DE102007027206A1 - Immobilization of enzymes on bleaching earths - Google Patents

Abstract

The invention relates to a process for the preparation of a solid enzyme complex. In this case, a pulverulent granulation mixture, which consists of at least 50% of an acid-activated phyllosilicate, first granulated and then calcined to obtain a water-resistant granules. Enzymes can then be immobilized on the water-resistant granules. Furthermore, a solid enzyme complex obtainable by the process and its use in enzymatically catalyzed reactions are described.

Description

The Invention relates to a process for the preparation of a solid enzyme complex, a solid enzyme complex as obtained by this method and its use in enzyme-catalyzed reactions.

Because of their high selectivity and relatively low energy requirements Enzymatically catalyzed reactions are becoming increasingly important also for industrial scale Syntheses. However, when the production cost of the enzymes is high, it is it requires that these can be used repeatedly. They therefore need to be inexpensive from the reaction mixture let disconnect. Methods such as chromatography or ultrafiltration However, they are generally used in industrial processes expensive. It is therefore attempted to immobilize the enzymes in a suitable manner. Essentially two methods are available for this purpose.

At the In the first method, the enzymes are bound on a carrier. The binding can be done in different ways. A physical Binding can be achieved by adsorption of the enzyme on the surface of the Carrier can be achieved. The binding takes place over hydrophobic interactions or by ionic forces, wherein charged groups of the enzyme are oppositely charged Groups interact on the surface of the carrier to step. The advantage of this method is its ease of execution as well as the relatively small influence on the activity of the Enzyme. The disadvantage, however, is that the enzymes are relatively easily recovered displaced from the surface of the carrier can be. An irreversible binding of the enzyme can be achieved by a covalent bond between enzyme and Carrier is formed. Here, however, must be accepted that may be the activity of the Enzyme is lowered, for example, because the enzyme on the surface It can be fixed that the active center is no longer accessible is. Finally, the stability of the enzyme / carrier complex can still be increased by the enzymes by at least bifunctional Molecules are crosslinked. There are larger ones Aggregates that have a lower solubility. The However, immobilization control is very important in this process difficult. Furthermore, usually a significant deactivation of the enzyme be accepted as its conformation changes greatly or the active center is no longer freely accessible is.

At the second method, the enzyme is in a spherical or tubular Matrix included. The matrix must be for the educts and Products of the catalyzed reaction are permeable, not however, for the enzymes. For this technique will be natural polymers, such as. As alginates, gelatin or Agar, or syn thetische polymers such as polyacrylamide or polyvinyl alcohol used. In this method, the enzyme in reactions in organic Media protected from inactivation by the solvent. As a disadvantage, however, must be accepted that the matrix as a diffusion barrier for the educts and products of enzyme-catalyzed reaction can act.

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

In the US 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. Drying under reduced pressure largely maintains the activity of the enzyme, whereas freeze drying results in marked deactivation. Macropores form in the solid which allow diffusion of the substrate to the active sites of the enzyme molecules.

In the US 5,279,948 describes an immobilized on a polymer enzyme, which can be used for example in columns. The polymer is either a homopolymer of 1-aminoethyl len or a copolymer of 1-aminoethylene and N-vinylformamide used. 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 to give a paste-like mass which can be made into particles of a desired size. However, the preparation of such an immobilized in a solid matrix enzyme is relatively expensive.

AK Bajpai et al, Colloid. Polym. Sci. (2002) 280; 892-899 report the immobilization of diastase on acid-activated bentonites. For immobilization, the acid-activated bentonite was suspended at a certain temperature in a solution of diastase adjusted to a pH of 6.9. The enzyme was then adsorbed on the support for 90 minutes and the complex of immobilized enzyme and acid-activated bentonite separated from the suspension by centrifugation. The adsorbed amount of enzyme was determined in each case by determining the amount of enzyme remaining in the supernatant from the difference to the amount of enzyme used.

H. Yağar et al., Turk. J. Chem. 26 (2002) 751-758 , report the non-covalent immobilization of quince-isolated polyphenol oxidase on bentonite. Immobilization was achieved by simple adsorption at a pH of 6.8.

PV Lara et al., Enzymes and Microbial Technology 34 (2004) 270-277 , investigated the catalytic activity of various lipases in the processing of used bleaching earths. The bleaching earths still contained about 40 wt .-% of oil, which could be almost completely removed by the enzymatic treatment, so that the reprocessed bleaching earth can be used again for bleaching oils or can be deposited without problems.

S. Gopinath et al., React. Kinet. Catal. Lett. Vol 88, No. 1, 3-9 (2006) , investigated the performance of montmorillonite-immobilized enzymes, either as a packing in a reactor or batchwise. For immobilization, the enzymes were either adsorbed on the montmorillonite or covalently bound to this. The carrier used was a montmorillonite K10, which corresponds to a highly active acid bleaching earth.

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 clay was reacted with glucose oxidase in a sodium acetate buffer at 25 ° C. 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 immobilization of alkaline phosphatase on sodium sepiolite. 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 soils. For the preparation of the immobilized enzyme, sepiolite having a particle size of <2 mm was first converted to the sodium form by repeatedly slurrying the sepiolite in a sodium chloride solution. For the immobilization, the enzyme was reacted in a phosphate buffer (pH 7) at 4 ° C. with the sepiolite present in the sodium form and the solid was separated by centrifuging. The separated pellet was washed with buffer, sodium chloride being added in the second washing step to separate weakly bound enzyme from the clay. Subsequently, the enzyme activity was determined.

T. Tietjen and RG Wetzel, Aquatic Ecology, 37, 331-339, 2003 , investigated the influence of immobilization of enzymes on clay minerals on their activity. For this purpose, various enzymes were adsorbed on montmorillonite and on a clay from a natural source, with a reduction in 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.

Report on the immobilization of cellulase on silicate clay minerals AAS Sinegani, G. Emtiazi and H. Shariatmadari, J. Colloid Interface Sci. 290, 2005, 39-44 , The clay minerals used were illite, kaolinite, montmorillonite, palygorskite and an undefined soil sample. 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. Yeşiloğlu, Process Biochemistry, 40, 2005, 2155-2159 reports on the adsorptive immobilization of the enzyme lipase from Candida rugosa to 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 ° 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 citations mentioned, the enzymes on clay minerals, in particular Bentonite immobilized in an aqueous buffer solution in colloidal form. Such a finely divided suspension However, it is not suitable for technical applications because the fine particles after the reaction is difficult from the reaction medium can be separated. Furthermore, it is not possible to pack such fine clay particles in the form of a column, which can be used in a continuous flow process.

In the US 5,342,768 For example, a particulate immobilized lipase consisting essentially of a thermostable lipase isolated from a microorganism selected from the group of species of the genus Humicola, Candida antarctica and Rhizomucor miehei and adsorbed on particles of a macroporous support is described. The support 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 lipase molecule and wherein the water content of the particulate immobilized lipase is between 1 and 20%. For adsorption, the particulate carrier is added to an aqueous solution of the lipase. The loaded carrier particles are separated, optionally washed and dried to the specified water content.

Report the use of layered silicates to separate enzymes from the filtered juice of homogenized sugar beet C. Buttersack, K. Novikow, A. Schaper and K. Buchholz (Zuckerind 119 (1994) No. 4, pp. 284-291) , 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. However, the handling of the layered minerals is complicated as they result in a large increase in the viscosity of the slurry in water and the layered 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 exited 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 bentonite, which is fixed in a calcium alginate as described by the authors, is unsuitable because of the high costs involved.

Of the Invention was based on the object, a process for the preparation to provide a solid enzyme complex which be carried out easily and inexpensively can and in which a solid enzyme complex is obtained, the can be used in technical processes and at the Apply a high activity and a long half life having.

These The object is achieved by a process for producing a solid enzyme complex solved with the features of claim 1. advantageous Further developments of the method according to the invention are the subject of the dependent claims.

The suitability of phyllosilicates as a carrier material for the immobilization of enzymes is already good known. In order to avoid the disadvantages of this support material, its high swellability or the delamination into fine particles, an acid-activated phyllosilicate is used in the process according to the invention for the preparation of a solid enzyme complex, wherein the phyllosilicate is provided in the form of a water-resistant granules. Such acid-activated phyllosilicates are also referred to as bleaching earths. The stability of the waterproof granules is achieved by calcining the granules after shaping. As a result, the swelling capacity of the layered mineral for disintegration into primary particles is lost, so that the granules or the solid enzyme complex prepared therefrom can be readily prepared, for example, in the form of a column or packing, which can then be passed through by an aqueous solution of the enzyme substrates. In a batchwise procedure of the reaction catalyzed by the immobilized enzyme, the solid enzyme complex can be easily removed from the liquid reaction medium, for example by allowing it to settle within a short period of time and decanting off or sucking off the supernatant liquid phase.

• – eine pulverförmige Granuliermischung bereitgestellt wird, welche zu zumindest 50 Gew.-% aus einem sauer aktivierten Schichtsilikat gebildet ist;
• – die pulverförmige Granuliermischung zu einem weichen Granulat granuliert wird;
• – das weiche Granulat calciniert wird, wobei ein wasserfestes Granulat erhalten wird; und
• – auf dem wasserfesten Granulat zumindest ein Enzym immobilisiert wird.

The process according to the invention for the preparation of a solid enzyme complex is carried out by

• - Providing a powder granulating mixture, which is formed to at least 50 wt .-% of an acid-activated phyllosilicate;
• Granulating the powdered granulation mixture into a soft granulate;
• - The soft granules are calcined, whereby a water-resistant granules is obtained; and
• - At least one enzyme is immobilized on the waterproof granules.

First a powdered granulation mixture is provided, the at least 50 wt .-% of an acid-activated sheet silicate is formed. Such acid activated phyllosilicates are also known as bleaching earths. These acid activated phyllosilicates have as a suspension in water (10 wt .-%) a pH of less as 7.0, preferably less than 6.0, more preferably less than 5.0 on. The pH can, for example, by means of a pH electrode be determined. Preference is given to the amount of acid-activated Phyllosilicate high chosen. Preferred is the proportion of the acid-activated phyllosilicate on the granulation mixture at least 70% by weight, more preferably at least 90% by weight. Especially Preferably, the granulation consists of 100% of the acid-activated Phyllosilicate. In addition to the acid-activated phyllosilicate can still contain other phyllosilicates in the granulation be, z. B. non-activated phyllosilicates. These are not activated Layered silicates can serve as binders and cause a higher strength of the granules. As such phyllosilicates For example, bentonites can be used. It can both phyllosilicates in the alkali form, in particular sodium form, as well as with alkaline earth ions as exchangeable cations, in particular Calcium ions, can be used. Exemplary smectic sheet silicates are bentonites, saponites, hectorites, attapulgites or sepiolites. The proportion of these non-activated phyllosilicates is preferably in the range of 1 to 50% by weight, preferably in the range of 2 to 40 Wt .-%, particularly preferably 5 to 30 wt .-%, particularly preferably Chosen from 15 to 25 wt .-%. In addition, you can For example, still granulating or pore forming in the be contained in powdered granulation. The percentages to the powder granulating mixture refer to a dry free-flowing granulating mixture, d. H. without an addition of liquid.

The powdery granulation mixture preferably has a mean Particle size in the range of less than 100 μm, more preferably in the range of 5 to 80 microns. The Particle size can be, for example by sieve analysis. The sieve residue is preferably the powdered granulation mixture on a sieve of Mesh 63 microns less than 40 wt .-% and is preferred in the range of 20 to 30 wt .-%. The dry sieve residue on a sieve of mesh size of 25 microns is preferably in Range of 50 to 65 wt .-%.

The powdered granulation mixture then becomes a soft Processed granules. Under a soft granules is doing a Granules understood, which is not stable in water but again decomposes into its powdery constituents. To check the stability, the soft granules can be introduced in water. After a certain lifetime, for example of one hour at room temperature, then the condition of the granules examined. Soft, non-waterproof granules form a cloudiness or a fine sediment and the granules partially or completely disintegrates. At a soft granules is the wet sieve residue after a lifetime of one hour in water at most 50 wt .-% of the amount of the granules used, preferably less as 20% by weight, preferably less than 10% by weight. Especially preferred no wet sieve residue remains after one hour in water.

For the granulation, arbitrary molding processes can be used. For example, the dry powder granulating mixture can be pressed under high pressure to form bodies the, which can be shredded to the desired size after shaping if necessary. Such a highly compacting shaping can be carried out, for example, in a tabletting press, an annular disc press or a briquetting press. However, it is also possible, for example, to moisten the dry pulverulent granulation mixture 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.

Preferably the granulation of the granulation mixture takes place in such a way that presented the granulation in a fast-running mixer and that an aqueous binder, such as. B. water or water glass, within a short period of time in its entirety Amount is added. The granulation can be done both in a batch process as well as in a continuous process become. For granulation in a batch process, a so-called Eirichmischer and for continuous granulation for example, a continuously operating plowshare mixer, as he z. B. offered by the company Lödige, or a ring layer mixer such as a Lödige CB mixer become.

The Energy intake when kneading is generally 0.01 to 1 kWh / kg, in particular 0.05 to 0.5 kWh / kg of moistened granulation mixture.

Becomes moistening the powder granulating mixture for granulation, so the water content of the wet granulation mixture is preferably adjusted to 35 to 55 wt .-%, preferably 40 to 50 wt .-%.

The binder used is preferably pure water. But it is also possible to use binders that remain in the granules after drying. A suitable binder is, for example, water glass. The proportion of the water glass, based on its solids content and on the dry pulverulent granulation mixture, preferably in the range of 0.5 to 20 wt .-%, particularly preferably selected in the range of 1 to 10 wt .-%. The modulus SiO ₂ / M ₂ O, with M = Na, K, is preferably selected in the range from 2.0 to 3.5. The solids content of the water glass is preferably 1 to 50 wt .-%, preferably 3 to 45 wt .-%, particularly preferably 5 to 40 wt .-%, particularly preferably 30 to 38 wt .-%.

at Use of water glass as a binder may be granulation be guided so that by the acidic layer silicate a condensation of the water glass is initiated, resulting in Silica gel forms, which for a cohesion between Primary particles of the acidic layer silicate provides.

to Stabilization, the soft granules are then calcined. Preferably, the calcination is carried out at temperatures in the range of 300 to 900 ° C, preferably 400 to 800 ° C, especially preferably carried out 500 to 700 ° C. The duration Calcination depends on the amount of granules to be calcined as well as the device used. A suitable device For example, is a rotary kiln, a uniform Heating the granules allows. The duration of calcination is preferably in the range of 5 minutes to 5 hours, preferably 10 minutes to 4 hours, more preferably 15 minutes to 2 hours.

After calcination, the water-resistant granules preferably have a specific surface area of more than 50 m ² / g, preferably in the range of 70 to 300 m ² / g, particularly preferably 80 to 260 m ² / g. The pore volume of the water-resistant granules, determined by the BJH method, is preferably more than 0.25 cm ³ / g and is preferably in the range of 0.07 to 0.7 cm ³ / g, particularly preferably in the range of 0.1 to 0.6 cm ³ / g.

To Calcination, the waterproof granules should be sufficient have high stability, so it even with a longer Use in water does not decompose. Preferred is the wet sud residue is more than 90% by weight, preferably more than 95 wt .-%, more preferably more than 98 wt .-%. One Method for determination of wet sieve residue is included given the examples.

If the powdered granulation mixture was moistened before granulation, the soft granules are preferably dried before being calcined. For this purpose, conventional devices or methods can be used. For example, drying in an oven or in a stream of hot air is possible. For drying, the temperature is preferably selected in the range of 80 to 120 ° C, particularly preferably 90 to 110 ° C. After drying or before calcining, the soft granules preferably have a water content of less than 35% by weight. Preferably, the water content is in the range of 5 to 30 wt .-%, especially preferably 10 to 20 wt .-%.

To the preparation of the water-resistant granules an enzyme is provided, which is to be immobilized on the waterproof granules, to obtain a solid enzyme complex. There are none Restrictions with respect to the enzyme, which is in the form a solid enzyme complex is to be provided. It can in itself all enzymes are incorporated into the solid enzyme complex, for the enzyme-catalyzed reactions are known. Preferably, the enzyme is selected from the group which is formed from lipases, oxidoreductases, such as glucose oxidase or pyruvate dehydrogenase, transferases, such as hexokinase or glycogen phophorylase, Hydrolases, such as amylases, chymotrypsin, peptidases or esterases, Isomerases, such as glucose isomerase, ligases, such as carboxylases, lyases, like aldolase or catalase. Most preferably, the enzyme is a Lipase, in particular from the enzyme class of hydrolases.

lipases are enzymes that release lipids, such as triglycerides or diglycerides Fatty acids and glycerol split. Lipases (triacylglycerol hydrolases) catalyze both the hydrolysis and the synthesis of esters from glycerol and long-chain fatty acids. More from Lipases catalyzed reactions are 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 produced annually Added to detergents. In the cosmetics industry Lipases used for the production of 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 used. Another great application for Lipases is the production of bulk and fine chemicals. there wins the use of the enzymes for stereoselective reactions more and more important. Lipases are also used in the Production of lubricants, hydraulic oils and biodiesel.

Around a denaturation of the enzyme during immobilization to avoid or to have a suitable pH for a most efficient adsorption of the enzyme on the waterproof To provide granules, the waterproof granules before the Task of the enzyme preferably equilibrated to a suitable pH. For this purpose, the waterproof granules are preferably in a suitable Buffer slurried. The pH of the buffer is preferred chosen in a range of 3.0 to 7.0. The buffer becomes preferably chosen equal to the buffer in which the enzyme solved or recorded. The duration for the equilibration of the water-resistant granules is preferably in the range of 5 minutes chosen up to 3 hours, preferably 30 minutes to 2 hours. Before immobilization, the one used for equilibration If necessary, replace buffer with fresh buffer.

The Enzyme is preferably in the form of an aqueous solution provided. To stabilize the enzyme, the enzyme is preferred provided in a buffered solution. The pH of the buffer becomes dependent on the one to be immobilized Enzyme selected and is preferably in the range of 3 to 9. The ideal range for the pH depends from the specific enzyme. For lipases, the pH is the buffer is preferably selected in the range of 3 to 5. Generally the buffer is suitably selected in such a way that its Buffering at a pH value is as large as possible, which is about one unit below the isoelectric Point of the enzyme in question. The enzyme then has one positive total charge on and can be due to an ionic bond on be fixed to the carrier. The concentration of the buffer is suitable in the range of 10 to 300 mmol / l, preferably 50 to 200 mmol / l set. The concentration of the enzyme in the solution is preferably chosen in the range of 1 to 10 mg / ml. Around To prevent premature deactivation of the enzyme, the Immobilization preferably at a temperature in the range of 0 to 37 ° C, particularly preferably 4 to 10 ° C. Enzyme and water-resistant granules can be used in any way be brought into contact. So can the wearer in one Solution of the enzyme to be suspended. It is also possible, the solution of the enzyme on the carrier spraying while this is moving, for example becomes. The duration necessary for the immobilization of the enzyme needed depends on the carrier used and the enzyme used. The reaction time is preferred in the Range from 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 centrifugation. 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 water-resistant granules. If only relatively little solvent, especially water in the solid enzyme complex, for example, because the enzyme has been sprayed onto the waterproof granules, the solvent can also be evaporated. This can, for example, under reduced pressure the solvent is distilled off.

Also here the temperature is chosen as low as possible, to avoid premature deactivation of the enzyme.

Prefers Although the enzyme is formed by non-covalent bonds on the surface immobilized the waterproof granules. However, it is also possible the enzyme via covalent bonds on the surface of the waterproof granules to fix. These are the waterproof Granules and the enzyme reacted with a coupling agent, which has at least two reactive groups, so that one of the groups with, for example, hydroxy groups on the surface of the waterproof granules and the other group with a suitable Group can react on the enzyme, such as a hydroxy, an amino or a thiol group. A suitable coupling agent is for example Cyanuric chloride.

Particularly suitable as coupling agents are functionalized silanes, as are commonly used for fixing enzymes on inorganic supports. This binds first functionalized alkoxy or chlorosilanes on the support. 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 functionalized silanes are used which carry functional groups which can react directly with groups on the enzyme, in a second step the enzyme can be bound directly to the carrier. A typical example of this is epoxysilane compounds, such as epoxyalkyltrialkoxysilanes. Here, the epoxy group of the silane after application to the carrier can react directly with free NH ₂ groups of the enzyme.

In In a second variant, the functionalized silane is replenished the application on the carrier first with a Activation agent order. With the Activation Agent, groups in the silane bound on the surface of the support which react with a group on the enzyme can, for example, an amino group. Only after the activation agent has reacted with a group on the silane can the enzyme in a further reaction step to a group of Activation agent be tethered. For example, the above described carrier obtained by thermal treatment of a clay mineral be reacted with an aminoalkoxysilane. The aminoalkoxysilane For example, it reacts with a hydroxy group on the surface of the carrier. By fixing the aminoalkoxysilane become on the surface of the carrier amino groups provided. These amino groups can then with a bifunctional activating agent, for example a dialdehyde or a diisocyanate. It reacts a functional Group with the on the surface of the carrier provided amino group and the other functional group is available for reaction with a group on the enzyme. A suitable bifunctional aldehyde is, for example, glutaraldehyde.

It it is also possible to have the enzyme by appropriate at least To crosslink bifunctional molecules, thereby reducing the molecular weight increase the enzyme and thus its solubility in the reaction medium to deteriorate. This measure can be taken when the enzyme on the waterproof granules physically is adsorbed. For the networking can usual bifunctional molecules are used.

Prefers is the enzyme via a non-covalent bond to the bound waterproof granules. Such non-covalent bonds are, for example, ionic bonds or non-specific bonds, like van der Waals ties. Due to the non-covalent binding of the Enzymes on the waterproof granules will change the structure of the enzyme less disturbed, so that the immobilization is not excessive affects the activity of the enzyme.

The Amount of the enzyme immobilized on the carrier, is preferably chosen to be sufficiently high in activity of the solid enzyme complex. Preferred is 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 .-%, in particular preferably 8 to 15 wt .-%.

The Enzyme contained in the solid enzyme complex is as already explained in itself is not subject to any particular restrictions. Prefers the enzyme has a molecular weight in the range of 10 to 200 kDa on.

At the The process according to the invention becomes an acid-activated Layered silicate used to produce the waterproof granules.

According to a first embodiment, the acid-activated phyllosilicate is obtained by adding a be preferably obtained from a natural source or a synthetic phyllosilicate is coated with an acid. Such acid-activated phyllosilicates are also known as surface-modified bleaching earths.

When Starting material can be used all phyllosilicates per se become. Particular preference is given to phyllosilicates from the montmorillonite / beidellite series used, such. As montmorillonite, bentonite, soda, saponite and hectorite.

The Activation can be carried out by conventional methods. The Starting material can be soaked with the acid or also be suspended in the acid. Activation can For example, also take place by an aqueous solution the acid is sprayed onto the layered silicate, wherein the layered silicate is preferably moved. excess Water that acts as a solvent for the acid can be removed after activation. The on the sheet silicate applied amount of acid is calculated as anhydrous acid, preferably in the range of 1 to 10 Wt .-%, more preferably 2 to 6 wt .-% selected.

According to a preferred embodiment, the starting materials used are weathering products of sheet silicates which have virtually no detectable layer structure and are therefore X-ray amorphous. These weathering products have a very high specific surface area of preferably more than 200 m ² / g, preferably 210 to 280 m ² / g, more preferably 220 to 260 m ² / g, and a high pore volume of preferably more than 0.5 ml / g, preferably 0.7 to 1.1 ml / g, more preferably 0.8 to 1.0 ml / g. These weathering products preferably still have a relatively high ion exchange capacity. The ion exchange capacity is preferably more than 40 meq / 100 g and is more preferably in the range of 50 to 90 meq / 100 g, particularly preferably 55 to 85 meq / 100 g. Furthermore, the weathering product preferably has a very low swelling capacity. After the weathering product has been allowed to stand in water for three days at room temperature, the sediment volume is preferably less than 15 ml / 2 g, more preferably less than 10 ml / 2 g. Room temperature is understood as meaning a temperature of about 15 to 25 ° C, in particular about 20 ° C.

To the surface activation of the layered silicate is preferred the phyllosilicate is not washed but merely dried. When drying is preferably a water content in the range of less than 20% by weight, preferably less than 15% by weight, especially preferably a water content in the range of 5 to 10 wt .-% set. If necessary, the acid-activated phyllosilicate can be adjusted to the desired Grit size are ground.

By Cover with acid surface-activated phyllosilicates still possess a significant cation exchange capacity. The se facilitates non-covalent attachment of enzymes to the surface activated phyllosilicates, as a bond of the enzyme via electrostatic interactions possible is, the enzyme so for example via ionic interactions is bound to the surface of the carrier. This especially applies when the enzyme is supported at a pH which is below the isoelectric point of the enzyme. The enzyme has cationic groups that can be exchanged with in the structure of the surface activated phyllosilicate existing protons or, for example, still existing alkali or alkaline earth ions can form an ionic bond. In addition to these ionic bonds can also be hydrophobic Interactions in the binding of the enzyme to the carrier play a role.

According to one second embodiment, the acid-activated phyllosilicate obtained by a preferably from a natural source obtained phyllosilicate with an acid extracted hot becomes. Such acid activated phyllosilicates are also considered to be high active bleaching earths known. As starting material you can all acid-activatable phyllosilicates are used become. Preference is given to di- and trioctahedral phyllosilicates the serpentine, kaolin and talc pyrophyllite group, smectites, vermiculites, Illites and chlorites as well as those of the sepiolite palygroskite group, such as As montmorillonite, soda, saponite and vermiculite or hectorite, Beidellit, Palygorsikt and Alternate Deposition Minerals (mixed layer mineral) are used. Of course you can too Mixtures of two or more of these minerals are used.

For activation, the phyllosilicate is mixed with strong acid and heated. Suitable acids are, for example, sulfuric acid, hydrochloric acid, phosphoric acid or nitric acid, which are preferably used in concentrated form. Salt acid and sulfuric acid are particularly preferred. The amount of acid is preferably chosen in the range from 50 to 200% by weight, based on the phyllosilicate used. For extraction, the suspension is heated to a temperature of more than 80 ° C, preferably more than 90 ° C, preferably at boiling heat. The duration of the extraction depends on the starting material and the ge Wanted activation level. The suspension is preferably heated for 3 to 20 hours, more preferably for 4 to 15 hours. Due to the amount of acid, the extraction temperature and the extraction time, the porosity of the acid-activated phyllosilicates can be influenced. By a more intensive extraction, in particular, the porosity in the range of pores with a diameter of less than 50 nm can be increased. After extraction, the solid is separated off, for example by filtration of the hot suspension. The acid-activated phyllosilicate is then preferably washed free of acid with water and then dried. Possibly. For example, the acid-activated phyllosilicate can be ground again.

The degree of activation of the acid-activated phyllosilicate is preferably selected such that at least 10% by weight, preferably at least 20% by weight, particularly preferably 40 to 80% by weight, of the aluminum present in the starting material is removed. The acid-activated phyllosilicate obtained by extraction with hot acid preferably contains from 2 to 15% by weight, preferably from 5 to 12% by weight, of aluminum, calculated as Al ₂ O ₃ . The silicate content, calculated as SiO ₂ , of the acid-activated phyllosilicate is preferably from 85 to 98% by weight, preferably from 88 to 95% by weight.

The acid-activated phyllosilicate produced by extraction with hot acid preferably has an average pore diameter, determined by the BJH method ( DIN 66131 ) between 2 and 25 nm, more preferably between 4 and 10 nm.

Preferably, the pore volume of the acid-activated phyllosilicate, determined by the CCl ₄ method, of pores having a diameter of up to 80 nm is between 0.15 and 0.8 ml / g, particularly preferably 0.2 to 0.7 ml / g, of pores with a diameter of up to 25 nm between 0.15 and 0.45 ml / g, particularly preferably 0.18 to 0.4 ml / g, for pores with a diameter of up to 14 nm between 0, 02 and 0.3 ml / g, particularly preferably 0.05 to 0.2 ml / g, and pores having a diameter of 25 to 80 nm between 0.02 and 0.3 ml / g, particularly preferably 0.05 to 0.2 ml / g.

The BET surface area of the acid-activated phyllosilicate prepared by extraction with hot acid, determined by DIN 66131 , is preferably 50 to 800 m ² / g, particularly preferably 100 to 600 m ² / g, particularly preferably 130 to 500 m ² / g.

By the extraction with acid decreases the ion exchange capacity from the phyllosilicates. The ion exchange capacity is preferably less than 30 meq / 100g. Particularly preferred is the ion exchange capacity of the acid-activated phyllosilicate in the range of 5 to 25 meq / 100 G.

at obtained by extraction with hot acid acid-activated phyllosilicates is the cation exchange capacity only small. Will therefore the enzyme on such a carrier Adsorbed, the bond is probably primarily by unspecific interactions, for example van der Waals bonds, as well as hydrogen bonds between groups provided on the enzyme and on the surface of the carrier Si-OH groups.

Of the produced by the method according to the invention solid enzyme complex is particularly suitable for an application as a column packing and for use in batches guided procedure. The invention is therefore also a solid enzyme complex, as with the one described above Procedure is obtained.

Of the solid enzyme complex comprises a water-resistant granules of an acidic activated phyllosilicate on which immobilized at least one enzyme is. The particle size of the waterproof granules is preferably selected in the range of 100 microns to 1.2 mm. On the waterproof granules can be any Enzymes immobilized. Exemplary enzymes have already been included the description of the manufacturing process discussed. Prefers are the enzymes non-covalently bound to the water-resistant granules, d. H. the immobilization takes place via physical interaction between the surface of the waterproof granules and the enzyme, for example via ionic bonds, hydrogen bonds or via hydrophobic interactions. details Details on the properties of the solid enzyme complex have already been made discussed during the procedure.

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, for which purpose the solid enzyme complex is slurried in a, preferably buffered, solution of the substrate to which other conventional components such as coenzymes, ATP, etc. may be added. But it is also possible to carry out the enzyme-catalyzed reaction continuously by the solid enzyme complex is packed, for example in the form of a column, through which then a solution of the substrate is passed continuously.

The The invention will be further described by way of examples explained.

It the following analytical methods were used:

BET surface area / pore volume after BJH and BET:

The Surface and pore volume were combined with a fully automatic Nitrogen porosimeter from the company Mikromeritics, type ASAP 2010 determined.

The Sample is heated to the temperature of liquid under high vacuum Nitrogen cooled. Subsequently, it becomes continuous Nitrogen dosed into the sample chamber. By capturing the adsorbed amount of gas as a function of pressure becomes more constant Temperature determined an adsorption isotherm. After a pressure equalization the analysis gas is gradually removed and a desorption isotherm added.

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

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

Determination of the pore volume by the CCl ₄ method

The Micropore volume is determined by adding carbon tetrachloride vapor act on the clay and the weight gain determined by the adsorption of carbon tetrachloride becomes.

Around To detect pores of a certain diameter, the vapor pressure of the carbon tetrachloride by adding certain quantities of paraffin lowered to the liquid carbon tetrachloride.

reagents

• Carbon tetrachloride (CCl ₄ )
• Paraffin (liquid), Fa. Merk KgaA, Darmstadt, DE, Best. No. 7160.2500

execution

1 to 2 g of the material to be tested are dried in a small weighing jar at 130 ° C in a drying oven. It is then cooled in a desiccator, weighed accurately and the vial placed in a vacuum desiccator containing, depending on the micropore volume to be measured, the following paraffin / carbon tetrachloride mixtures: Paraffin (ml CCl ₄ micropores (Å) 26 184 800 47.9 162.1 390 66.5 143.5 250 82.5 127.5 180 96.4 113.6 140 108.7 101.3 115

Of the associated with graduated cold trap, pressure gauge and vacuum pump Desiccator is now evacuated to boiling the contents. 10 ml Carbon tetrachloride are vaporized and in the cold trap collected.

thereupon Leave the desiccator contents at room temperature for 16 to 20 hours balance, then slowly let air in the desiccator. After removing the desiccator lid, the weighing jar becomes immediately closed and weighed back on the analytical balance.

evaluation

(Dichte Tetrachlorkohlenstoff bei 20°C: d = 1,595 g/cm³)The values are calculated in milligrams of carbon tetrachloride adsorption per gram of substance by weight gain. Dividing with the density of the carbon tetrachloride gives the pore volumes in ml / g of substance

(Density carbon tetrachloride at 20 ° C: d = 1.595 g / cm ³ )

Particle Size Determination by means of dynamic light scattering (Malvern)

The Measurements are made with a device "Mastersizer" of the company Malvern Instruments Ltd., UK, according to the manufacturer's instructions carried out. The measurements are provided with the Sample chamber ("dry powder feeder") performed in air and determines the values related to the sample volume.

Water content:

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

Determination of the pH of a bentonite

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

Elemental analysis:

These Analysis is based on the total digestion of the clay materials or the appropriate product. After dissolving the solids The individual components are made with conventional specific ones Analysis methods, such. For example, ICP is analyzed and quantified.

Ion exchange capacity:

To determine the cation exchange capacity, the clay material to be examined is dried at 105 ° C. over a period of 2 hours. 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 is filtered, whereupon the filter cake is washed, dried and ground and determined the NH ₄ content in the clay by nitrogen determination (CHN analyzer "Vario EL III" from Elementar in Hanau) according to the manufacturer. The proportion and type of exchanged metal ions is determined in the filtrate by ICP spectroscopy.

Determination of sediment volume:

A graduated 100 ml graduated 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 on the graduation of the measuring cylinder. To determine the sediment volume after storage in water for 3 days, the sample ^{batch is} sealed with Parafilm® and allowed to stand vibration-free for 3 days at room temperature. Then the sediment volume is read off the graduation of the measuring cylinder.

Determination of montmorillonite content via the methylene blue adsorption

Of the Methylene blue value is a measure of the inner surface the clay materials.

a) Preparation of a tetrasodium diphosphate solution

5.41 tetrasodium diphosphate is reduced to 0.001 g in a 1000 Weigh out the volumetric flask and shake to the calibration mark with dist. Water filled up.

b) Preparation of a 0.5% methylene blue solution

In In a 2000 ml beaker, 125 g of methylene blue in about 1500 ml least. Water dissolved. The solution is decanted off and on 25 1 with dist. Water filled up.

0.5 g of wet test bentonite with a known internal surface are weighed to the nearest 0.001 g in an Erlenmeyer flask. 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 Methylenblaulösung and repeated the dot sample. The addition takes place until the yard turns slightly light blue, so that the added Methylenblaumenge is no longer absorbed by the test bentonite.

c) Testing of clay materials

The Testing of the clay material is carried out in the same way as for the test bentonite. From the amount consumed Methylene blue solution allows the inner surface of the clay material.

381 mg methylene blue / g clay are equivalent to this method of 100% montmorillonite.

Determination of dry residue

Approximately 50 g of the examined air-dry clay material on weighing a sieve of the appropriate mesh size. The sieve is connected to a vacuum cleaner, which has an under the sieve bottom circular suction slot all shares, the finer than the strainer are sucked out through the strainer. The sieve comes with a Covered plastic lid and turned on the vacuum cleaner. To 5 minutes, the vacuum cleaner is turned off and the amount of up the coarser portions remaining by sieving by differential weighing determined.

Determination of wet sieve residue

It First, a 5% suspension is prepared by an appropriate amount of the clay material to be examined about 930 rpm is stirred for about 5 minutes in water. The Suspension is for about 15 minutes at about 1865 rpm stirred and then the suspension through a sieve of the desired Mesh size cast. The residue is with so long Washed tap water until the wash water runs clear. The sieve with the residue is then left for 5 minutes placed in an ultrasonic bath to remove residual fines. The remaining residue is briefly with tap water washed and if necessary the sonication repeated until while the ultrasound treatment no more fines pass into the water. The sieve is then dried to constant weight. For weighing the residue remaining on the sieve is weighed Porcelain bowl transferred.

Determination of mechanical stability

105 to 115 g of the granules are placed on a sieve of mesh size 0.15 mm given and sieved finely divided portions of the granules.

100 g of the freed of fines granules are placed on a sieve of mesh size 0.15 mm, which is mounted on a drip tray. On the granules 3 rubber balls are given, which have a diameter of 2.9 mm. The sieve is covered with a lid, between sieve and cover a distance of 25 mm is provided. The device composed of drip tray, sieve and lid is placed on a rotary shaker and shaken there for 15 min. Subsequently, the collected in the drip tray granules is weighed. This number corresponds to the refractive index for 15 min. Shaking time.

The Sieve is shaken for another 15 minutes and again the material balanced, which has accumulated in the drip tray. Out the sum of the sieve passes results Brechbarkeitszahl for 30 minutes

Determination of bulk density

One at the 1000 ml mark truncated graduated cylinder is weighed. Then the sample to be examined is determined by means of a powder funnel so filled in the measuring cylinder in a train that above the completion of the measuring cylinder a cone of material formed. The pour cone is made with the help of a ruler, that passed over the opening of the measuring cylinder is stripped off and the filled measuring cylinder again weighed. The difference corresponds to the bulk density.

Determination of protein concentration according to the Lowry method

The Protein quantification is based on the modified Lowry protocol on the reaction of proteins with copper sulfate and copper tartrate in alkaline solution, resulting in the formation of a tetradentate Copper-protein complex leads.

in the next step is a reduction by adding the Folin-Ciocalteau phenol reagent (contains phosphomolybdate-phosphotungstic acid), wherein an intense blue coloration occurs. The concentration of resulting reaction product is determined photometrically at 750 nm.

Execution:

The Determination was carried out with reagents used by the Sigma-Aldrich GmbH, Taufkirchen, DE ready to use were.

100 μl the protein / enzyme solution will be in a cavity a 96-well plate pipetted. 100 μl of Lowry's reagent pipetted and the mixture homogenized. After 20 minutes will be Add 50 μl Folin-Ciocalteu reagent and mix Homogenized again. 30 minutes after adding the Folin Ciocalteu reagent the extinction is against a blank at 750 nm in the plate photometer measured.

Determination of protein concentration according to the BCA method

Bicinchoninic acid (BCA) serves as a detection system in the BCA method. First of all, the 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, which from Pierce, Illinois, US.

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

Example 1: Preparation of the granules

Two commercially available (Süd-Chemie AG, Munich, DE) bleaching earths were used for the production of the granules. Bleaching earth 1 is a 5% H ₂ SO ₄ surface-activated bleaching earth. Bleaching earth 2 is a highly activated bleaching earth digested by extraction with hot HCl. The properties of the bleaching earths are summarized in Table 1. Table 1: Physical properties of sour activated bleaching earths used as starting material parameter Bleaching earth 1 Bleaching earth 2 Sieve residue> 63 μm (%) 41 29 BET surface area (m ² / g) 190 200 CCl ₄ -sorosimetry 0-800 Å (Ml / g) 0.64 0.29 0-250 Å (Ml / g) 0.37 0.25 0-140 Å (Ml / g) 0.21 0.23 BJH porosimetry Cumulative pore volume for pores with diameters of 1.7-300 nm (Ml / g) 0.7 0,376 Average pore diameter according to BJH [nm] 16, 2 6, 2 pH (-) 2.3 3.3 CEC (meq / 100g) 46 32 Silicate SiO ₂ % 70.2 66.8 Fe ₂ O ₃ % 2.8 3.7 Al ₂ O ₃ % 9.4 14.2 CaO % 2.3 1.1 MgO % 2.5 2.3 Na ₂ O % 0.4 0.8 K ₂ O % 1.5 2.2 TiO ₂ % 0.3 nb Loss on ignition (2 h, 1000 ° C) % 9.5 8.0 total % 98.9 99.1

a) shaping by wet granulation

• Laundrosil^® DGA: alkalisch aktivierte Bleicherde (Süd-Chemie AG, München)

Tabelle 3: Eigenschaften der Granulate vor dem Calcinieren Bleicherde 1 Bleicherde 1+ 10% DGA Bleicherde 1 + 20% DGA Schüttgewicht (g/l) 383 359 394 pH-Wert 3,7 4,1 4,8 Quellvolumen (ml) 6 6 7 Stabilität in Wasser nach 1 Tag moderat stabil nicht stabil nicht stabil Tabelle 4: Eigenschaften der Granulate nach dem Calcinieren Bleicherde 1 Bleicherde 1+ 10% DGA Bleicherde 1 + 20% DGA Schüttgewicht (g/l) 361 325 358 pH-Wert 4,69 4,96 5,34 Quellvolumen (ml) 6 7 7 Stabilität in Wasser nach 1 Tag stabil stabil stabil BET-Oberfläche m²/g 181 - 160 Kumuliertes Porenvolumen nach BJH für Poren mit Durchmessern von 1,7–300 nm (cm³/g) 0,689 - 0,643 Mittlerer Porendurchmesser nach BJH [nm] 15 - 16,5 Tabelle 5: Nassgranulation hoch aktivierter Bleicherden Bleicherde 2 (g) Wasser (g) DGA (g) 600 278 0 540 280 60 480 293 120 Tabelle 6: Eigenschaften der Granulate vor dem Calcinieren Bleicherde 2 Bleicherde 2 + 10% DGA Bleicherde 2 + 20% DGA Schüttgewicht (g/l) 712 649 671 pH-Wert 4,3 6,8 7,3 Quellvolumen (ml) 3 5 6 Stabilität in Wasser nach 1 Tag moderat stabil nicht stabil nicht stabil Tabelle 7: Eigenschaften der Granulate nach dem Calcinieren Bleicherde 2 Bleicherde 2+ 10% DGA Bleicherde 2+ 20% DGA Schüttgewicht (g/l) 620 588 566 pH-Wert 3,87 4,97 6,03 Quellvolumen (ml) 4 4 3 Stabilität in Wasser nach 1 Tag stabil stabil stabil BET-Oberfläche m²/g 210 - 176 Kumuliertes Porenvolumen nach BJH für Poren mit Durchmessern von 1,7–300 nm (cm³/g 0,335 - 0,292 Mittlerer Porendurchmesser nach BJH [nm] 5,75 - 5,98 The quantities of bleaching earths 1 and 2 indicated in Table 2, 5 were initially introduced into an Eirich Intensive Mixer R02E and water was metered in via a funnel as agglomerating agent. It was the low setting for the rotational speed of the plate as well as the maximum rotational speed for the swirler. Unless otherwise indicated, the agglomeration parameters were each chosen below such that more than 50% of the granules were in a particle size range of 0.4 to 1.4 mm. For this purpose, the granules were sieved off appropriately after granulation and drying. The granules were then dried in a drying oven to a maximum water content of 15% by weight. The dried granules were transferred to an oven and calcined at 600 ° C for one hour. The properties of the soft granules obtained before the heat treatment and the water-resistant granules obtained after the calcination are summarized in Tables 3 and 4 for the surface activated bleaching earths and in Tables 5 and 6 for the highly activated bleaching earths. Table 2: Wet granulation of surface-activated bleaching earths Bleaching earth 1 (g) Water (g) DGA (g) 400 378 0 360 372 40 320 347 80

• Laundrosil ^® DGA: alkaline activated bleaching earth (Süd-Chemie AG, Munich)

Table 3: Properties of the granules before calcination Bleaching earth 1 Fuller's earth 1+ 10% DGA Fuller's earth 1 + 20% DGA Bulk density (g / l) 383 359 394 PH value 3.7 4.1 4.8 Swelling volume (ml) 6 6 7 Stability in water after 1 day moderately stable not stable not stable Table 4: Properties of the granules after calcining Bleaching earth 1 Fuller's earth 1+ 10% DGA Fuller's earth 1 + 20% DGA Bulk density (g / l) 361 325 358 PH value 4.69 4.96 5.34 Swelling volume (ml) 6 7 7 Stability in water after 1 day stable stable stable BET surface area m ² / g 181 - 160 Cumulative pore volume according to BJH for pores with diameters of 1.7-300 nm (cm ³ / g) 0.689 - 0.643 Mean pore diameter according to BJH [nm] 15 - 16.5 Table 5: Wet granulation of highly activated bleaching earths Bleaching earth 2 (g) Water (g) DGA (g) 600 278 0 540 280 60 480 293 120 Table 6: Properties of the granules before calcination Bleaching earth 2 Bleaching earth 2 + 10% DGA Bleaching earth 2 + 20% DGA Bulk density (g / l) 712 649 671 PH value 4.3 6.8 7.3 Swelling volume (ml) 3 5 6 Stability in water after 1 day moderately stable not stable not stable Table 7: Properties of the granules after calcination Bleaching earth 2 Fuller's earth 2+ 10% DGA Fuller's earth 2+ 20% DGA Bulk density (g / l) 620 588 566 PH value 3.87 4.97 6.03 Swelling volume (ml) 4 4 3 Stability in water after 1 day stable stable stable BET surface area m ² / g 210 - 176 Cumulative pore volume according to BJH for pores with diameters of 1.7-300 nm (cm ³ / g 0.335 - 0.292 Mean pore diameter according to BJH [nm] 5.75 - 5.98

b) shaping by extrusion

For the production of granules became commercially available Bleaching earths 3 and 4 and a Na / Ca bentonite (Süd-Chemie AG, Munich, DE). The properties of the starting materials are summarized in Table 8.

60 kg bleaching earth 3, 32 kg bleaching earth 2 and 8 kg Ca / Na bentonite are homogenized in a mixer. Thereafter, 35 kg of water are added and the mixture kneaded for another 30 minutes. The plastic mass is shaped into extruded products in a circular disk press (flat die press) by Amandus Kahl GmbH & Co KG, Reinbek, DE with a hole diameter of 3 mm under highly compacting conditions. A running cutting tool shortens the pellets to a length of 2 to 3 mm. The moist extrudates are dried in a drum dryer and screened to a particle size of 0.5 to 2 mm. Oversized grain is broken and returned to the sieve. The resulting granules are calcined for one hour at 600 ° C. The properties of the granules are also listed in Table 8. On the one hand, the granules have a high mechanical stability and, on the other hand, still have a high porosity. Table 8: physical properties of acid-activated bleaching earths used as starting material and the granules produced therefrom Bleaching earth 3 Bleaching earth 4 Ca / Na bentonite Granules (uncalcined product BET surface area m ² / g 361.75 277.02 63.7 270.8 270 Cumulative pore volume according to BJH for pores with diameters of 1.7-300 nm [cm ³ / g] .4535 0.316 0.0743 .3136 .3645 Mean pore diameter according to BJH [nm] 4.9 4.8 7.4 4.9 5.7 Wet sieve residue (%) 63 μm 10.1 21.5 15.7 Granules break down 99 45 μm 15.7 30.0 41.8 Granules break down 99d Dry residue (%) 63 μm nb nb nb 99 99 45 μm nb nb nb 100 100 PH value 3.04 3.74 6.54 3.92 4.19 Bulk density (g / l) - - - 720 555

A granulate of bleaching earth 2 was prepared analogously to the abovementioned conditions. The properties of the granules are summarized in Table 9 Table 9: Physical properties of a granulate made from bleaching earth 2 Granules (uncalcined) Product (according to the invention) BET surface area m ² / g 285.8 215.4 Cumulative pore volume according to BJH for pores with diameters of 1.7-300 nm [cm ³ / g] .4244 0.3354 Mean pore diameter according to BJH [nm] 5.7 5.8 pH (2% by weight in water) 3.01 3.85 Bulk density (g / l) 552 515

Around the stability of the granules in aqueous media These were tested in distilled water as well as in 7 days stored in different buffers. In each case 0.5 g of the granules in 20 ml of liquid.

The following buffers were used: buffer PH value citrate 3 Na acetate 5 HEPES 7 TRIS 9

To one week storage time were the invention Granules unchanged. It was not a decay by the Formation of turbidity or fine sediment is displayed, recognizable. In contrast, the starting granules which do not decompose decay were treated at 600 ° C, under these conditions in fine particles

Example 2: Supports lipase on thermally treated clay minerals

The Lipase was in each case once on the powdered and once immobilized on the granulated bentonite.

For the support on the thermally treated clay minerals was a lipase from Candida rugosa (Sigma-Aldrich GmbH, Taufkirchen, DE). Lipase from an organism Candida rugosa, a Yeast mushroom, has a size of approx. 60-65 kDa and an isoelectric point of 5.2. Lipases from the organism Candida rugosa are among the non-specific lipases that have no regiospecific properties and thus all ester bonds hydrolyze.

a.) Determination of the enzyme binding capacity in the static system

Preparation of the carrier

Each 25 mg of the powdery starting material used in Example 1 Bentonites 1 and 2 are equilibrated in 10 ml of 50 mM Na-acetate buffer (pH 4). The carrier suspended in the buffer is sonicated for 30 minutes treated and at 15 rpm for 30 min in an overhead mixer shaken. The suspension is subsequently centrifuged at 3219 g and 10 ° C for 10 minutes and the supernatant discarded.

at immobilization of lipase on those from bentonites 1 and 2 granules were prepared analogously as described above, however, each used four times the amount of all components and the loaded carrier by decating from the protruding Enzyme solution was separated.

Preparation of the enzymes

For the examples will be a stock solution of the lipase with a Concentration of 2 mg / ml in 50 mM sodium acetate buffer (pH 4) produced.

Investigation of enzyme adsorption

To 25 mg each of the equilibrated carriers Add 10 ml of the enzyme stock solution and add the suspension for 30 min at 4 ° C and 15 rpm in the overhead shaker incubated. Thereafter, the solid at 3219 g (10 ° C) Centrifuged for 10 min and the protein content in the supernatant determined by the Lowry and the BCA method. Subsequently the solid is resuspended in 10 ml of the appropriate buffer, for 5 min at 4 ° C and 15 rpm in the overhead shaker and re-centrifuged at 3219 g (10 ° C). In the case of the granules investigated, the separation from the aqueous phase in each case by decantation. The protein content the wash water is also determined. The on the carrier adsorbed amount of enzyme was used from the difference between Enzyme amount and the sum of the supernatant or in the wash water calculated amount of enzyme calculated. All experiments are called triple determination carried out. After the supernatant completely removed is, the loaded with the enzyme carrier in 1-3 ml of distilled water and the suspension for used the activity tests.

Activity determination of the supported Lipase (Candida rugosa)

i. Reading lipase solution

In a 50 ml Erlenmeyer flask
2.5 ml of distilled water
1.0 ml TRIS buffer 200 mM (pH 7.7)
3.0 ml of olive oil
pipetted. The samples are preheated for about 5 minutes with stirring in a water bath at 37 ° C. Then 1.0 ml of lipase solution (stock solution) is pipetted in and incubated with stirring in a water bath at 37 ° C for 30 minutes. Thereafter, 3.0 ml of ethanol (95%) are pipetted in and after addition of 50 .mu.l of thymolphthalein solution is titrated with 0.1 M NaOH standard solution until the envelope to blue.

ii. Blank value of lipase solution

In a 50 ml Erlenmeyer flask, in each case 25 mg of the carrier described in Examples 1 to 4 are weighed in and then, after equilibration with buffer
3.5 ml of distilled water
1.0 ml 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 ° C. Thereafter, 1.0 ml of lipase solution (stock solution) and 3.0 ml of ethanol (95%) are pipetted in, and after adding 50 μl of thymolphthalein solution, titrated with 0.1 M NaOH standard solution until blue.

iii. Measured value of lipase

In a 50 ml Erlenmeyer flask
2.5 ml of distilled water
1.0 ml TRIS buffer 200 mM
3.0 ml of olive oil
pipetted. The samples are preheated for about 5 minutes with stirring in a water bath at 37 ° C. 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 adding 3.0 ml of ethanol (95%) and 50 μl of thymolphthalein solution, titrate with 0.1 M NaOH standard solution until blue.

iv. Blank value-supported lipase

In a 50 ml Erlenmeyer flask
2.5 ml of distilled water
1.0 ml 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 ° C. The enzyme adsorbed on the respective carrier is taken up in 1 ml of distilled water and added to the solution in the Erlenmeyer 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 above the NaOH consumption determined.

Unit-Definition

A Unit hydrolyzes 1.0 μM fatty acid (from triglycerides) in one hour at pH 7.7 and 37 ° C.

Results

In Table 10 is the results of adsorption and activity of the supported enzyme lipase (Candida rugosa).

As As shown in Table 10, the acid activated Phyllosilicates are suitable for supporting lipase. The (Non-inventive) powder show a high Binding capacity for the lipase. So tie 25 mg the powdered bleaching earth 2 13.9 mg lipase. The relative Although activity of the lipase on the carriers is reduced in activity over the activity. However, it is still over 30% of the two powders Activity in solution.

By Granulation can be made from these powders stable granules which has a reduced, but still high binding capacity for lipase. This is caused by that the enzymes usually only on the outer shell the granules are adsorbed and the interior of the granules for Adsorption and diffusion are inaccessible. So shows in particular by extrusion and subsequent calcination at 600 ° C granules produced a binding capacity of more than 20% enzyme based on the carrier material. Furthermore, the granules still show a good activity of adsorbed lipase.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (14)

Process for the preparation of a solid enzyme complex, in which - A powdery granulation which is at least 50 wt .-% of an acid-activated Layered silicate is formed; - the powdery Granulating mixture is granulated to a soft granules; - the soft granules are calcined, with a water-resistant granules is obtained; and - on the waterproof granules at least one enzyme is immobilized. The method of claim 1, wherein the acid activated Phyllosilicate by applying a layered silicate with a Acid is obtained. Method according to one of the preceding claims, wherein the acid-activated phyllosilicate is obtained by a Phyllosilicate is extracted with hot acid. A process according to any one of the preceding claims, wherein the acid-activated phyllosilicate has an aluminum content, calculated as Al ₂ O ₃ , of less than 17% by weight. A method according to any one of the preceding claims, wherein the stabilized granules have a cumulative pore volume for pores in the range of 1.7-300 nm diameter as determined by the BJH method in the range of 0.5 to 0.7 cm ³ / g. Method according to one of the preceding claims, the soft granules being calcined to a temperature of at least 400 ° C is heated. Method according to one of the preceding claims, wherein the waterproof granules have a mean diameter in the range from 100 μm to 2 mm. Method according to one of the preceding claims, wherein the enzyme is selected from the group formed is composed of lipases, reductases, transferases, hydrolases, isomerases, Ligases, lyases. Method according to one of the preceding claims, wherein the enzyme for immobilization via a Covalent bond is bound to the stabilized granules. Method according to one of the preceding claims, the proportion of the enzyme, based on the weight of the solid Enzyme complex, between 3 and 35 wt .-% is. A method according to any one of the preceding claims, wherein the stabilized granules have a specific surface area of 50 to 280 m ² / g. Solid enzyme complex obtained by a process according to one of the claims 1 to 11 with a core of a waterproof granules of an acidic activated phyllosilicate on which immobilized at least one enzyme is. Use of a solid enzyme complex according to claim 12 in an enzymatically catalyzed reaction. Use according to claim 13, wherein the solid enzyme complex before use in the enzymatically catalyzed reaction to a pH is equilibrated within a range of ± 1 to a pH at which the enzyme is the highest Sales.