mg / ml

Standard 5 1.5 mg / ml

Standard 6 2.0 mg / ml

Blank value only buffer Example 1: Heat treatment of bentonites

Two different bentonites were used for the heat treatment. Bentomt 1 is a natural calcium / Natn-umben- tonit. Bentonite 2 was prepared by blending Bentomt 1 with 4.3% by weight soda, then kneading, drying and milling. The bentonites had a dry sieve pressure of <15 wt .-% on a sieve of mesh size 45 microns and a residue of <7 wt .-% on a sieve of mesh size 75 microns. The properties of the bentonites 1 and 2 used as starting materials are shown in Table 1.

Table 1: Properties of bentonites 1 and 2 used as starting materials

The bentonites 1 and 2 used as starting material were heated in an oven for 1 hour each to the temperatures indicated in Table 2. The heat-treated bentonites 1 and 2 were taken out of the oven and cooled to room temperature while in the air. Thereafter, the swelling volume was determined for each of the heat-treated bentonites. The values found are summarized in Table 2. Table 2: Swelling volumes of thermally treated and treated bentonites 1 and 2

Due to the thermal treatment, the swelling capacity of the bentonites is reduced with increasing temperature. At 500 ⁰ C, the swelling volume of the bulk density of the Bentomts in water, so the sediment volume corresponds. Bringing bentonites, which have been treated at these high temperatures, into aqueous or other liquids, no swelling of the bentonites is evident even after prolonged storage. The typical gelation of benotite can not be observed. The particles rapidly sink to the bottom of the vessels.

In Tables 3a, b, the properties of an untreated Bentomts 1 are compared to a thermally treated at 500 ⁰ C Bentomt l _5O o.

Table 3a: Physical properties of the thermally treated and untreated bentonite 1

Table 3b: Screen pressure levels of the thermally untreated and treated bentonite 1

Further, the cation exchange capacity of the thermally treated and untreated bentonites 1 and 2 was determined. The corresponding values are given in Table 4. Table 4: Cation exchange capacity of thermally treated (500 ⁰ C) and untreated Bentomten 1 and 2

Example 2: Investigation of further Bentomts

Analogously to Example 1, a bentonite 3 and a bentonite 4 were thermally treated at 500 ^° C. for 1 hour. Bentonite 4 is obtained from bentonite 3 by a stochiometπsche activation with soda. Bentonite 3 is a natural Ca / Na bentonite. The properties of Bentomte 3 and 4 used as starting material are summarized in Table 5. The swelling volumes for the thermally untreated and the treated at different temperatures Bentomte 3 and 4 are summarized in Table 6.

Table 5: Properties of Bentonites 3 and 4 used as starting material

Table 6: Swelling volumes of thermally treated and untreated bentonites 3 and 4

The physical data for the thermally untreated and treated at 500 ° C and 600 ⁰ C bentonite 3 are in Table 7, the physical data for the thermally untreated and the treated at 500 ⁰ C bentonite 4 are shown in Table.

Table 7: Physical properties of the thermally treated and untreated bentonite 3

Table 8: Physical properties of the thermally treated and untreated bentonite 4

The data obtained for bentonites 3 and 4 show, as observed with bentonites 1 and 2, that the temperature of the heat treatment in a range of about 500 ^° C. leads to an almost complete suppression of swelling in water. The activated by an exchange with sodium ions bentonite 4 shows in comparison to the natural Na / Ca bentonite 3 after the temperature treatment, a much lower BET surface area. An analogous behavior is shown in the cumulative pore volume data. Example 3: Studies of saponite malaria

A saponite was used which was thermally treated analogously to Examples 1 and 2. The properties of saponite 1 used as the starting material are summarized in Table 9.

Table 9: Properties of saponite used as the starting material 1

The saponite characterized in Table 9 was subjected to a temperature treatment at 500 ^° C. (saponite I ₅₀₀ ) and at 600 ^° C. (saponite I ₆₀₀ ) analogously to Examples 1 and 2. The physical properties of the thermally untreated and thermally treated saponite are shown in Table 10. Table 10: Physical properties of the thermally treated and untreated saponite 1

Example 4: Granulation of the carrier material

In an Eirich Intensive Mixer R02E (Eirich, Hartheim, DE), 3,000 parts of the clay mineral were initially introduced and 400 ml of water were metered in via a funnel as agglomeration agent. The low setting for the rotational speed of the plate as well as the maximum rotational speed for the whirl beer were chosen. The wet granules were first dried at 70 ⁰ C and then subjected to a thermal treatment at the indicated temperature for one hour. The granules were then screened in a wide range of 0.1 to 1.5 mm. The physical properties of the granules are summarized in Table 11. Table 11: physical properties of granules

Bentonite l ₆ oo Bentonite 3600 Saponite Iβoo

Dust weight [g / i] 780 + 100 1060 + 150 835 + 100 pH (5 wt% in water) 8.5 9.3 8.3

BET surface area [m ² / g] 58 67 45

Water content (wt.%) <3 <4 <3

To test the stability of the granules in aqueous media, they were stored for 7 days in distilled water and in different buffers. For this purpose, in each case 0.5 g of the granules were added in 20 ml of liquid.

The following buffers were used:

Buffer pH

Citrate 3

Na acetate 5

HEPES 7

TRIS 9

After one week of storage, the granules were unchanged. It was not a decay, which is indicated by the formation of turbidity or fine sediment, recognizable.

Example 5: Support of lipase on thermally treated clay marl

For the support on the thermally treated clay minerals a lipase from Candida rugosa (Sigma-Aldrich GmbH, Taufkirchen, DE) was used. The lipase from the organism Candida rugosa, a yeast fungus, has a size of about 60-65 kDa and an isoelectric point of 5.2. Lipases from the organism Candida rugosa belong to the nonspecific lipases, which have no regiospecific properties and thus hydrolyze all ester bonds.

a) Determination of the enzyme Bmdekapazitat in the static system

Preparation of the carrier

In each case 25 mg of m prepared in Examples 1 to 4 carriers are equilibrated in 10 ml of the listed in Table 12 50 mmol buffer. The carrier suspended in the buffer is sonicated for 30 minutes and shaken at 15 rpm for 30 mm in an overhead mixer. The suspension is then centrifuged at 3219 g and 10 ⁰ C for 10 minutes and the supernatant discarded.

Table 12: Buffers used for the equilibration (50 mM) (Sigma-Aldrich GmbH, Taufkirchen)

pH buffer

3 citrate

4 Na acetate

5 Na acetate

6 BISTRIS

7 HEPES

8 tris

9 Tris

Preparation of the enzymes

For the examples, a stock solution of the lipase is made at a concentration of 2 mg / ml in the appropriate buffer. Investigation of the pH-dependence of enzyme adsorption

To 25 mg of equilibπerten at a specified in Table 13 pH Tragers 10 ml of the enzyme stock solution is added and the suspension for 30 min at 4 ⁰ C kopfschuttler mkubiert and 15 rpm in Uber-. Thereafter, the solid is at 3219 g (10 ⁰ C) for 10 minutes centrifuged and the protein content in the supernatant liquid U as determined by the Lowry and BCA method. The solid is resuspended in 10 ml of the appropriate buffer, for 5 mm at 4 ° C and 15 rpm in Uber- kopfschuttler mixed and 3219 g (10 ⁰ C) centrifuged again. The protein content of the wash water is also determined. The amount of enzyme adsorbed on the carrier was calculated from the difference between the amount of enzyme used and the sum of the amount of enzyme measured in the supernatant or in the wash water. All experiments are carried out in triplicate. The loadings determined for bentonite ₅ oo are given in Table 13.

Table 13: pH dependence of the adsorption of lipase (Candida rugosa) on thermally treated bentonite I ₅₀₀

After the supernatant is completely removed, the carrier loaded with the enzyme is taken up in 1 ml of distilled water and the suspension used for the activity tests. Activity determination of the stored lipase (Candida rugosa)

i. Measured value lipase solution

In a 50 ml Erlenmeyer flask

2.5 ml of distilled water 1.0 ml of TRIS buffer 200 mM 3.0 ml of olive oil

pipetted. The samples are preheated for about 5 minutes while stirring in a water bath at 37 ⁰ C. Then, 1.0 ml Lipaselosung (stock solution) is pipetted and 30 minutes while stirring in a water bath at 37 ⁰ C mcubiert. Thereafter, 3.0 ml of ethanol (95%) are pipetted in and, after addition of 50 μl of thymolphthalein solution, titration is carried out with 0.1 M NaOH, until blue.

ii. Blank value lipase solution

In a 50 ml Erlenmeyer flask 25 mg each of the carriers shown in Examples 1 to 4 are weighed and then

3.5 ml of distilled water 1.0 ml of TRIS buffer 200 mM 3.0 ml of olive oil

pipetted. The samples are incubated for 30 minutes with stirring in a water bath at 37 ⁰ C. Thereafter, 1.0 ml of lipase solution (stock solution) and 3.0 ml of ethanol (95%) are pipetted in and, after addition of 50 μl of thymolphthalein solution, titration is carried out with 0.1 M NaOH, until blue. iii. Measured value of lipase

In a 50 ml Erlenmeyer flask

2.5 ml of distilled water 1.0 ml of TRIS buffer 200 mM 3.0 ml of olive oil

pipetted. The samples are preheated with stirring in a water bath at 37 ⁰ C for about 5 minutes. The activity determination is scheduled at intervals of 1 minute. The enzyme adsorbed on the carrier is taken up in 1 ml of distilled water and added to the solution in the Erlenmeyer flask. The further samples follow at intervals of 1 minute. After addition of 3.0 ml of ethanol (95%) and 50 .mu.l Thymolphthaleinlosung is titrated with 0.1 M NaOH Maßlόsung until the turn to blue.

iv. Blank value-supported lipase

In a 50 ml Erlenmeyer flask

2.5 ml of distilled water 1.0 ml of TRIS buffer 200 mM 3.0 ml of olive oil

pipetted. The samples are incubated for 30 minutes with stirring in a water bath at 37 ⁰ C. The enzyme adsorbed on the respective carrier is taken up in 1 ml of distilled water and added to the solution in the conical flask together with 3.0 ml of ethanol (95%). After addition of 50 μl thymolphthalein solution, titrate with 1.0 M NaOH standard solution until blue.

The activity of the enzyme is determined by the NaOH consumption. Unit-Definition

One unit hydrolyzes 1.0 μM fatty acid (from triglycerides) for one hour at pH 7.7 and 37 ^° C.

The lipase activities determined are given in Table 14 in comparison to the unlabelled lipase.

Table 14: Comparison of lipase activity (Candida rugosa) of non-stored and stored enzyme

Absorption of adsorption isotherms

The measured values from Tables 13 and 14 show that at pH 4, the loading of the carrier with the enzyme is higher. Further, higher activity is measured in the adsorbed enzyme. For the following measurements, therefore, the enzyme was adsorbed on the carriers at a pH of 4.

In the same way as described above, adsorption isotherms for the adsorption of the lipase to various supports of thermally treated clay minerals were determined. In each case, 25 mg of the carrier equilibrated with 50 mM Na acetate buffer at a pH of 4 in 10 ml of enzyme solution were added. The enzyme solutions were prepared by dilution of the enzyme stock solution (Na acetate 50 mM, pH 4) with Na acetate buffer (50 mM, pH 4). The following concentrations were provided: 0.25 mg / ml 0.5 mg / ml 1.0 mg / ml 2.0 mg / ml 3.0 mg / ml 4.0 mg / ml

The determinations were carried out with bentonite isocu bentonite 3soo and saponite I ₅ 0 ₀ . The values for the adsorption isotherms are shown in Table 15 and in Figures 1 to 3.

Table 15: Adsorption of lipase on thermally treated benitites

Enzyme Conc. Loading Lipase (mg / mg _Carrier r) (mg / ml)

Bentonite lone bentonite 3500 saponite l ₅₀ o (Fig. D (Fig. 2) (Fig. 3)

0.25 0.05 0.10 0.09

0.5 0.12 0.18 0.13

1 0.29 0.25 0.13

2 0.44 0.63 0.30

3 0.34 0.88 0.31

4 0.48 1.18 0.49

Furthermore, the activity was determined in the manner described above for the supported lipase. The values are shown in Table 16. Table 16: Lipase activity as a function of enzyme concentration

Results

Tables 13 to 16 show the results for the adsorption and activity of the supported enzyme lipase {Candida rugosa). Surprisingly, it has been found that the enzyme can be adsorbed with very good binding capacities of up to 1.1 mg of protein per mg of carrier. Usually, protein binding capacities or enzyme binding capacities of commercially available carrier materials or chromatography materials are in the range from 0.1 mg to 0.3 mg per milligram of carrier. In addition, it has been shown that the enzyme activity of the supported enzymes is still high, ie only slightly reduced with respect to activity in solution or to solution activity. This is especially true for bentonite I500, bentonite

as well as bentonite 3soo and bentonite 3 ₆ oo the case.

Claims

claims

A solid enzyme complex comprising at least one enzyme immobilized on a carrier obtained by heating a clay mineral to a temperature and for a period of time that the carrier has a swelling capacity in water of less than 15 ml / 2 g.

2. Solid enzyme complex according to claim 1, characterized in that the clay mineral has been heated to a temperature of at least 400 ⁰ C.

3. Solid enzyme complex according to claim 1 or 2, characterized in that the carrier has a Ionenaustauschkapazitat of at least 5, preferably at least 10, more preferably at least 15 meq / 100 g.

4. Solid enzyme complex according to one of the preceding claims, characterized in that the carrier has a specific surface area of less than 300 m ² / g, preferably less than 200 m ² / g, particularly preferably less than 120 m ² / g.

5. Solid enzyme complex according to one of the preceding claims, characterized in that the solid enzyme complex is formed as a shaped body, in particular as granules.

6. Solid enzyme complex according to one of the preceding claims, characterized in that the enzyme is selected from the group which is formed from lipases, reductases, transferases, hydrolases, isomerases, ligases, lyases.

7. Solid enzyme complex according to one of the preceding claims, characterized in that the enzyme is bound via a non-covalent bond to the carrier.

8. Solid enzyme complex according to one of the preceding claims, characterized in that the proportion of the enzyme, based on the weight of the solid enzyme complex, between

3 and 35 wt .-% amounts.

9. Solid enzyme complex according to one of the preceding claims, characterized in that the enzyme has a molecular weight in the range of 10 to 200 kDa.

10. A process for preparing a solid enzyme complex according to any one of claims 1 to 9, characterized in that

providing a carrier obtained by heating a clay mineral to a temperature and for a period of time that the carrier has a swelling capacity in water of less than 15 ml / 2 g;

an enzyme is provided;

- The enzyme and the carrier are brought into contact in a reaction medium, wherein the enzyme is immobilized on the carrier; and

the solid enzyme complex is optionally separated from the reaction medium.

11. The method according to claim 10, characterized in that the clay mineral is heated to a temperature of at least 400 ⁰ C.

12. The method according to claim 10 or 11, characterized in that the carrier has a ratio of exchangeable divalent to exchangeable monovalent cations of at least

4: 1.

13. The method according to any one of claims 10 to 12, characterized in that the carrier has a proportion of divalent cations to the Ionenaustauschkapazitat of at least 50%.

14. The method according to any one of claims 10 to 13, characterized in that the carrier has a specific surface area of less than 300 m ^? / g, preferably less than 200 m ² / g, particularly preferably less than 120 m ² / g.

15. The method according to any one of claims 10 to 14, wherein the carrier before the immobilization of the enzyme to a Formkόr- by, in particular a granule is formed.

16. The method according to any one of claims 10 to 15, characterized in that the carrier after storage in water for 3 days, a sediment volume of less than 15, preferably less than 10 ml / g.

17. The method according to any one of claims 10 to 16, characterized in that the carrier is equilibrated to a pH of 3.5 to 6.0 prior to the immobilization of the enzyme.

18. The method according to any one of claims 10 to 17, characterized in that the enzyme is fixed with a binder on the carrier.

19. The method according to any one of claims 10 to 18, characterized in that the reaction medium is formed by an aqueous buffer system.

20. The method according to any one of claims 10 to 19, characterized in that the reaction medium for the immobilization is heated to a temperature of 0 to 30 ⁰ C.

21. Use of a solid enzyme complex according to any one of claims 1 to 9 for enzyme-catalyzed reactions.