DE2717965A1 - POROESE CELLULOSE BEADS - Google Patents

Description

The invention relates to porous cellulose beads which are suitable as carriers for enzymes and other biologically active agents, and a method for their production. The invention also relates to the use of those produced according to the invention Cellulose beads for immobilizing enzymes, removing metal ions from solutions, and for separation and purification of enzymes, proteins, nucleic acids and similar compounds.

Porous cellulose beads make a relatively inexpensive stable one Material with versatile chemical properties. For example, they are suitable as carriers for immobilized enzymes and other active biological agents.

Albeit common cellulose particles and regenerated cellulose powder meet most of the requirements placed on good supports for immobilized enzymes, they are structural Disadvantages that cause the packing of the reactors to become too tight, which in turn leads to a reduction in the flow rate and sometimes channeling and hence insufficient contact between the immobilized enzymes and the flowing one Reaction medium leads. Immobilization of enzymes on an insoluble support is a common practice for the practical use of enzymes to avoid having to use fresh enzymes each time they are used.

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sen. The immobilization of the enzymes provides a stabilization achieved, which allows an effective use of enzymes and the use of continuously operating enzyme reactors.

The successful application of immobilized enzymes depends in large part on the properties of the agent used for immobilization used carrier. Accordingly, a good carrier should be inexpensive and have a shape suitable for the Use in reactors is suitable. In view of these requirements, spherical beads are particularly desirable because they are particularly suitable for fixed beds, fluidized beds, expanded beds, stirred tanks and other common types of reactors. Such a The carrier should also have adequate physical and mechanical strength so that it does not break or deform when packed in a tall column. The disintegration and deformation of the support in the column lead to one too tightly packed bed, which hinders the flow of liquid reagents through the column and reduces the effectiveness of the Reactor decreases. Suitable carriers should also have versatile chemical properties, so that the immobilization of enzymes and other biological agents on the carrier by ionic or covalent bonding or by Surface adsorption can be achieved easily. Accordingly, the carrier should have a large capacity to form a large one Have number of bonds so that each carrier unit can immobilize large amounts of the desired enzyme. Particularly

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a carrier with high porosity and uniformity is therefore suitable distributed inner pores. Such porosity allows good diffusion of chemical reagents or reaction products into and out of the inner pores of the cellulose beads. Carriers should be chemically stable, physically be solid and made of an inert material that is microbiological attacking a destruction of the wearer effect, resist, to provide an immobilized enzyme system with an extended lifespan.

For the immobilization of enzymes, porous glass and ceramic particles are used. While such particles meet most of the requirements, they are proportionate expensive. In addition, the number of chemical reactions necessary for the immobilization of enzymes on glass and ceramic substrates can be used, limited.

In U.S. Patents 3,947,325, 3,905,954, 3,573,277, 3,505,299, 3,501,419, 3,397,198, 3,296,000, 3,251,824, 3,236,669, 2,843,583, 2,773,027, 2,543,928 and 2,465,343 will manufacture a variety of cellulosic materials in a variety of forms described, some of which are used for the fixation of biologically active materials such as enzymes or of ion exchange groups should be suitable. However, these methods also have the disadvantage that they are expensive and the products are one have undesirable body shape for use in such chemical reactors as fixed beds and fluidized beds. In particular

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However, the known processes do not produce spherical cellulose beads with a uniform pore distribution over the entire surface and a large one with uniform pores existing, internal pore volume. In addition, the known cellulose particles and powders generally have such small particle sizes that they are unsuitable for use in chemical reactors. In addition, the well-known Cellulose powder and particles often have a hard surface layer, which is a serious obstacle to diffusion and an ineffective one Use in chemical reactors causes.

The invention is now based on the task of cheap, highly porous, stable particles with versatile chemical properties, which make them suitable as carriers for immobilized enzymes or other biologically active materials. Another object of the invention is to provide porous cellulose beads with improved physical and mechanical properties To provide stability and a sufficiently large surface area for a high immobilization capacity for enzymes.

The invention accordingly relates to a method of production of porous cellulose beads, which are suitable as carriers for enzymes and other biologically active agents, characterized in, that he

(a) a hydrolyzable cellulose derivative in an inert, water-miscible solvent in a ratio of

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1:20 to 1: 3 weight / volume dissolves to a solution with a greater density than the precipitation solution,

(b) the solution thus prepared is distributed in the form of droplets in a precipitation solution and thereby the cellulose derivative precipitates in the form of uniform porous beads,

(c) separating the precipitated beads from the solution,

(d) washing the separated porous beads with water,

(e) the washed beads to convert to cellulose and to increase the active sites for coupling with Enzymes and other biologically active agents hydrolyzed and

(f) the hydrolyzed beads to give porous cellulose beads Washes with evenly distributed pores and a pore volume of more than 50% by volume.

The highly porous cellulose beads according to the invention with a uniform porosity are particularly suitable for immobilization of enzymes. In addition, they are also suitable for cleaning and separating enzymes, proteins and nucleic acids and similar compounds as well as for the separation of metal ions from dilute solutions. Because of their porosity and their good mechanical stability are provided by those according to the invention Cellulose beads provide excellent passage of liquids when used in fixed bed reactors and are not subject to any signs of disintegration or deformation.

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The method according to the invention provides porous cellulose beads, which are suitable as carriers for enzymes and other biologically active agents. The method according to the invention also enables the change in the chemical and physical properties of porous beads made from cellulose derivatives. Although usually microcrystalline cellulosic and other particles made from cellulose, many of the essentials However, when meeting requirements for suitable enzyme carriers, these particles tend to pack too closely under pressure and do not provide sufficient porosity to couple sufficiently large amounts of enzyme thereto. Cellulose derivatives are generally inexpensive and, when processed according to the invention, provide a very versatile, generally speaking biologically inert material for chemical reactions. Accordingly, the cellulose beads of the present invention have many desirable properties for use as a carrier for immobilized enzymes.

By dissolving a cellulose derivative according to the invention in a selected solvent and the distribution of the same in a selected precipitation solution, it is possible Manufacture cellulose beads with very uniform porosity and superior chemical and physical properties. The beads produced according to the invention are highly porous, with the pores generally being uniform over the surface and distributed inside the pearls. By suitable selection of the solvents and the precipitation solutions, the

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Pore size can be regulated. A particular advantage of the invention The method is that both the pore size and the pore distribution can be controlled. From the figures 2, 4a and b it can be seen that the pore openings are evenly distributed over the pearl surface and are about 1000 8 in size, what is a suitable size for the movement of enzyme and reagent molecules in the pores.

The inert, organic, water-miscible solvent can be a single liquid or a combination of liquids be. It is important to use the correct combination of inert organic solvent and precipitation solution, to obtain porous cellulose beads with the desired shape and porosity. The inert, organic, water-miscible Solvent can be a combination of liquids that, together with the cellulose derivative, provide a solution that when mixed with the precipitation solution leads to a phase inversion, whereby the cellulose derivative in the form of porous Pearls coagulate. The inert organic solvent accordingly contains a component (a) which is identified as a liquid capable of dissolving the cellulose derivative such as cellulose acetate and in the precipitating solution is soluble.

A second component (b) of the solvent system is one Liquid which is soluble in component (a) and also in the precipitation solution and which is in the solvent solution in is present in sufficient quantity so that the density of the

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finished solvent solution (including the cellulose derivative) is sufficiently higher than the density of the precipitation solution and when distributing the solvent solution in the form of Droplets in the precipitation solution coagulate the cellulose and precipitate as porous beads with the desired size and porosity. Component (b) of the solvent is used to regulate the surface activity of the solvent solution, so that the solvent solution droplets keep their shape upon contact with the precipitating solution. Component (b) serves also to regulate the pore size and the porosity of the precipitated pearls. In some cases the component (a) and component (b) be the same. In other cases it can be advantageous to prepare component (a) and / or component (b) to use one or more liquids.

The term "precipitation solution" is defined as a liquid one Solution that is not a solvent for the cellulose derivative and with the above inert, organic, water-miscible solvent is miscible. The precipitation solution can be, for example, water or an aqueous solution. The precipitation solution is therefore miscible with both component (a) and component (b). So if you put the cellulose derivative in dissolves the organic solvent and then one drop of the resulting solvent solution to the precipitation solution there, the cellulose derivative coagulates and falls due to the phase inversion to form the desired porous cellulose bead.

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Of course, the method described above for making porous cellulose beads can be varied in various ways. In addition to cellulose acetate, other cellulose derivatives can also be used as a starting material for the production of porous beads, such as e.g. cellulose nitrate and methyl cellulose can be used. The terms "cellulose derivative" and "hydrolyzable cellulose derivative" include those materials from which cellulose is regenerated, for example by hydrolysis or hydrogenation can.

The organic solvent components (a) and (b) for the cellulose derivative should be chemically inert towards the cellulose derivative and should be completely or essentially miscible with the precipitation solution. It is of particular importance that the density of the solvent solution formed by adding the cellulose derivative to the inert solvent is greater than that of the precipitation solution in which it is distributed, so that when the solvent solution droplets are distributed in the precipitation solution, the droplets sink down when the aqueous Solution is not stirred. Suitable individual solvents when using an aqueous precipitation solution include dimethyl sulfoxide and methyl acetate. It should be noted that commercially available materials can be used as solvent components (a) and / or (b) and that these materials may contain moisture, which has proven advantageous in some cases.

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When using an aqueous precipitation solution, acetone, formamide, a mixture of acetone and methanol or ethanol, methyl acetate, a mixture of methylene dichloride and methanol, methyl ethyl ketone and / or dimethyl sulfoxide can advantageously be used as solvent component (a). The solvent component (b) can accordingly be selected from the following
Solvents can be selected: dimethyl sulfoxide, formamide,
Methyl acetate, cyclohexanone, methylene dichloride, ethylene dichloride, a mixture of methylene dichloride and methanol and / or a mixture of ethylene dichloride and methanol.

A preferred solvent component (a) is acetone, however
other solvents are also suitable. When using an aqueous precipitation solution, one solvent component (a) can be selected from the following materials (the ratios of the mixtures are the minimum desired ratio on a volume basis):

Component (a) Minimum Ratio

(Volume)

acetone

Acetone + methanol 60:40

Acetone + ethanol 60:40

Methyl acetate

Methylene dichloride + methanol 80:20

Dimethyl sulfoxide

Methyl ethyl ketone

Formamide

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As stated above, the main function is the component (a) In dissolving the cellulose derivative. The addition of component (b) is necessary to make a solvent solution with the required density so that the cellulose derivative precipitates in the precipitation solution. The component (b) also allows control of the pore size and the uniform porosity of the beads.

The solvent component (b) thus provides the specific desired Weight of the solvent solution. When using an aqueous precipitation solution is preferred as dimethyl sulfoxide Component (b) used. In some cases, Component

(a) and component (b) be the same, i.e. dimethyl sulfoxide, formamide or methyl acetate if aqueous precipitation solutions are used will. Various materials that can be suitably used as a component when an aqueous precipitating solution is used

(b) are shown in the following table:

Component (b) Minimum Ratio

(Volume)

Dimethyl sulfoxide

Ethylene dichloride + methanol 60:40

Methylene dichloride + methanol 60:40

Ethylene dichloride

Methylene dichloride

Formamide

Cyclohexanone

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The solution of cellulose derivative and inert solvent should have a controlled cellulose derivative-solvent ratio as this has an effect on the final porosity of the pearls produced. In general, a little leads Ratio (greater solvent content) to beads with greater porosity. A cellulose to solvent ratio 1:20 and 1: 3 (weight / volume; including components (a) and (b)) has been found to be suitable for manufacture of cellulose beads have been shown to have various specific uses. A cellulose derivative / solvent ratio is preferred from 1:10 to 1: 6 (weight / volume) used to get an easy to use solution that to cellulose beads with the desired properties and with a pore volume of at least 50% by volume, preferably 75% Up to 95 and in particular 75 to 80% by volume leads. Higher porosity beads generally have a greater proportion of uniformity distributed inner pores and thus less diffusion hindrance, however, are somewhat weaker in terms of the physical strength than pearls with lower porosity.

The preferred precipitation solution in which the solution of the cellulose derivative is distributed, consists generally of water, but can also be an aqueous solution, the appropriate amounts of Contains nonionic or ionic surfactants in order to reduce the surface tension and the formation of the porous beads to facilitate. The precipitation solution can also be a mixture of water and methanol or ethanol (volume ratio

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50: 5O) included. It is also possible that the precipitation solution is not aqueous as long as the cellulose derivative is insoluble therein and meets the density requirements are. Accordingly, hydrocarbon solutions such as cyclohexane, Hexane, decane, benzene and the like are used as long as they are liquid, a lower density than the inert one Have organic solvents and are miscible with the latter. If the cellulose derivative solution is sprayed by means of a suitable device such as a spray nozzle, the pressure drop and the miscibility of the inert solvent result with the aqueous solution to a dispersion and finally to the precipitation of porous cellulose derivative beads.

Of course, there must be a sufficient amount of solvent component (b) present for that containing the cellulose derivative Solvent has the required higher density than the precipitation solution. In Table 1 are a number of inert organic Solvents indicated for the precipitation of a cellulose derivative in an aqueous solution. The specified ratios are the minimum ratios required to obtain a solvent solution that has a greater density than water Has. The greater the specific gravity of component (b), the less component (b) is required to achieve the minimum To achieve density.

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Table 1

solvent

Component (a) component (b)

Minimum volume
ratio
away 30th 70 2O 80 : 20 80 25th 75 55 45 65 35

Acetone dimethyl sulfoxide

Acetone ethylene dichloride

Acetone methylene dichloride

Acetone formamide

Acetone cyclohexanone

Acetone methyl acetate

After the production of the porous beads, the cellulose is hydrolyzed regenerated from the derivative to create more active sites for enzyme coupling. To regenerate the cellulose the substituted groups (like acetate in cellulose acetate) can be removed from the cellulose derivative after the formation of the beads, to regenerate all hydroxyl groups normally present in the cellulosic material. The higher the degree of regeneration the more stable the resulting pearls will be. In some cases where enzymes are on the cellulose bead supports are to be immobilized, it is desirable to convert the hydroxy or other substituent groups into functional chemical groups how to convert amino groups that facilitate the coupling of enzymes.

In the following, the invention will be explained in more detail with reference to the figures will; show it:

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FIG. 1 is a graph showing the particle size distribution of the porous beads;

FIG. 2 shows an electron microscopic raster image of a porous Cellulose bead;

Figure 3 is a graph showing the pressure drop properties of the porous cellulose beads;

FIG. 4 (A) is an electron microscopic raster image of the surface a porous cellulose bead (20,000 x)

FIG. 4 (B) is an electron microscopic raster image of the interior a porous cellulose bead (20,000 x).

The graph in FIG. 1 shows the particle size distribution of the finished porous beads which are obtained when a cellulose derivative solution is distributed by means of a spray nozzle according to the method described in detail below. Beads that are too large or too small depending on the intended use can be collected and, if necessary, redissolved in the appropriate solvent. For use in a column reactor, beads of uniform size are generally preferred. The desired particle size can vary depending on the intended use of the beads, for example depending on the enzyme to be immobilized.

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The porous cellulose beads produced by the process described above generally have a very high porosity and a controlled pore size ranging from 0.05 to 30, u. If a cellulose / solvent ratio of 1:10 (weight / Volume) is used to produce the cellulose / solvent solution, the finished beads have a high porosity with a pore volume of about 90%. An electron microscopic raster image of a according to the method according to the invention produced porous cellulose bead is shown in Figures 2, 4 (A) and 4 (B). There are several of these recordings important features of the cellulose beads according to the invention can be seen. First, the beads are generally spherical in shape and the pore openings are uniform across the bead surface distributed. For most applications this is desirable because you can use an immobilized enzyme catalyst with more uniformity Activity receives. The void phase of the cellulose beads is continuous. This is a desirable feature because a discontinuous, discrete "bubble" to an unusable and inaccessible dead space in an immobilized enzyme system would lead. Third, there is no hard layer on the pearl surface. A hard surface layer would seriously hinder diffusion. After all, the pore sizes are very uniform, so that the entire inner surface of the inner pore volume of the beads is accessible for enzyme immobilization and for enzyme-catalyzed reactions. Both the high porosity and the other features mentioned give the porous cellulose beads according to the invention a unique ability to immobilize enzymes and other biologically active agents.

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An important property of an enzyme carrier is the pressure loss, which it produces at different liquid throughputs through an enzyme reactor containing the carrier. Industrial For example, DEAE cellulose is currently used as an enzyme carrier in the conversion of glucose to fructose. With DEAE cellulose, however, the pressure drop is very high and accordingly only flat beds can be used to obtain a reasonable fluid flow rate. The pressure drop properties of the porous cellulose beads according to the invention when used in a packed column are shown in FIG. Curve A, reproduced. The nominal linear flow rate is calculated by dividing the volumetric flow rate of feed liquid through the column by the column cross-sectional area. In practice, the nominal linear flow rate in industrial column reactors is less than 0.5 cm / second. A linear flow rate corresponds to a 60.96 cm diameter reactor of 0.5 cm / second, for example, a volumetric throughput of 5254 1 / hour. In a typical industrial process implementation for the production of fructose from glucose, the sugar concentration in the feed stream is about 0.5 kg / l. The above Flow rate results in more than 27 million kg of product per 61 cm column per year. Because of the dwell time required in the enzymatic reaction the linear flow rate is usually less than 0.5 cm / second. From it is It can be seen that the porous cellulose beads of the present invention do not have serious procedural issues with respect to pressure drop

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Pose problems when used as a carrier for enzymes and other biologically active agents in column reactors will. If the porous cellulose beads according to the invention after appropriate derivative formation for other possible applications (e.g. removal of tannin from fruit juice, wine or beer as well as metal ions from dilute solutions), the flow rate through a column reactor can be very much greater than the previously specified 0.5 cm / second.

The flow properties and other physical and mechanical Properties of the porous cellulose beads can be improved by crosslinking with bi- and / or multifunctional compounds will. Curve B in Figure 3 shows the pressure drop in the porous cellulose beads after treatment with tolylene-2,4-diisocyanate and subsequent enzyme immobilization. Above a nominal linear flow rate of 2 cm / Second, the untreated cellulose beads (curve A) are compressed and significantly deformed, resulting in a drastic Increase in pressure drop. Curve B, on the other hand, shows only a slight curvature, which, if present at all, shows a indicates only slight deformation of the treated beads.

The treatment of the porous cellulose beads with a crosslinking agent, either before or after hydrolysis of the pearls, leads to an increase in their physical strength. Also the Coupling of enzymes increases their physical strength. After treatment with, for example, a diisocyanate (e.g.

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Tolylene-2,4-diisocyanate or hexamethylene diisocyanate) the pearls are in fact very hard and firm. Crosslinking with epichlorohydrin improves the physical properties the porous cellulose beads as well. The chemistry of the crosslinking of polysaccharides, including cellulose and Strength is well known. Other suitable crosslinking agents include formaldehyde in hydrochloric acid solution or glutaraldehyde. Many other crosslinking agents for carbohydrates are known and are described, for example, in U.S. Patent 3,905,954.

In general, the porous cellulose beads according to the invention are produced according to the following process steps:

a) cellulose in a hydrolyzable form is dissolved in an inert, organic, water-miscible solvent, where the ratio of cellulose derivative to solvent is generally in the range from 1:20 to 1: 3 (weight: Volume) to make a solvent solution. The solvent used should be wholly or essentially miscible be with the precipitation solution, and the density of the solvent solution should be sufficient so that upon contact with the precipitation solution easily dissolves the solvent with this is miscible and the cellulose derivative precipitates.

b) A solvent solution is in the form of droplets (e.g. by spraying) distributed in a precipitation solution. Upon contact with the precipitation solution, which may contain a surfactant,

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the solvent is dispersed in the precipitating solution and porous cellulose beads are formed by coagulation. These sink to the bottom of the container for the precipitation solution. The cellulose derivative solution can suitably under Pressure can be sprayed through a spray nozzle into a precipitation solution bath. If desired, the bath can be used to speed up the bead formation are stirred.

c) After washing, the precipitated beads are hydrolyzed to regenerate cellulose, being porous Cellulose beads with active sites for enzyme coupling can be obtained. If desired, the pearls can be used for enhancement the stability or to obtain suitable reaction sites chemically modified in various ways will. For example, the pearls can be crosslinked for greater stability and increased physical strength to obtain. One can also change the surface absorption properties of the cellulose beads either positively charged or negatively charged groups by substitution insert into the pearls. The cellulose itself is generally hydrophilic and, accordingly, one can change it of the reaction sites change their hydrophilic properties.

The invention also relates to a method by which enzymes and other biologically active agents are immobilized by coupling to the porous cellulose beads of the invention

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can. For example, one can use the porous cellulose beads of the present invention Convert cellulose to diethylaminoethyl (DEAE) by treating the beads with N, N-diethyl-2-chloroethylamine hydrochloride implemented in a conventional manner. The pearls obtained in this way contain DEAE cellulose and are successful for coupling of glucose isomerase obtained from Streptomyces cultures. It is also one Procedure using cyanogen bromide for immobilization of glucose isomerase has been used.

Another procedure for enzyme immobilization tolylene 2,4-diisocyanate is used on porous cellulose beads. Diisocyanate was used to crosslink the cellulose in order to increase the physical strength of the porous beads to enhance. However, it has been found that porous cellulose beads according to the invention after treatment with diisocyanate enzymes Can immobilize on their surface by simply adding the diisocyanate treated beads and an enzyme solution mixes together. For example, when glucoamylase was used, more than 1000 international units of the Enzyme coupled and immobilized per g of dry beads on the beads treated with diisocyanate. Given the improved The physical strength of the beads is believed to result in significant crosslinking when dry porous cellulose beads in dry acetone in the presence of a catalyst (e.g. triethylamine) with tolylene-2,4-diisocyanate brings together. After a sufficiently long reaction time

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the beads were washed with dry acetone to remove the free Remove diisocyanate residues. The cellulose beads appear to have a large number of isocyanate groups attached. When the treated beads are mixed with an aqueous enzyme solution, the enzyme molecules appear through the isocyanate groups to be covalently bound to the cellulose beads. It was also found that when the pearls were washed with water the isocyanate groups are converted to amino groups. In this way the immobilization of an enzyme, glucoamylase, on aminocellulose beads with glutaraldehyde, which is known for its ability to interact with amino groups (on the beads and in the enzymes) to react and to network them.

The porous cellulose beads according to the invention are also suitable for the separation and purification of enzymes, proteins, nucleic acids and similar compounds. The invention Cellulose beads can be derivatized to produce porous DEAE cellulose beads. Own such pearls excellent flow properties and yet are able to effectively separate enzymes, proteins, nucleic acids and similar compounds. The effectiveness of such procedures corresponds to the currently customary column chromatographic procedures.

The porous cellulose beads of the invention can also be used derivatize in situ with groups other than DEAE. Through this the cellulose beads according to the invention are suitable for a large number of specific applications. For example, you can use a

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attach special functional groups to the porous cellulose beads and then use the beads derivatized in this way, e.g. to remove tannin from fruit juice by passing fruit juice through a bed of derivatized, porous cellulose beads with protein.

Similarly, one can get metal ions from dilute solutions remove. Such processes can be used, for example, to obtain valuable metal ions (e.g. copper and gold ions) from dilute Mine solutions and wastewater are used and were particularly suitable for solution techniques that are common today where metals are extracted from ores using acid solutions.

example 1

50 g of cellulose acetate (Vise 3 from Eastman Kodak Chemicals) was added in 4OO ml of solvent A (composed of acetone and dimethyl sulfoxide in a volume ratio of 6: 4) under Dissolved to form a 12.5% (weight / volume) solution. The cellulose solution was then sprayed with a spray gun (paint spray gun from Sears Roebuck & Co.) under an air pressure of 1.41 kg / cm in the form of fine droplets in a water tank sprayed, the 151 1 water and 4 drops of a common household detergent contained. When they come into contact with the surface of the water, the cellulose acetate droplets coagulate to form porous beads and sink to the bottom of the container. The porous pearls were

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then collected and washed. The washed beads were then washed overnight at room temperature with an approximately 0.15 η sodium hydroxide solution deacetylated. The deacetylated beads were then washed and suction dried. The so obtained porous cellulose beads had a pore volume of more than 50% by volume and were ready for enzyme immobilization. FIG. 1 shows the particle size distribution of the porous beads obtained. Electron microscope images showed that the beads were generally spherical in shape and that the inside and the surface of the balls had the same structure. The pore sizes were very uniform, and the pores were uniform over the entire beads, as in Figures 2, 4 (A) and 4 (B). The pore size of the pearls was determined with the help of electron microscopic raster images. The recording of electron microscopic raster images requires dry samples, and since air drying the beads causes shrinkage, the beads were dried using liquid carbon dioxide using the critical point technique. The pore size was determined to be about 10OO 8.

Example 2

Following the procedure of Example 1 were using a 10% (weight / volume) cellulose acetate solution in solvent A also made porous beads. Even these beads worked well for enzyme immobilization.

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- 3ο -

Example 3

A 10% (w / v) cellulose acetate solution (Vise 3 from Eastman Kodak Chemicals) in solvent B (acetone and formamide in a volume ratio of 7: 3). The cellulose acetate solution was then made according to the procedure sprayed according to Example 1 and hydrolyzed. The result was highly porous cellulose beads with a pore volume of more than 50 Vol.% Preserved.

Example 4

Example 2 was repeated with the difference that a solution with cellulose acetate of the Vise 45 type (available from Eastman Kodak Chemicals). The porous beads obtained were excellent in enzyme immobilization properties.

Example 5

The procedure of Example 2 was carried out using a 10% (weight / volume) solution of cellulose triacetate (available from Eastman Kodak Chemicals) in Solvent A repeated. The resulting beads had excellent porosity for enzyme immobilization.

As already mentioned several times, cellulose can be used as a carrier material for the immobilization of enzymes and other biologically active substances

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Agents are used. Cellulose has often been chosen as a carrier because it is cheap, chemically stable, and more microbiological Contamination is resistant. In addition, cellulose possesses three hydroxyl groups in each glucose anhydride unit giving the versatility of the cellulose and also the great capacity supply for the immobilization of a desired substance.

The main disadvantage of using cellulose as a carrier material is that cellulose is fibrous in shape and lacks the required mechanical strength. Reactors packed with cellulose have poor flow properties, have a high pressure drop and lead to channel formation. These problems are solved by the cellulose beads according to the invention, which have greater mechanical strength and improved flow properties than known materials. However, since the structure of the cellulose beads according to the invention is different from normal cellulose, and the loading of enzymes and the stability of the immobilized enzymes may be different than in normal cellulose. The chemical processes involved in the production of immobilized enzy- j

i men not only influence the loading and stability of the I.

i enzyme on the cellulose beads, but also their mechanical j strength. Any chemical method for immobilizing enzymes that increases the mechanical strength of cellulose beads also improves the flow properties in a reactor. This also follows from the following

Examples: j

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Example 6

1 g of porous cellulose beads produced according to Example 1 were dispersed in 15 ml of water, which with sodium hydroxide on a pH of 11.5 had been adjusted and kept at a constant Temperature of 20 C was maintained. 1 g of cyanogen bromide was added to this dispersion. The pH was adjusted to 11.5 with 1 η NaOH held. After 15 minutes the beads were filled with a phosphate buffer (0.1N) at pH 7.0 and 0 ° C. Then 15 ml of glucoamylase solution (30 mg / ml) was added to the beads. The mixture was left to stand overnight. The pearls produced in this way contained maltose at 60 ° C. and using 5% Substrate 1830 enzyme activity units per g dry weight of cellulose beads. One enzyme activity unit is defined as that amount which produces 1 micromole of product per minute.

Example 7

Porous cellulose beads (0.2 g) prepared according to Example 1 were dispersed in 5 ml of acetone. To this dispersion were 0.2 ml of triethylamine and 0.2 ml of tolylene 2,4-diisocyanate were added. After 30 minutes the beads were washed with acetone and then with an acetate buffer at pH 4.75. The pearls 5 ml of glucoamylase solution (25 mg / ml) were then added. The enzyme immobilized on the beads as a result gave an activity of 2000 units / g cellulose beads.

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Example 8

200 mg of glucose isomerase in maleic acid buffer solution were added 2 g of cellulose beads produced according to Example 7 immobilized. The cellulose beads contained at 60 ° C using from 9% fructose as substrate 90 enzyme activity units per g cellulose beads.

Example 9

300 mg of invertase in 5 ml of acetate buffer were immobilized on 0.5 g of porous cellulose beads prepared according to Example 7. The cellulose beads contained 3000 units of activity per g of cellulose used.

Example 10

50 mg of lactase in a phosphate buffer (pH = 7.0) were added
0.5 g of cellulose beads produced according to Example 6 immobilized. The resulting cellulose beads contained about 80 units of enzyme activity per gram of cellulose beads at 30 ° C. using 1% lactose as substrate.

Example 11

500 mg of glucose isomerase were added to 150 ml of maleic acid buffer
(0.01 m, pH = 5.5) dissolved. The enzyme solution was replaced by 5 g
porous crosslinked cellulose polymer produced according to Example 16

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len pumped. The DEAE cellulose beads then contained Enzyme activity units per gram of cellulose beads.

Example 12

0.25 g of porous cellulose beads produced according to Example 1 were placed in a 3% glutaraldehyde and 0.1 M MgCl solution soaked. After drying by suction in a BÜchner funnel, the samples were heated to 80 ° C. for 30 minutes. then 5 ml of glucoamylase solution (25 mg / ml) was added to the beads. After standing overnight, those so prepared contained Beads about 200 units of enzyme activity per gram of dry cellulose beads.

Example 13

1 g of porous cellulose beads were cyanoethylated at 50 ° C. with 10 ml of acrylonitrile (C = CCSN). The cellulose beads treated in this way were then at a pH of 6.5 to 6.7 at 50 to 100 ° C

Treated with hydroxylamine for 4 hours. The resulting

modified porous bead product

NH-
-C = NOH groups and is suitable for the absorption of heavy

Ions such as iron (II), iron (III) and copper (II) ions.

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Example 14

A suspension of 2.5 g of porous cellulose beads was treated with 2.5 ml of hexamethylene diisocyanate and triethylamine and then hydrolyzed in water. The product was then at a pH of 5 with 50 ml of 0.5M O-methylisourea treated. The product obtained has the following functional group:

porous cellulose beads

-N-C-NH

2 '

which is suitable as an anion exchanger.

Example 15

5 g of porous cellulose beads produced according to Example 1 were added to 100 ml of 36% formaldehyde and 200 ml of 37% hydrochloric acid. After standing for 11/2 hours at room temperature, the beads were filtered off and then washed with water and 0.2% sodium carbonate solution. The beads were then dried at 75 to 80.degree. The resulting crosslinked, porous cellulose beads exhibited great physical strength.

Example 16

3 g of porous cellulose beads were made according to Example 15 with formaldehyde networked. The beads were then washed with 3 g of 2-chlorotriethylamine treated. After heating the mixture to a

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Temperature of 80 to 85 ° C, the beads were successively with sodium chloride solution, sodium hydroxide solution, hydrochloric acid, water and washed with ethanol. The crosslinked porous DEAE cellulose beads thus obtained were excellent in porosity a pore volume of more than 50% by volume.

Example 17

0.5 g of porous cellulose beads were dispersed in 5 ml of 0.2 η sodium hydroxide and 5 ml of epichlorohydrin. The dispersion was then heated to a temperature of 80 ° C. for several minutes. The pearls were then washed. The crosslinked porous beads thus treated had greater strength than the porous beads before crosslinking. Wet cellulose beads produced according to Example 1 were washed in acetone. The washed beads were then suspended in dry acetone containing 0.6 ml of triethylamine per g of cellulose. 1.6 ml of tolylene-2,4-diisocyanate per g of cellulose beads were added to this suspension at ^{0 ° C.} After 30 minutes the beads were washed with dry acetone and then filtered off. The resulting porous cellulose beads contained reactive isocyanate groups which could then be hydrolyzed to amino groups by adding water.

Example 18

0.2 g of cellulose beads produced according to Example 1 were in

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Suspended 10 ml of distilled water, the pH was at 20 ° C. adjusted to 11.5 by adding 1 η NaOH. 0.2 g of CNBr was added to the cellulose bead suspension. this happened in small portions, and the pH was checked with the aid of an automatic titration device using 1 η NaOH kept constant. After 20 minutes, the beads were washed with ice cold distilled water and an appropriate buffer solution washed. Enzymes dissolved in a suitable buffer solution were added to the washed cellulose beads. The one with this one Method used cellulose (Solka floc) was 4 hours with 18% (weight / volume) NaOH mercerized and then washed with distilled water.

Example 19

2 g of suction-dried cellulose beads produced according to Example 1 were washed with acetone to remove moisture and suspended in 10 ml of acetone. Then 0.1 ml of triethylamine or dibutyltin diacetate were added as a catalyst. 0.1 ml of tolylene-2,4-diisocyanate or hexamethylene diisocyanate were then added to the cellulose bead suspension. After 45 minutes of reaction at room temperature, the cellulose beads were used to remove excess diisocyanate Washed acetone and then washed with water to remove the acetone. Dissolved in a suitable buffer solution Enzymes were added to the cellulose beads. The cellulose beads were stored at 4 ° C. overnight.

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Example 20

Aryl diisocyanate was applied to cellulose beads produced according to Example 19 coupled. Before the enzyme solution was added, the cellulose beads were suspended in distilled water. 0.1 ml of triethylamine was added to cause the reaction between isocyanate and water to form arylamine cellulose beads to catalyze. The arylamine derivative was then diacetated with NaNO- in HCl. Suspended in a suitable buffer solution Enzymes were then coupled to the cellulose beads.

Example 21

Diisocyanate coupled to cellulose according to Example 19 reacts with water with or without the addition of a tertiary amine Catalyst to amino groups. Glutaraldehyde is used to make the enzyme by crosslinking the amino groups of the enzyme and the To couple cellulose beads to the cellulose beads.

Example 22

2 g of suction-dried cellulose beads produced according to Example 1 were suspended in 10 ml of 3% glutaraldehyde. The concentration of MgCl _{2 in} the suspension was 0.1 M. The suspension was then heated to 100 ° C. for 30 minutes. Then the cellulose beads were washed with distilled water. The washed cellulose beads were treated with enzymes dissolved in a suitable buffer solution. The reaction mixture was left to stand at 4 ° C. overnight.

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Example 23

1 g of cellulose beads produced according to Example 1 were treated with 10 ml of 10% 3-aminopropyltriethoxysilane in toluene for 4 hours Refluxed. The cellulose beads were then filtered and washed with acetone. The cellulose beads were then made with a 2.5% (weight / volume) glutaraldehyde solution in 0.1 M phosphate buffer (pH = 7.0) and occasionally Stirred for 1 hour at room temperature. Then the cellulose beads were carefully washed with water and an appropriate one Washed buffer solution. Enzymes dissolved in a suitable buffer solution were then added to the cellulose beads. the The reaction mixture was left to stand at 4 ° C. overnight.

Example 24

Porous cellulose beads produced according to Example 1 were first crosslinked with 36% formaldehyde and 37% HCl in a volume ratio of 5: 1. The crosslinked cellulose beads (5 g dry weight) were suspended in 50 ml of cold 1.5 η NaOH solution. Then 6 g of 2-chlorotriethylamine hydrochloride was added and the mixture was heated to 80-85 ° C for 35 minutes. Then the mixture was cooled in an ice bath and filtered. The cellulose beads were then washed 3 times alternately with 500 ml of 2 M NaCl solution, 200 ml of 1 η NaOH and 200 ml of 1 η NaOH. After washing again with 200 ml of 1 η NaOH, the cellulose beads were washed with distilled water until the pH of the wash water was neutral. the

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Reaction with 2-chlorotriethylamine hydrochloride was repeated, to achieve a higher degree of substitution. Enzymes dissolved in a suitable buffer solution were added to the cellulose beads. The reaction mixture was left at 4 ° C overnight ditched.

Example 25

Hexamethylene diisocyanate was attached to cellulose beads as in Example 14 described, coupled, and then the isocyanate groups, as described in Example 20, to amino groups hydrolyzed. O-methyl isosulfite became the cellulose beads given to introduce the guanidine function into the derivatized beads.

The enzyme immobilization in Examples 18 to 23 takes place through covalent bonds, while in Examples 24 and 25 ionic adsorption takes place. The beads according to Example 23 to 2 5 were with glucoamylase, glucose isomerase and Loaded invertase, and the respective enzyme load was measured.

The enzymes immobilized by covalent bonds were treated with 2 M NaCl solution to remove the adsorbed enzymes washed. Some properties of the immobilized enzymes are shown in Table 2. It follows that the chemistry used for normal cellulose also for cellulose

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beads can be used. The fact that the cellulose beads have a higher enzyme loading capacity than normal cellulose indicate that the porous cellulose beads have a larger surface area.

Example 26

Proteins and enzymes can be prepared using the following procedure separated and cleaned. 2 g of glucose isomerase (Strep, albus from Miles Laboratory) were added to 20 ml of a 0.01 m Phosphate buffer solution (pH = 7.0) was suspended, and the suspension was centrifuged. The supernatant solution was in a with Porous DEAE cellulose beads produced according to Example 16 given packed column. The bed volume was 30 ml and the column diameter was 1.5 cm. The column was filled with 0.01 M phosphate buffer (pH = 7.0) washed. The column was then eluted with an NaCl gradient solution in 0.01 M phosphate buffer. the The concentration of glucose isomerase in the fractions emerging from the column was 0.25 to 0.45 m.

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Tabel Enzyme loading of porous cellulose beads

O CD OO

Enzymes Immobilization
procedure: Enzyme loading of cellulose,
IU / g (calculated from the An
catch reaction speed
speed) porous cell
loose beads Test conditions Glucoamylase
(A. Oryzae) example normal cellu
loose 800 1800
550 10% maltose, 60 ° C
Il 18th
19th 275 10% maltose, 40 ° C 20th 530 5% maltose, 60 ^° C 21 80 190 10% maltose, 60 ° C 22nd 200 Il Glucoamylase
(A. Niger) 23 3000 9000
1000 M.
II Glucose isomerase
(Strep, albus) 24
25th 90
300 0.5 M fructose, 60 ^° C
Il 19th
24 160 M. 25th 1140 0.125 M sucrose, 45 ° C Invertase
(Candida utilis) 19th Il
Il 24
25th 2000
1840

IU = international units

Example 27

The porous cellulose beads according to Example 13 were added to a 0.05 M sodium acetate solution (pH = 5.2), the 1600 ppm
Contained copper (II) ions. After 1 hour, the cellulose beads had taken up 6.3% of copper (II) ions, based on their weight.

It has also been found that when the porous cellulose beads of the present invention are dried and / or heated (e.g. to 100 ° C) prior to use, the resulting beads
have increased physical strength.

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Claims (19)

UEXk ^ LL Λ STOLBtRG PATENTANWÄLTE 2 HAMBURG 52 BESELERSTRASSE 4 DR. J.-D. FRHR. von UEXKÜLt "Λ · DR. ULRICH GRAF STOLBERG 2-7 1 7965 DIPL.-ING. JÜRGEN SUCHANTKE, PURDUE RESEARCH FOUNDATION (Prio: April 22, 1976 ,, i. τ cu ^ TJ- /, τ oi. Λ US 679 497 and West Lafayette, Indiana / V.St.A. March 21, 1977 - US - 13904) Hamburg, April 19, 1977 Porous cellulose pearl claims 1. Process for the production of porous cellulose beads, which can be used as a carrier for enzymes and other biological Active agents are suitable, characterized in that one (a) a hydrolyzable cellulose derivative in a 42 ^he "water-miscible solvent th in a ratio of 1:20 to 1: 3 w / v to a solution with higher density than dissolving the precipitation solution, 7 0 9 045/0887 2717865 (b) the solution thus prepared is distributed in the form of droplets in a precipitation solution and thereby the cellulose derivative precipitates in the form of uniformly porous beads, (c) separating the precipitated beads from the solution, (d) washing the separated porous beads with water, (e) the washed beads to convert to cellulose and to increase the active sites for the Coupling with enzymes and other biologically active agents hydrolyzes and (f) the hydrolyzed beads to give porous cellulose beads with evenly distributed pores and a pore volume of more than 50% by volume washes. 2. The method according to claim 1, characterized in that in step (b) the solution is distributed by spraying. 3. The method according to claim 1, characterized in that the precipitation solution is water, mixtures of water and ethanol or methanol, hexane, cyclohexane, octane and / or benzene are used. 4. The method according to claim 3, characterized in that there is used as the precipitation solution water. 709845/0887 5. The method according to claim 1, characterized in that there is used as the cellulose derivative and cellulose acetate carries out the hydrolysis in a caustic solution. 6. The method according to claim 1, characterized in that a mixture of: (a) acetone, a mixture of acetone and methanol or ethanol, methyl acetate, methylene dichloride and methanol, Methyl ethyl ketone, formamide and / or dimethyl sulfoxide and (b) dimethyl sulfoxide, formamide, methyl acetate, cyclohexanone, Methylene dichloride, ethylene dichloride, a mixture of methylene dichloride and methanol and / or a mixture of ethylene dichloride and methanol used. 7. The method according to claim 6, characterized in that the solvent is dimethyl sulfoxide, formamide or
Methyl acetate used. 8. The method according to claim 1, characterized in that beads with a pore volume of about 75 to 95% manufactures. 9. The method according to claim 1, characterized in that the porous cellulose beads for the production of crosslinked porous cellulose beads with at least one cross-linking 709845/0887 medium networked. 10. The method according to claim 9, characterized in that the beads are crosslinked before the hydrolysis. 11. The method according to claim 9, characterized in that the beads are crosslinked after hydrolyzing. 12. The method according to claim 9, characterized in that there is used as the crosslinking agent diisocyanate. 13. The method according to claim 12, characterized in that the diisocyanate is tolylene-2,4-diisocyanate or hexamethylene diisocyanate used. 14. The method according to claim 9, characterized in that the crosslinking agent used is epichlorohydrin in a sodium hydroxide solution used. 15. The method according to claim 9, characterized in that the crosslinking agent is formaldehyde in a hydrochloric acid solution used. 16. The method according to claim 9, characterized in that there is used as the crosslinking agent glutaraldehyde. 17. Porous cellulose beads, characterized in that they are produced by the method according to claim 1. 709845/0887 -S- 18. Porous crosslinked cellulose beads, characterized in that they are produced by the method according to claim 9 are. 19. A method for immobilizing enzymes, characterized in that that the enzymes are coupled to the porous cellulose beads according to claim 17. 20. A method for immobilizing enzymes, characterized in that that the enzymes are coupled to the porous crosslinked cellulose beads according to claim 18. 21. The method according to claim 19, characterized in that the enzymes used are glucoamylase, glucose isomerase, invertase or lactase is used. 22. A method for removing metal ions from dilute solutions, characterized in that the diluted Bringing solution with the porous cellulose beads according to claim 17 in contact, which contain groups for coupling these ions are capable of attaching to the pearls. 23. A method for the separation and purification of enzymes, proteins, nucleic acids and similar compounds, characterized in that that one brings enzymes, proteins or nucleic acids with the porous beads according to claim 17 in contact, the Have groups that are capable of coupling enzymes, proteins or nucleic acids. 709845/0887