DK158005B - PROCEDURE FOR THE PREPARATION OF POROUS CELLULOSE PEARLS AND THE USE OF THESE - Google Patents

Description

DK 158005B

The invention relates to a process for the preparation of porous cellulose beads suitable for use as carriers of enzymes and other biological agents and to the use of the porous cellulose beads made by the process.

5

Porous cellulose beads constitute a relatively inexpensive stable material having versatile chemical properties so that they can be useful as carriers for immobilized enzymes and other active biological agents.

Ordinary cellulose particles and regenerated cellulose powders satisfy most of the desired requirements for good carriers to which enzymes can be immobilized, but they suffer from formulas that cause column reactors to become tightly packed, resulting in flow reduction and some sometimes duct formation and therefore insufficient contact between the immobilized enzyme and the reaction liquid or gaseous. Immobilization of enzymes on an insoluble carrier is a widely used technique for the practical use of enzymes which avoids the necessity of using fresh enzymes for any desired use. Through the immobilisation of the enzyme, stabilization is achieved which provides efficient enzyme use and enables the construction and operation of enzyme reactors in a continuous manner.

A successful result of an immobilised enzyme for use in practical application depends to a large extent on the properties of the carriers used for the immobilization. A good carrier must therefore satisfy the requirements to be cheap and must be of such physical shape that it is easy to use in reactors. In this regard, the shape of a spherical bead is particularly desirable because it is useful in a packed bed, a fluidized bed, an expanded bed, a stirred tank or other common types of chemical reactors. Such a carrier must also have the necessary physical and mechanical strength so that it is not crushed or deformed when packed in a high column. Crushing and deformation results in the column being tightly packed, thereby blocking the flow of liquid reagents through the column and therefore reducing the efficiency of the chemical reactor.

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put. Suitable carriers must also have versatile chemical properties so that the immobilization of the enzymes and other biological agents on the carrier through ionic or chemical covalent bonding and surface adsorption can be readily achieved. In this regard, the carrier must have a high capacity to form a large number of bonds, so that each unit of the carrier can immobilize large amounts of the desired enzyme. Therefore, a carrier having a high degree of porosity and uniformly distributed internal cavities is particularly desirable. Such porosity provides good diffusion of chemical reagents and reaction products into and out of the internal cavities of the cellulose beads. Carriers must be chemically stable, physically strong and made of inert material that resists microbiological attack causing destruction of the carrier in order to obtain an immobilised 15 enzyme system with a long active life.

Currently, porous glass particles and porous ceramic particles are commonly used for the immobilisation of enzymes, and these particles satisfy most of the above requirements for an acceptable particle, but are relatively expensive. Furthermore, the number of chemical reactions that can be used to immobilize the enzymes for glass and ceramic carriers is limited.

In U.S. Patents Nos. 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 describe the preparation of various cellulosic materials in various forms, some of which are described as suitable for use in the fixation of biologically active materials, such as enzymes or ion exchange groups thereto. . However, these methods also appear to suffer from the disadvantage of being costly, and the resulting products generally have an undesirable physical form for use in such chemical reactors as packed beds and fluidized beds. In particular, the prior art cannot provide a means for producing spherical cellulose beads having a uniform distribution of pores through the surface and a large uniform porous internal cavity. The cellulose particles and 3

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Furthermore, the powders of the prior art are generally of such small particle size that they are not suitable for use in chemical reactors. In addition, the prior art cellulose powders and particles often have a hard surface skin which causes severe diffusion prevention and inefficient use in chemical reactors.

The object of the present invention is to provide inexpensive, highly porous, stable particles with versatile chemical properties that make them suitable as carriers of immobilized enzymes and other biologically active materials, and to provide porous cellulose beads with improved physical and mechanical stability and sufficient size. surface for a high immobilization capacity for enzymes.

15

The process of the invention is characterized in that a) dissolves a hydrolyzable cellulose derivative in an inert organic water-miscible solvent at a ratio of from 20 1:20 to 1: 3 w / v to form a solution having a density greater than than the density of the precipitate solution used in (b), (b) distributes this solution in the form of small droplets in a precipitate solution which is miscible with the solvent used in (a) and which consists of water, an aqueous solution of ionic or ionic surfactants, a mixture of water and ethanol or methanol or a hydrocarbon or hydrocarbon mixture, whereby the cellulose derivative is precipitated in the form of uniformly porous beads; c) separates the precipitated beads from the solution; e) hydrolyzes the washed beads to convert the beads to cellulose and to increase the active sites for the binding of enzymes and other biologics. no funds, 4

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f) washing the hydrolyzed beads to obtain porous cellulose beads having a uniformly distributed cavity greater than 50% by volume, and 5 h) optionally cross-linking the porous cellulose beads with at least one crosslinking agent to produce crosslinked porous cellulose beads before or after the hydrolysis.

The invention also relates to the use of the porous cellulose beads prepared by the process according to the invention, optionally after a pretreatment, for the immobilization of enzymes and other biologically active agents and for the separation and purification of enzymes, proteins and nucleic acids.

The highly porous cellulose beads of uniform porosity of the invention are particularly suitable for the immobilization of enzymes. In addition, they are also suitable for purification and separation of enzymes, proteins, nucleic acids and similar compounds and for the separation of metal ions from dilute solutions. Due to their porosity and their good mechanical stability, the cellulose beads made according to the invention, when used in fixed bed reactors, provide excellent fluid passage and do not undergo decomposition and deformation in any phenomena.

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The process of the invention provides porous cellulose beads suitable as carriers for enzymes and other biological agents. Furthermore, the process of the invention enables the chemical and physical properties of porous beads 30 made from cellulose derivatives to be changed. Conventional microcrystalline cellulose and other particles made of cellulose satisfy many of the general requirements for a suitable carrier for enzymes, but such particles suffer from a tendency to pack tightly under pressure and also cannot provide sufficient porosity to attach. a sufficient amount of enzymes thereto. Cellulose derivatives are generally inexpensive and when treated according to the present invention provide a very versatile chemical reaction material.

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which are generally biologically inert. Thus, the cellulose beads prepared according to the invention have many desirable properties for use as carriers of immobilized enzymes.

5

By dissolving a cellulose derivative according to the invention in a selected solvent and distributing it in a selected precipitation solution, it is possible to prepare cellulose beads with a high uniform porosity and improved chemical and physical properties. The beads made according to the invention are highly porous. The pores are generally uniformly distributed over the surface and through the interior of the bead. By appropriate choice of solvents and precipitation solutions, the pore size of the beads can be adjusted. It is particularly advantageous that in accordance with the method it is possible to control both the pore size and the pore distribution. Referring to FIG. 2, 4 · (A) and (B), it will be seen that the pore openings are uniformly distributed over the surface of the bead and were judged to be approx. I'IO-? m, which is an appropriate size for the movement of enzyme and reagent molecules in the pores.

The inert organic water-miscible solvent may be a single liquid or a combination of liquids.

It is important to use a correct combination of inorganic solvent and precipitate solution to obtain porous cellulose beads of the desired shape and porosity.

The inert organic water-miscible solvent may be a combination of liquids which together with the cellulose derivative provide a solution which, when mixed with the precipitate solution, results in a phase reversal whereby the cellulose derivative is coagulated in the form of a porous bead . Thus, the inert organic solvent contains a component (a) characterized as a liquid capable of dissolving the cellulose derivative, such as cellulose acetate 35, and being soluble in the precipitating solution.

Another component (b) of the solvent system is a liquid which is soluble in component (a) and also in the precipitate.

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the solution, which is present in the solvent in a sufficient quantity that the density of the final solvent solution (together with the cellulose derivative) is sufficiently much higher than the density of the precipitate solution, so that when distributing the solvent solution in the form of small droplets in the precipitate solution, the cellulose will coalesce and precipitate as a porous bead of desired size and porosity. The component (b) in the solvent is used to regulate the surface activity of the solvent solution so that the small droplets of the solvent solution will retain their shape upon contact with the precipitate solution. The component (b) also serves to regulate the pore size and porosity of the precipitated beads. In some cases, component (a) and component (b) may be the same. In other cases, it may be convenient to use one or more liquids to produce component (a) and / or component (b).

As used in the present specification, the term "precipitate solution" is defined as a liquid solution which is not a solvent for the cellulose derivative and is miscible with the aforementioned inert organic water-miscible solvent. By way of illustration, the precipitation solution may be water or an aqueous solution. Thus, the precipitation solution is miscible with both the solution component (a) and (b).

Thus, it will be understood that when dissolving the cellulose derivative in the organic solvent and then adding a drop of the resulting solvent solution to the precipitate solution, the cellulose derivative will coagulate and precipitate due to the phase reversal the cellulose derivative undergoes, thereby forming the desired porous cell. .

As will be apparent from the specification, various variations are possible in the process described above for preparing the desired porous cellulose beads. In addition to cellulose acetate 35, other cellulose derivatives can be used as starting material for preparing the porous beads, e.g. cellulose nitrate and methyl cellulose. The terms cellulose derivative and hydrolyzable cellulose derivative are to be understood in the present specification 7

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as including materials from which cellulose can be regenerated, e.g. by hydrolysis or hydrogenation.

The organic solvent components (a) and (b) of the cellulose derivative may vary but must be chemically inert to the cellulose derivative and fully or substantially miscible with the precipitate solution. It is important that the density of the solvent solution formed by adding the cellulose derivative to the inert solvent is 10v greater than the density of the precipitating solution in which it is distributed so that when small droplets of the solvent solution are distributed in the precipitating solution, the droplets of solution will sink when do not stir. Suitable single solvents when an aqueous precipitate solution is used include eg. dimethyl sulfoxide and methyl acetate. It is to be understood that industrially available materials can be used as the solvent components (a) and / or (b) and that these materials may contain moisture, which in some cases has proved advantageous.

When an aqueous precipitate solution is used, it is convenient to use as a solvent component (a) a representative of the group consisting of acetone, formamide, a mixture of acetone and methanol or ethanol, methyl acetate, a mixture of methylene dichloride and methanol, methyl ethyl ketone and dimethyl sulfoxide. Thus, the solvent component (b) may conveniently be selected from representatives of the group consisting of dimethyl sulfoxide, formamide, methyl acetate, cyclohexanone, methylene dichloride, ethylene dichloride, a mixture of methylene dichloride and methanol, and a mixture of ethylene dichloride and methanol.

A preferred solvent component (a) is acetone, but other solvents may be used, and when an aqueous precipitate solution is used, a component (a) may be selected from the following materials (the mixing ratio in the case of mixtures, the minimum desirable ratio of volume basis): DK 158005 B_ 8 /

Component (a) Minimum ratio (volume)

Acetone -----

Acetone + methanol 60; 40 5 Acetone + ethanol 60:40

Methyl acetate -----

Methylene dichloride + methanol 80:20

Dimethylsulfoxide -----

Methylethyl ketone ----- Formamide -----

As mentioned above, the primary function of component (a) is to dissolve the cellulose derivative. The addition of component (b) is necessary to create a solvent solution having the necessary density so that the cellulose derivative will precipitate into the precipitate solution. Component (b) also provides control of pore size and uniform porosity of the beads.

The solvent component (b) therefore provides the desired density of the solvent solution and when using an aqueous precipitate solution it is preferred to use dimethyl sulfoxide as component (b). It will be understood that in some cases component '(a) and component (b) may be the same, dsv. dimethyl sulfoxide, formamide or methyl acetate when used with aqueous precipitate solutions. Various materials that can be used as component (b) when using an aqueous precipitate solution are listed below: 30 35 9

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(· * Λ: 4

Component (b) Minimum ratio (volume)

Dimethylsulfoxide ____ 5 Ethylene dichloride + methanol 60:40

Methylene dichloride + methanol 60:40

Ethylene dichloride _____

Methylene dichloride _____

Formamide _____ 10 Cyclohexanone _____

The solution of cellulose derivative and inert solvent must have a regulated ratio of cellulose derivative to solvent because this will have an effect on the final porosity of the beads produced. Generally, a small ratio (greater solvent content) results in beads having greater porosity. A ratio of cellulose to solvent (including components (a) and (b)) from 1:20 to 1: 3 (w / v) has been found suitable for the preparation of cellulose soaps 1 having various special applications. Preferably, a cellulose derivative to solvent ratio of 1:10 to 1: 6 (w / v) is used to provide a readily manageable solution resulting in porous cellulose beads having desired properties having a void 25 of at least 50% by volume. preferably 75 to 95% and most conveniently about 75 to 80%. Beads having greater porosity will generally have a greater amount of uniformly distributed inner voids, providing less diffusion impediment, but will be somewhat weaker in physical strength than beads 30 having lower porosity.

The preferred precipitation solution in which the cellulose derivative solution is to be distributed generally consists of water, but may be an aqueous solution containing appropriate amounts of nonionic or ionic surfactants to reduce its surface tension and facilitate the formation of the porous beads. Conveniently, the precipitate solution may also contain a mixture of water and methanol or ethanol (

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capture ratio 50:50). The precipitate solution may be non-usual, provided that the cellulose derivative is insoluble therein and the required density requirement is satisfied. Hydrocarbon solutions can thus be used, such as cyclohexane, hexane, decane, benzene, provided they are in liquid form, have a density less than the density of the inert organic solvent and are miscible therewith. When the cellulose derivative solution is dispensed by spraying via a suitable means such as a nozzle, the pressure drop and miscibility of the inert solvent in the aqueous solution results in a dispersion and eventually the precipitation of porous beads of the cellulose derivative.

As will be appreciated by those skilled in the art, when precipitating the cellulose beads, a sufficient amount of solvent component (b) must be present for the solution containing the cellulose derivative to have the necessary higher density than the density of the precipitate solution. Table 1 lists some inert organic solvents for precipitation of a cellulose derivative in an aqueous solution. The stated proportions are the minimum required to give a solvent solution having a higher density than water. The greater the density of component (b), the less of this component is required to obtain the minimum density.

Table 1 -

Solvent Minimum volume;

Component (a) _Component (b) · _ ratio a: b_i

Acetone Dimethylsulfoxide 70:30

Acetone Ethylene Dichloride 80:20

Acetone Methylene Dichloride 80:20 30 Acetone Formamide 75:25

Acetone Cyclohexanone 45:55

Acetone Methyl Acetate 35:65 35

After precipitation of the porous beads, cellulose is regenerated by the derivative by hydrolysis to create more active sites for enzyme binding. In the regeneration of cellulose by its derivative 11

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after formation of the beads, the substituent groups (such as acetate from cellulose acetate) may be removed to regenerate all the hydroxyl groups normally found in the cellulose material. The higher the degree of regeneration, the more stability can be found in the resulting beads. In some cases where the enzymes are to be immobilized on the cellulose bead carriers, it is desirable to convert the hydroxy groups or substituent groups to functional chemical groups such as amino groups which facilitate enzyme binding.

10 In the drawing, FIG. 1 shows a partial distribution of the cell size distribution in the porous beads; FIG. 2 is a scanning electromicrograph of a porous cellulose bead; FIG. 3 is a graph of the pressure drop properties of the porous cellulose beads; FIG. Figure 4 (A) is a scanning electron micrograph of the surface of a porous cellulose bead (20,000 x); 4 (B) is a scanning electron micrograph of the interior of a porous cellulose bead (20,000 x).

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FIG. 1 illustrates the size distribution of some of the final porous beads prepared by distributing (e.g., by spraying) a solution of cellulose derivative through a spray nozzle in accordance with the process described in detail below. Beads that are either too large or too small, depending on the intended final use, can be collected and redissolved in the desired solvent. Generally, beads of uniform size are preferred if used in a column-type chemical reactor. The desired particle size may vary with the intended use of the beads, e.g. the type of enzyme to be immobilized.

The porous cellulose beads prepared by the process described above generally have very high porosity and a

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regulated pore size ranging from 0.05 to 30 microns.

When a cellulose-solvent ratio of 1:10 (w / v) is used to prepare the cellulose-solvent solution, the resulting finished beads 5 can have a high porosity of approx. 90% cavity. A scanning electron micrograph of a porous cellulose bead made by the method is shown in FIG. 2, 4 (A) and 4 (B). From these pictures one can see several important peculiarities of the beads made. First, they will be seen that the beads are generally spherical and porous openings are uniformly distributed over the surface of the beads. For most applications, this is desirable because it can provide an immobilized enzyme catalyst with uniform activity. The void phase in the cellulose period is continuous. This is a desirable feature because discontinuous separated bubbles would result in a useless and unavailable dead space in an immobilized enzyme system. Third, there is no hard skin on pearl surface 1 aden. A hard skin would cause severe diffusion obstruction. Finally, the pore sizes are completely uniform. As a result, as much as 20 of the inner surface area of the inner cavities of the beads will be available for enzyme immobilization and for enzyme catalyzed reactions. Both the high porosity and the other features mentioned make the porous cellulose beads of the invention exceptionally suitable for use in immobilizing enzymes and other biologically active agents.

An important property of an enzyme carrier is the pressure drop that it causes at different fluid flow rates through an enzyme reactor containing the carrier. Eg. DEAE-30 cellulose is currently used in the industry as an enzyme carrier to convert glucose into fructose. For DEAE cellulose, the pressure drop is very large and consequently only low bearings can be used to obtain a reasonable rate of fluid flow. The pressure drop properties of the porous cellulose beads of the present invention when operating in a packed column are shown by curve A of FIG. 3. The nominal linear flow rate is calculated by dividing the volumetric flow rate of the liquid fed to the column by the cross-sectional area of the column.

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In practical operation, the nominal linear flow rate in industrial column reactors will be less than 0.5 cm / sec. With a reactor column with an internal diameter of 60.96 cm, a linear velocity of 0.5 cm / sec. eg. equivalent to 5 volumetric flow rate of 5254 liters / hour. In a typical industrial operation for the production of glucose fructose, the sugar concentration in the liquid supplied is approx. 0.59 kg / liter. The above flow rate will yield more than 27,000,000 kg of product per 61 cm column per unit. year. Due to the residence time requirements of the enzymatic reaction, the linear flow rate is usually less than 0.5 cm / sec. Therefore, it can be seen that the porous cellulose beads prepared according to the present invention present no serious pressure drop problems when used in column-type chemical reactors as carriers to which enzymes and other biologically active agents can be immobilized. . When the porous cellulose beads, after appropriate derivative formation, are used for other purposes (e.g., removing tanning from fruit juice, wine or beer, and metal ions from dilute solutions), the fluid flow rate through the reactor column may be much greater than the 0.5 cm mentioned herein. /SEC.

The flow properties and other physical and mechanical properties of the porous cellulose beads can be enhanced by cross-linking 25 with bi- and / or multifunctional compounds. The curve B in FIG. 3 shows the pressure drop requirement for porous cellulose beads after treatment with tolylene-2,4-diisocyanate and enzyme immobilization.

Above a nominal linear velocity of 2 cm / sec. For example, the untreated cellulose beads (curve A) become significantly compressed and deformed, resulting in a drastic increase in pressure drop. Curve B is only slightly concave, showing no or slight deformation of the treated beads.

Treatment of the porous cellulose beads with a crosslinking agent either before or after hydrolysis of the beads results in an increase in their physical strength. Binding of enzymes to the beads will also increase their physical strength. After treatment with, e.g. a diisocyanate (e.g., tolylene-2,4-diisocyan 14)

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(or sodium hexamethylene diisocyanate), the beads actually become quite stiff and strong. Cross-linking with epichlorohydrin also improves the physical properties of the porous cellulose beads. The chemistry of cross-linking polysaccharides, including cellulose and starch, is a well-developed branch of physics. Other suitable crosslinking agents include, e.g. formaldehyde in hydrochloric acid solution or giutaraldehyde. Many other carbohydrate cross-linking agents are well known, e.g. disclosed in U.S. Patent No. 3,905,954.

10

Generally, the porous beads are prepared by the process of the invention by the following steps: a) A hydrolysable form of cellulose is dissolved in an inert organic water-miscible solvent in a controlled ratio of cellulose derivative to solvent which is generally in the range of 1:20 to 1: 3 (weight: volume) to prepare a solvent solution. The solvent must be completely or substantially miscible with the precipitate solution, and the density of the solvent should not be sufficient for the solvent to become easily miscible with the precipitate solution and the cellulose derivative to precipitate therein.

B) The solvent solution is distributed (e.g. by spraying) in the form of small droplets in a precipitate solution. Upon contact with the precipitate solution which may contain a surface active agent, the solvent is dispersed within the solvent medium and porous beads of the cellulose material are formed as they coagulate and precipitate to the bottom of the container containing the precipitate solution. The cellulose derivative solution may conveniently be sprayed under pressure through a nebulizer nozzle into a precipitate solution bath. If desired, the bath can be stirred to promote the formation of the beads.

c) After being washed, the precipitated beads are hydrolyzed to regenerate cellulose, thereby providing a polymer.

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rose cellulose bead having active sites for enzyme binding.

If it is desired to enhance the stability of the porous beads or provide proper response in onsite locations, the beads can be chemically modified in various ways. Eg. For example, the beads 5 can be crosslinked to provide greater stability and increased physical strength. Chemically, either positively charged or negatively charged groups can be chemically substituted to alter the surface absorption properties of the cellulose bead. The cellulose itself is generally hydrophilic, and by changing its reaction sites one can therefore change its hydrophilic properties.

The invention further relates to a method by which enzymes and other biologically active agents can be immobilized by binding to the porous cellulose beads. Eg. For example, 15 porous cellulose beads, as described above, can be converted to t-methylaminoethyl cellulose (DEAE) by reacting the beads with N, N-diethyl-2-chloroethylamine hydrochloride in the usual manner. Beads obtained in this way contain DEAE cellulose and; has been successfully used in T-at bNtrdf- ^ jjkoseisomerase, derived from a streptomyces culture. Also, a method involving cyanogen bromide has been used. to ^ Mmmob'f 1 in sere g 1 the ukose isomerase.

Another method of enzyme immobilization on the porous cellulose beads involves the use of tolylene-2,4-diisocyanate. The diisocyanate was used to crosslink cellulose to improve the physical strength of the porous beads, however, it has been found that the porous cellulose beads of the invention, when treated with diisocyanate, can immobilize enzymes on the surface thereof by mix the diisocyanate-treated beads together with an enzyme solution. For example, when was used giukoamylase, the diisocyanate beads bound more than 1000 international units of the enzyme per g dry beads. The invention is not limited to the following teas, but it appears that when dry porous cellulose beads are in dry acetone with two 1 µl of 2,4-di isocyanate in the presence of a catalyst (e.g. triethylamine), there is a considerable degree of cross-linking between cellulose molecules, as shown at 16

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the enhanced physical strength of the beads. After a sufficiently long reaction time, the beads were washed with dry acetone to remove free diisocyanate residues. The cel 1u1 oseps 1s appear to have a large number of bound isocyanate groups. By mixing 5 of the treated beads with an aqueous enzyme solution, enzyme molecules appear to be covalently bound to the cellulose beads through the isocyanate groups. It has also been found that washing the treated beads with water results in the conversion of isocyanate groups to amino groups. In this way, an enzyme, glucoamylase, is immobilized to the amino cellulose beads with glutaraldehyde, an agent well known for its ability to react and crosslink amino groups (on the beads and enzyme).

The porous compounds prepared according to the invention can also be used for the separation and purification of enzymes, proteins, nucleic acids and the like. The porous cell 1 beads prepared by the method of the invention can be derivatized in the form of DEAE porous cell 1, as well as excellent flow properties and excellent flow properties. capable of separating enzymes, proteins, nucleic acids, and the like, as effectively as existing "industrial products in accordance with the technique known as column 1 ecr oma-tography.

25

The porous cellulose beads of the invention can also be derivatized (in situ) with groups other than DEAE. Thus, the porous cellulose beads of the invention are useful for a variety of applications. Eg. For example, one may bind a specific functional group to the porous cellulose soaps 1, and the subsequently derivatized beads may be used e.g. for removing tanning substance from fruit juice by passing the fruit juice through a bed of the derivatized porous cellulose beads with protein n.

Similarly, metal ions can be removed from dilute solutions containing these. Such a method could enable the extraction of valuable metal ions (ie, copper ions 17)

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and gold ions) of dilute solutions occurring in the mining industry and could find particular applicability to existing mining techniques with solutions whereby metals are extracted from ores with acidic solutions.

5

The invention is further illustrated by the following examples, of which Examples I-V illustrate the preparation of porous cellulose beads, Examples VI-XII and XVIII-XXVII illustrate the use of porous cellulose beads, and Examples XIII-XVIII illustrate the cross-linking of porous cellulose beads.

Example I

50 g of cellulose acetate (Vise 3 from Eastman Kodak Chemicals) was dissolved in 400 ml of solvent A (composed of acetone and dimethyl sulfoxide in a volume ratio of 6 to 4) to form a 12.5% (w / v) solution. With a spray gun (paint sprayer from Sears Roebuck & Co.) t>! © v cellulose solution at no time, sprayed at an air pressure of U ,, 4 fcg / cai * as fine small 20 drops ii / nd ii © m ' vawdtan'k containing 152 liters of water and 4 drops of one ordinary household cleaner. Upon contact with the surface of the water, the cellulose acetate drops coagulate into porous beads and sink to the bottom. The porous beads were collected and washed. was then deacetylated with about a 0.15 N solution of sodium hydroxide overnight at room temperature.The deacetylated beads were then washed and suction dried to give a porous cellulose bead with a cavity larger than 50 volume percent ready for use in enzyme immobilization. Electron micrographs revealed that the beads were generally spherical and the interior and surface thereof had the same structure. The pore sizes were completely uniform and pores ne were distributed uniformly throughout the bead as shown in FIG. 2, 4 (A) and 4 (B). The pore size of the beads was determined by scanning electron micrographs. Scanning micrographing requires dry samples, and as drying the beads in air results in a reduction in size, the beads were dried by the critical point technique of liquid carbon dioxide. The pore size was determined to be approx. 1 * 10- ^ m.

Example II

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ιβ

Using a 10% (w / v) cellulose acetate solution in solvent A by the procedure of Example I, 5 porous beads suitable for use in enzyme immobilization were also formed.

Example III

A 10% (w / v) cellulose acetate solution (Vise 3 from Eastman Kodak Chemicals) was prepared in solvent B (acetone and formamide in a volume ratio of 7 to 3). The cellulose acetate solution was then sprayed and hydrolyzed by the procedure of Example I. Highly porous cellulose beads with a cavity greater than 50% by volume were obtained.

Example IV

The procedure described in Example II was repeated 20 using a solution prepared with cellulose acetate of type 45 (from Eastman Kodak Chemicals) - Porous beads with excellent properties for enzyme immobilization were also obtained.

Example V

The procedure described in Example II was carried out using a 10% w / v solution of cellulose etria etate (from Eastman Kodak Chemicals) in solvent 30 a. The resulting beads showed excellent porosity for enzyme immobilization. Cellulose can be used as a support material for immobilization of enzymes and other biologically active agents. Many have chosen cellulose as support because cellulose is cheap, chemically stable and resistant to microbiological contamination. Cellulose also has three hydroxyl groups on each anhydro glucose unit, providing great versatility as well as great capacity to immobilize a desired substance.

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The main disadvantage of using cellulose as a backing material is that cellulose has a fibrous form and lacks the necessary mechanical strength. Reactors packed with cellulose have poor flow properties, develop a severe high pressure drop and sometimes duct formation. In order to overcome these problems, according to the invention, cellulose was prepared in pearl form which exhibited better mechanical strength and gave improved flow properties compared to known materials. However, since the structure of the cellulose beads of the invention is different from the structure of regular cellulose, the loading of enzymes and the stability of the immobilized enzymes may be different from that obtained with regular cellulose. The chemistry in the preparation of immobilized enzymes not only affects the loading and stability of the enzyme on the cellulose beads, but also affects the mechanical strength of the cellulose beads. Any mechanical method for immobilizing enzymes which increases the mechanical strength of cellulose beads would improve the flow characteristics of a reactor as will be apparent from the examples.

20

Example VI

1 g of porous cellulose beads prepared according to Example I was dispersed in 15 ml of water which was adjusted to pH 11.5 with 25 sodium hydroxide and kept at a constant temperature of 20 ° C. One gram of cyanogen bromide was added to this dispersion. The pH was maintained at 11.5 with 1N NaOH. After 15 minutes, the beads were washed with a phosphate buffer (0.1 M) at pH and 0 ° C. 15 ml of glucoamylase solution (30 mg / ml) was then added to the beads.

The mixture was left to stand overnight. The beads thus prepared contain 1830 units of enzyme activity per ml. g dry weight of the cellulose bead at 60 ° C using 5% maltose as a substrate. One unit of enzyme activity is defined as that which produces one micromole of product per minute.

35 20

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Example VII

0.2 g of porous cellulose beads obtained as in Example I were dispersed in 5 ml of acetone. 0.2 ml of triethyl amine was added to the dispersion, and the same was 0.2 ml of tolylene-2,4-diisocyanate. After 30 minutes, the beads were washed with acetone and then an acetate buffer at pH 4.75. 5 ml of glucoamylase solution (25 mg / ml) was added. The enzyme was thereby immobilized on the beads with an activity of 2000 units per ml. g cello 1 ulos beads.

Example VIII

200 mg glucose isomerase in a maleic acid buffer solution was immobilized on 2 g of cellulose beads in the same manner as described in Example 7. The cellulose beads contained 90 units of enzyme activity per ml. g cellulose beads at 60 ° C using 9% fructose as substrate.

Example IX

300 mg of invertase in 5 ml of acetate buffer was immobilized on 0.5 g of porous cellulose beads using the procedure described in Example VII. The cellulose beads contained 25,000 units of activity per g of cellulose.

Example X

50 mg of lactase in phosphate buffer (pH 7.0) was immobilized in sera 30 on 0.5 g of cellulose beads using the procedure described in Example VI. The resulting cellulose beads contained approx. 80 units of enzyme activity per g cellulose beads at 3 ° C using 1¾ lactose as substrate.

35

Example XI

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500 mg of glucose glucose isomerase was dissolved in 150 ml maleic acid buffer (0.0 M, pH = 5.5). The enzyme solution was pumped through 5 g of porous cross-linked cellulose beads prepared as described in Example XIV. Thus, the DEAE cellulose beads contained 100 units of enzyme activity per ml. g beads.

Example XII

10 0.25 g of porous cellulose beads prepared as in Example I were soaked in 3% giutaraldehyde and 0.1 M MgCl 2 · After drying using vacuum suction on a Buchner funnel, the samples were heated to 80 ° C for 30 minutes. 5 ml of glucoamylase 15 (25 mg / ml) was added to the beads. After standing overnight, the beads thus prepared contained approx. 200 units of enzyme activity per g of dry cellulose beads.

Example XIII

20 1 g of porous cellulose beads were cyanethylated with 10 ml of acrylonitrile (C = CCsN) at 50 ° C. The cellulose beads thus treated were then treated with hydroxylamine at a pH of 6.5-6.7 at 50-100 ° C for 4 hours. The resulting modifier 25 NH 2 the porous beads contained - C = Ν0Η groups and is suitable for absorbing heavy ions such as ferric, ferrous and copper.

Example XIV

A suspension of 2.5 g porous cellulose beads was treated with 2.5 ml hexamethylene than isocyanate and triethylamine, followed by hydrolysis in water. The product was then treated with 50 ml of 0.5 35 M O-methyl isourine at pH 5. The resulting product had the following functional groups: 22

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porous m © cellulose ΪΗ2 beads ---- - C - 5 useful as anion exchanger.

Example XV

10 g of porous cellulose beads prepared according to Example I were added to 100 ml of 36% formaldehyde and 200 ml of 37% hydrochloric acid. After standing for 1 hour at room temperature, the beads were filtered and then washed with water and 0.2% sodium carbonate solution. The beads were then dried at 75 to 80 ° C. The resulting cross-linked cellulose beads showed great physical strength.

Example XVI

3 g of porous cellulose beads were crosslinked with formaldehyde by the procedure of Example XV. The beads were then treated with 3 g of 2-chlorotriethylamine. After heating the mixture for 35 minutes to a temperature of 80 to 85 ° C, the beads were washed sequentially with sodium chloride, sodium hydroxide, hydrochloric acid, water and ethanol. The resulting cross-linked porous DEAE cellulose beads showed excellent porosity and had a void greater than 50% by volume.

Example XVII

A dispersion of 0.5 g of porous cellulose beads was formed in 5 ml of 0.2 N sodium hydroxide and 5 ml of epichlorohydrin. The dispersion was then heated for 5 minutes to a temperature of 80 ° C. Then, the beads were washed and the cross-linked porous beads exhibited greater strength than the porous cellulose beads prior to the cross-linking. Wet cellulose beads obtained by the procedure of Example I were washed in acetone.

The washed beads were then suspended in dry acetone containing 0.6 ml of triethylamine for each gram of cellulose. Tolylene 23

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2,4-diisocyanate in an amount of 1.6 ml. g of cellulose beads were added to the suspension at 0 ° C. After a period of 30 minutes, the beads were washed with dry acetone and then filtered. The resulting porous cellulose beads contained reactive 5 isocyanate groups which could then be hydrolyzed to an amino group by the addition of water.

Example XVIII

10 0.2 g of the cellulose beads prepared in Example I were suspended in 10 ml of distilled water, the pH was adjusted to 11.5 by the addition of 1 N NaOH at 20 ° C. 0.2 g CNBr was added to the suspension of cellulose beads, slightly at a time, and the pH was maintained by an autothe itrameter with 1 H 15 NaOH. After 20 minutes, the beads were washed with ice-cold distilled water and a suitable buffer solution. Enzymes dissolved in a suitable buffer solution were added to the washed cellulose beads. Cellulose (Solka floc) used in this procedure was mercerized with 18% (w / v) NaOH for four 20 hours and then washed with distilled water.

Example XIX

2 g of suction-dried cellulose beads of Example I were washed with 25 acetone to remove moisture and suspended in 10 ml of acetone. One tenth ml of triethylamine or dibutyltin diacetate was added as a catalyst. One-tenth ml of tolylene-2,4-d in isocyanate or hexamethyl than in isocyanate was added to the cellulose suspension. After 45 minutes of reaction at ambient temperature, the cellulose beads were washed with acetone to remove excess diisocyanate, and water was then used to wash the cellulose beads to remove acetone. Enzymes dissolved in a suitable buffer solution were added to the cellulose beads. The cellulose beads were stored at 4 ° C overnight.

Example XX

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Aryl diisocyanate was bound to cellulose beads as described in Example XIX. Before adding the enzyme solution, the cellulose beads were suspended in distilled water. One-tenth ml of triethylamine was added to catalyze the isocyanate-water reaction to form arylamine cellulose beads. The arylamine derivative was then diazotized with NaNO 2 in HCl. Enzymes suspended in a suitable buffer solution were then bound to the 10 cellulose beads.

Example XXI

Diisocyanate bound to the cellulose by the procedure of Example 15 XIX reacts with water to form amino groups with or without a tertiary amine as catalyst. Gutaraldehyde is used to link the enzyme to the cellulose beads by crosslinking amino groups on enzymes and on cellulose beads.

Example XXII

2 g of suction dried beads prepared in Example I were suspended in 10 ml of 3% glutaraldehyde which was 0.1 M in Mg Cl 2 · The suspension was heated to 100 ° C for 30 minutes. The cellulose beads were then washed with distilled water. Enzymes dissolved in a suitable buffer solution were added to the cellulose beads. The reaction was allowed to continue overnight at 4 @ C.

Example XXIII 30 g of cellulose beads prepared by the method of Example I were heated under reflux with 10 ml of 10% 3-amino-propyl triethoxysilane in toluene for 4 hours. The cellulose samples were then filtered and washed with acetone. 2.5% (w / v) of 35 glutaraldehyde solution in 0.1 M phosphate buffer (pH = 7.0) was added to the cellulose beads at ambient temperature for one hour with occasional stirring. The cellulose beads were then thoroughly washed with water and a suitable buffer solution.

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Enzymes dissolved in a suitable buffer solution were added to the cellulose beads. The reaction was allowed to continue overnight at 4 ° C.

Example XXIV

Porous cellulose beads prepared by the procedure of Example I were first cross-linked with 36% formaldehyde and 37% HCl with a volume ratio of 5 to 1. The cross-linked cellulose beads (5 g 10 dry weight) were suspended in 50 ml of cold 1.5 N NaOH solution.

6 g of 2-chlorotriethylamine hydrochloride were added to the cellulose beads. The mixture was then heated to 80-85 ° C for 35 minutes. The mixture was cooled in an ice bath and filtered. The cellulose beads were ashed with 500 ml of 2M NaCl and then washed with 200 ml of 1N NaOH and 200 ml of 1N NaOH alternately three times. After washing with an additional 200 ml of 1N NaOH, the cellulose beads were washed with distilled water until the pH of the wash water became neutral. The reaction with 2-chlorotriethylamine hydrochloride was repeated again to obtain a higher degree of substitution. Enzymes dissolved in a suitable buffer were added to the cellulose beads overnight at 4 ° C.

Example XXV

Hexamethylenedi isocyanate was bound to cellulose beads as described in Example 14, and then the isocyanate groups were hydrolyzed to form amino groups as described in Example XX. O-methyl in acid in substance was added to the cellulose beads to incorporate the guanidino function into the derivatizing beads.

Examples 18-23 describe immobilization of enzymes with covalent bond, whereas Examples 24 and 25 describe ionic adsorption. Glucoamylase, glucose isomerase and invertase were loaded onto various of the beads of Examples XVIII to XXV and the extent of enzyme loading was measured.

The enzymes immobilized in the sera by covalent binding were washed with 2M NaCl solution to remove the absorbed enzymes. Some 26

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properties of the immobilized enzymes are shown in Table 2. It shows that the same chemistry used for cellular cellulose can also be used for cellulose beads. The fact that the cellulose beads have higher capacity for loading with 5 enzyme than regular cellulose may indicate a larger surface area in the porous cellulose beads.

Example XXVI

Protein and enzymes can be separated and purified by the following procedure. 2 g of glucose isomerase (Strep, Albus from Miles Laboratory) was suspended in 20 ml of 0.01M phosphate buffer (pH = 7.0) and the suspension was centrifuged. The supernatant was added to a column of DEAE porous cellulose beads prepared according to Example XVI. The bearing volume was 30 ml and the column diameter 1.5 cm. The column was washed with 0.01 M phosphate buffer (pH = 7.0). The column was eluted with NaCl gradient solution in 0.01 M phosphate buffer. 61ucose isomerase begins to elute out of the column of the NaCl fractions at concentrations ranging from 0.25 to 0.45 M.

25 30 35 27

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Table 2. Enzyme loading on porous cellulose beads.

«R | SoLa: tnln8 on I

5 immobile! - IUX // g (designated)

Enzymes reaction rate) Sample conditions

Ex. Regular Porous cellulose-free beads

XVIII 820 1.800 10% maltose, 60 ° C

10 XIX 550 »

Glycoamylase XX 275 10% maltose, 40 ° C

(A. Oryzae) XXI 550 5% maltose, 60 ° C

XXII 80 190 10% maltose, 60 ° C

XXIII 200 "15 Glycoamylase XXIV 3,000 9,000" (A. Niger) XXV 1,000 "

Glucose XIX 90 0.5M fructose, 60 ° C

Isomerase XXIV 300 "20 (Strep. XXV 160" albus)

Invertase XIX 1.140 0.125M sucrose, 45 ° C

(Candida XXIV 2,000 "utilis) XXV 1,840" 25 * I-U - International Units.

Example XXVII

The porous cellulose beads of Example XIII are added to a 0.05 M sodium acetate solution (pH = 5.2) containing 1,600 parts of coprion per million parts. After one hour, the cellulose beads collected 6.3% of cuprion based on the weight of the beads.

They have also been found that when the porous cellulose beads of the invention are dried and / or heated e.g. to 100 ° C before use, the resulting beads exhibit increased physical strength.

Claims (11)

A process for the preparation of porous cellulose beads suitable for use as carriers of enzymes and other biological agents, characterized in that a) dissolves a hydrolyzable cellulose derivative in an inert organic water-miscible solvent in a ratio of 10 1:20 to 1: 3 w / v to form a solution having a density greater than the density of the precipitation solution used in (b), b) distributing this solution in the form of small droplets in a precipitation solution which is miscible with the solvent used in (a), consisting of water, an aqueous solution of nonionic or ionic surfactants, a mixture of water and ethanol or methanol or a hydrocarbon or hydrocarbon mixture, thereby precipitating the cellulose derivative in the form of 20 kinds of porous beads, c) separating the precipitated beads from the solution, d) washing the separated porous beads with water, 25 e) hydrolyzing the washed beads for to transform the beads into cellulose and to increase the active sites for binding enzymes and other biological agents, f) wash the hydrolyzed beads to obtain porous cellulose beads having a uniformly distributed cavity greater than 50% by volume, and h) optionally, crosslinking, either before or after the hydrolysis, the 35 porous cellulose beads with at least one crosslinking agent to produce crosslinked porous cellulose beads. Process according to claim 1, characterized in that the solution is distributed in step (b) by spraying. DK 158005 B Process according to claim 1 or 2, characterized in that cellulose acetate is used as a cellulose derivative and the hydrolysis is carried out in a caustic solution. Process according to claims 1-3, 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 is used as the solvent. and (b) dimethylsulfoxide, formamide, methyl acetate, cyclohexanone, methylene dichloride, ethylene dichloride, a mixture of methylene dichloride and methanol and / or a mixture of ethylene dichloride and methanol. Process according to claim 1, characterized in that beads having a pore volume of 75 to 95% are produced. Process according to claims 1-5, characterized in that diisocyanate is used as the crosslinking agent. Process according to claim 6 *, characterized in that as diisocyanate, tolylene-2,4-diisocyanate or hexamethylhylendi in socyanate is used. Process according to claims 1-5, characterized in that epichlorohydrin is used as a cross-linking agent in a sodium hydroxide solution. 30 Process according to claims 1-5, characterized in that formaldehyde in hydrochloric acid solution is used as a crosslinking agent. Process according to claims 1-5, characterized in that glutaraldehyde is used as a crosslinking agent. Use of the porous cellulose beads prepared by the process of claims 1-10, optionally after a pretreatment, for the immobilization of enzymes and other biologically active agents and for the separation and purification of enzymes, proteins and nucleic acids. DK 158005 B 5 10 15 20 25 35