FI67560B - FOERBAETTRAT FOERFARANDE FOER FRAMSTAELLNING AV AGGLOMERERAD FIBROES CELLULOSA - Google Patents

Description

67560

An improved process for making agglomerated fibrous cellulose

The invention relates to a process for the preparation of an agglomerated fibrous ion exchange cellulose product capable of adsorbing or binding charged macromolecules, for example enzymes, by agglomerating the fibrous cellulose with a hydrophobic polymer. The obtained products have a better ion exchange efficiency than the products prepared by hitherto known methods.

10 In food production and other commercial applications, the use of microbial or fungal enzymes adsorbed or bound as catalysts bound to inert supports has largely supplanted older methods using soluble enzymes or whole cells of microorganisms. The main advantage of bound enzymes is that they can be used in continuous processes. Thus, a more efficient use of the enzyme is achieved and the duration of action of the enzyme on the substrate is shortened, resulting in an improvement in the quality of the product and a reduction in the enzyme and production costs.

The following description and examples relate primarily to the use of agglomerated fibrous ion exchange celluloses for the adsorption and binding of glucose isomerase.

However, it is believed that the agglomerated material would also be able to adsorb other enzymes and charged macromolecules, such as other proteins, nucleic acids, etc., and that it would also be able to recover these molecules from various materials, such as organ waste products. This would involve, for example, the collection of proteins from milk whey, meat and vegetable treatment solutions and the reduction of the BHK value of waste solutions.

Because of the economics of glucose isomerase production 35, it is extremely important to use isomerase under conditions that produce fructose in the best possible yield of 2,67560 and with the lowest possible amount of enzyme. In addition, the isomerization conditions should be such that the smallest possible amounts of worthless by-products are formed.

5 In recent years, rather economical methods have been developed for the preparation of solutions containing fructose / using glucose isomerase bound to inert carriers. Such substances include many polymeric substances such as modified cellulose, ion exchange resins and synthetic fibers, glass, insoluble organic and inorganic compounds, etc. Glucose isomerase is also encapsulated or pelletized in suitable materials, but these preparations have the disadvantage that they are not usually used. can be reused.

Cellulose is naturally a linear polymer that already contains β-1,4-glycoside-linked glucose anhydride units. Each glucose anhydride unit contains three free hydroxyl groups which can react with suitable substances to form insoluble cellulose derivatives which, due to their relatively high inertness, wide surface area and open porous structure, have good adsorption and ion exchange capacity for protein molecules.

The preparation and use of the adsorbent-25 pathway of cellulose-based ion exchange enzymes is known. Peterson and

Soker, J. A. C. S. 78 (1956) 751 and Guthrie and Bullock, I / EC, 52 (1960) 935 have described methods for preparing adsorbent cellulose products that can be used to separate and purify enzymes and other proteins. Tsumura et al., Nippon Shokuhin Kogyo

Gakkaishi 14 (1967) 12, describe the binding of glucose isomerase to DEAL-Sephadex.

DS Patent 3,708,379 to Sipos discloses a process for binding glucose isomerase to basic 35 anion exchange celluloses. U.S. Patent 3,823,133,

Hurst et al., Discusses the method of cationic cellulose

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3,67560 for the preparation of ethers with good adsorption capacity for enzymes and other proteins. U.S. Patent No. 3,838,007 to van Welzen provides a method for obtaining an enzyme preparation in the form of bodies. U.S. Patent Nos. 3,788,945 and 3,909,354 to Thompson et al. Disclose a continuous process for converting glucose to fructose by passing a glucose-containing solution through solid or fluidized beds containing glucose isomerase bound to various cellulose products. U.S. Patent 3,947,325 to Dinelli et al. Relates to the preparation of a cellulose-containing pelletized enzymatic product. Cellulose consists of an emulsion of an aqueous solution of enzymes and nitrocellulose. U.S. Patent 3,956,065 to Idaszak et al. Discloses a continuous process for converting glucose to fructose, in which a glucose-containing solution is passed through a layer containing a cellulose derivative to which glucose isomerase is bound and solid surface spherical polystyrene granules. The spheres prevent the layer from condensing and channeling when used in flow reactors. Peska et al., "Ion Exchange Derivatives of Bead Cellulose", Die Angewandte Makromolekulare Chemie 53 (1976) 73-80, describe several cellulose derivatives prepared as spheres.

U.S. Patent Nos. 4,110,164 and 4,168,250;

Sutthoff et al., Refer to agglomerated fibrous ion exchange cellulose products and methods of making the same. Using these methods, the hydrophobic polymer is combined with fibrous cellulose that has been pretreated for ion exchange. Although these products are satisfactory in many applications, their ion exchange efficiency and ability to adsorb or bind glucose isomerase are not always of the desired magnitude. In addition, the production of products by these methods is not as economically advantageous as desired.

4,67560

The process according to the invention is characterized in that an agglomerate is first prepared from fibrous cellulose and a hydrophobic polymer and then the agglomerate is converted in an aqueous suspension to ion exchange properties under temporal conditions, whereby the hydrophobic polymer component of the granular product does not dissolve significantly.

The term "fibrous" in this specification and the appended claims means naturally occurring cellulose which has been chopped or converted into fibers by mechanical and chemical means; the designation does not include cellulose or its derivatives which have been chemically treated to result in the dissolution of the natural fibrous structure of the cellulose, which may occur when converting cellulose to a high number of substituents.

Fibrous cellulose can be modified to contain ion exchange materials with a high ability to adsorb or bind macromolecules. For this purpose, ion exchange materials which are either anionic or cation exchanging can be incorporated into the cellulose, depending on the charge of the material to be adsorbed. When the substance to be adsorbed is glucose isomerase, the cellulose is preferably converted to anion exchange, since in this form its enzyme-binding capacity is very high. In order to convert to the anion exchange form, the agglomerated fibrous cellulose is typically treated with suitable reagents which form e.g. di- and triethylaminoethylcellulose such as DEAE and TEAE cellulose, and cellulose derivatives of epichlorohydrin and triethanolamine such as ECTEOLA cellulose. 30 Background information and methods for converting cellulose are described in U.S. Patent 3,823,133 to Hurst et al.

The high loading capacity of glucose isomerase-containing fibrous ion-exchange cellulose products when used in industry allows the use of relatively small equipment to convert large amounts of glucose to fructose.

Il 5 67560

In addition, due to this high loading capacity, the substrate and the product to be obtained are only for a short time under the conditions under which the isomerization takes place. Under these isomerization conditions, small amounts of harmful by-products are generally formed due to the reactivity of fructose, and the longer fructose is under these conditions, the more harmful by-products are produced. Thus, the high loading capacity of fibrous ion exchange cellulose results in the substrate being isomerized to the desired degree in a short time, and fructose lingers for a short time under the isomerization conditions. Such preparations containing fibrous ion-exchange cellulose have the disadvantage that they "pack" and are usually used in thin layers in order to avoid the inconveniences caused by a large pressure drop. Even when thin layers are used, it is possible that channelization will occur, leaving the substrate in contact with the bound glucose isomerase to the desired extent. Although certain glucose isomerase preparations have been developed in which these problems are minimized, they generally have other disadvantages, for example, their enzyme capacity or activity per unit volume is not as desired and / or is not as economical as fibrous ion exchange cellulose.

Many polymers can be used to agglomerate fibrous cellulose in the practice of this invention. Examples of such are melamine-formaldehyde resins, epoxy resins, polystyrene, etc. The preferred polymer is polystyrene.

According to U.S. Patent Nos. 4,110,164 and 4,168,250, cellulose has the ability to bind glucose isomerase when an ion exchange material is added thereto and then agglomerated with a hydrophobic polymer under appropriate conditions. A preferred method of making the products involves treating the alkali cellulose with a solution of diethylaminoethyl chloride hydrochloride (DEC) and agglomerating the ion-exchanged cellulose derivative thus formed with polystyrene. However, since polystyrene is soluble in the DEC reaction mixture, it is expected that cellulose could not be efficiently converted if agglomeration were performed prior to cellulose conversion.

We have found that fibrous cellulose can surprisingly be efficiently converted in the presence of a hydrophobic polymer by adjusting the manufacturing conditions during the conversion so as to prevent the polymer 10 from dissolving in the conversion solution. Thus, it has been found that when the converting material is added to the aqueous suspension of the agglomerate at an appropriate rate under basic conditions, the hydrophobic polymer portion of the granular product is not significantly soluble.

Another surprising consideration is that when the cellulose is converted after agglomeration, the cellulose product can be converted to a greater extent and thus provide a better ion exchange capacity than the agglomerated cellulose product prepared by the hitherto known method of converting cellulose before agglomeration. The ion exchange capacity of the agglomerated fibrous cellulosic product of the invention can vary widely, typically the ion exchange capacity should be at least about 0.1 meq (milliequivalents) / g and preferably at least about 0.2 meq / g.

The agglomerated fibrous ion exchange cellulose products of the invention can also be regenerated, i. when the bound glucose isomerase activity has decreased due to a certain amount of denaturation or some other reason for prolonged use, the product-containing layer or column can be treated with a glucose isomerase solution so that its glucose isomerase activity rises again to the desired value.

However, prior to regeneration, it is preferable to hand-product the product with an alkaline solution to better obtain the ion exchange sites of the fibrous cellulose.

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7 67560 for isomerase adsorbing. While not wishing to be bound by any theory of such a mechanism, it is likely that substrate residues, denatured isomerase, or other proteinaceous materials attached to fibrous cellulose will be removed or soluble.

When fibrous cellulose is converted prior to agglomeration, the conversion agents tend to cause the cellulose to swell or partially dissolve and make it difficult to isolate by filtration.

The isolation of the product according to the invention is simplified by the fact that such swelling, which may occur, does not cause major filtration problems due to the granularity of the modified product. In addition, since the granular cellulose-15 products are not sensitive to compaction, they can be used in devices with thick layers without difficulty and with minimal channeling.

Depending on the specific gravity of the substrate, the agglomerated fibrous ion exchange cellulose product may tend to float to 20, and it is possible that the product will lose some of the inlet or outlet of the column type equipment. In addition, problems can occur when the column is filled with product. Therefore, in certain cases, it is preferable to add a density enhancing agent to the agglomerated fibrous ion exchange cellulose product.

Although many bulking agents can be used, they must, of course, be fairly inert to the substrate, nor must they inactivate glucose isomerase. Powdered metal oxides or silicates or mixtures thereof can be used as density enhancers.

When forming an agglomerated fibrous product, the fibrous cellulose must be implanted in the hydrophobic polymer so that the cellulose is not completely encapsulated or covered in the polymer. If this happens, the ability of the fibrous ion exchange cellulose to adsorb enzymes will be drastically impaired. The larger the cellulose free surface, the better the adsorption capacity of the product.

Many methods can be used to implant fibrous cellulose into a hydrophobic polymer. Typically, one of the methods used involves dissolving the hydrophobic polymer in an organic solvent and adding other materials to this solution, and the other method involves heating the polymer to softness and adding other materials thereto. The latter method is preferred because it does not require evaporation of the solvent.

The resulting material can then be comminuted by grinding, or the like, the granules can be sorted by suitable sieves, and the agglomerated fibrous cellulose converted.

The particle size distribution of the granules can vary quite a bit. Satisfactory results have been obtained using granules which pass through a 20-mesh U.S. standard (ASTM) sieve with a mesh width of 0.84 mm but stop at that temperature. a standard 60 mesh screen with a mesh width of 0.250 mm.

In order to more clearly illustrate the nature of the invention, more specific examples are given below.

Example I

This example illustrates a process for preparing an agglomerated fibrous ion exchange cellulose product such that the cellulosic component of the product is converted after agglomeration.

The agglomerate was prepared by mixing 25 parts of chemically pure cellulose (C-100, manufactured by International Filler Corp., North Tonawana, hY) and 25 parts of alum 30 and combining the mixture with 50 parts of polystyrene in a heated (180-200 ° C) twin drum mixer for about 10 min. . After cooling, the combined mixture was ground and screened to a size of 40-100 mesh. wherein the particles passed through a sieve having a mesh width of 0.42 mm but not a sieve having a mesh width of 0.1499 mm.

220 g of the screened product was slurried in water (616 mL) containing 176 g of Na 2 SO 4 and 26.4 g of NaOH. The slurry was heated

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9,67560 to 40 ° C, after which 57.2 g of a 50% aqueous solution of diethylaminoethyl chloride hydrochloride (DEC) was added thereto with stirring at a rate of 0.7 ml / min over 1 h. Next, 26.4 g of NaOH dissolved in water (26 mL) was again added to the slurry, followed by an additional 57.2 g of DEC solution at a rate of 0.7 mL / min.

The temperature of the slurry was then raised to 60 ° C and maintained at this temperature for 15 min. Water was added to the volume of the slurry and sieved through a 60 mesh sieve with a mesh width of 0.250 mm. The product was washed on the sieve with water and reslurried in the same amount of water as added above. The pH of this slurry was adjusted to 7 with HCl, washed and filtered through paper and dried.

The ion exchange capacity of the dried product was determined to be 0.84 meq / g for cellulose and 0.21 meq / g for agglomerated product.

The ion exchange capacity of the product was determined as follows: 1. Weigh 20 g of dry modified agglomerated cellulose (equivalent to 5-10 g of starting cellulose) 2. Slurry in water and adjust the pH to 12.5-13.0 with 1 N NaOH 3. Rinse the slurry a porous plate 4 is placed on the chromatography column and on top of the 25 layers. Add about 10 ml of 1 N NaOH to the column and lower the liquid level dropwise to the plate level, rinse the column with a wash bottle and lower the liquid level to the plate level 30 5. Wash with about 6 times the volume of the layer using about 1/2 the amount of water at a time. Allow the liquid surface to settle to the level of the plate after each rinse.

6. Add 25 ml of 1N HCl to the bed and rinse with about 10 ml of water using a wash bottle. Start collecting the effluent drop by drop at a rate of 1-1.5 ml / min. Rinse with a wash bottle when the liquid level drops to the level of the plate.

_ - n --------- 10 67560 7. Wash with about 6 times the volume of water as described in step 5. 8. Titrate the effluent to pH 7.0 with 1 N NaOH. The ion exchange capacity is calculated as follows: 5 Capacity (meq / g dry (mlHClxn) - (mlNaOHxn) product) = g dry adsorbent

From Example I, it can be calculated that the ratio of the conversion agent (DEC) to the dry weight of the cellulose was 1.04, while in the 10 previously known process described in U.S. Patent No. 4,110,164, the DEC / cellulose ratio was 0.7. This value corresponds to the maximum amount at which unagglomerated cellulose can be converted and isolated without difficulty by conventional means.

Example II

This example illustrates a process for preparing an agglomerated fibrous ion exchange cellulose product such that the cellulose is converted after agglomeration with a DEC / cellulose ratio greater than 2.

The agglomerated product prepared according to Example I 20 was ground and screened to a size of 40-80 mesh, whereby the particles passed through a sieve with a mesh width of 0.42 mm, but not through a sieve with a mesh width of 0.177 mm.

100 g of the screened product was slurried in water (280 ml) in which 80 g of Na 2 SO 4 and 24 g of NaOH had been dissolved. To the slurry at 40 ° C was added 55 g of a 50% DEC solution with stirring at a rate of 0.5 ml / min over about 1.5 h. Additional NaOH (26 g of a 50% solution) was added to the slurry and then 55 g of the DEC solution in the same manner as above. The reaction mixture was heated to 60 ° C and maintained at this temperature for 15 min.

Water was added to the volume of the slurry and the diluted slurry was dewatered and washed with a 60 mesh screen having a mesh width of 0.250 mm. The product was reslurried in water and the pH was adjusted to 6.5-7.0 with HCl, screened and washed as above.

The ion exchange capacity of the dry product was determined as mentioned and was 1.28 meq / g cellulose and 0.32 meq / g product. In order to achieve a comparable ion exchange capacity of 11,67560 using a known method of agglomerating modified DEAE cellulose with polystyrene, a degree of conversion would be required to make the cellulose gelatinous and difficult to isolate, filter and dry the cellulose without expensive or cross-linking.

Example lii This example describes the glucose isomerase adsorption capacity of the above-mentioned fibrous agglomerated ion exchange cellulose products 10 and the products described in the previous literature, and compares the nature and performance characteristics of said products.

Glucose isomerase obtained from Streptomyces with a capacity of about 20 IGIU / ml was added in equal amounts to products prepared according to U.S. Patent No. 4,110,164 and Examples I and II. The enzyme / product slurries were adjusted to pH 7 and stirred for 5 h at 25 ° C. The products were isolated by filtration and the amount of enzyme adsorbed on them was determined by measuring the residual glucose isoroerase activity of the respective filtrates by the method described in N. E. Lloud et al., Cereal Chem.

49 No. 5 (1972) 544.

The amounts of glucose isomerase adsorbed by each product and information describing specific performance characteristics are given in Table I.

Table I

Product Weight ratio Ion exchange Adsorption DEC: cellulose capacity capacity 30 _ (meq / q) _ (IGIU g ** 1) _

Example I 1.04 0.21 490

Example II 2.2 0.32 690 U.S. Patent 0.7 0.14 361 No. 4,110,164 12,67560

Example IV

This example describes the porosity of the agglomerated fibrous ion exchange cellulose products prepared by the process of the invention and compares the flow characteristics of said and previously known products and some non-agglomerated fibrous cellulosic products.

The porosity properties of the following materials were determined: 1. a & b. Crosslinked Whatman cellulose, manufactured by W&R Balston Ltd, England 2. Non-crosslinked DEAE cellulose (prepared according to U.S. Patent 3,823,133) 3. Product prepared by agglomeration 15 fibrous ion exchange cellulose and polystyrene (prepared according to U.S. Patent 4,110,164) 4. Product of Example 1 5. Product of Example IX

The porosity concentration for each material was determined as follows: 15-75 g of dry product was slurried in water and the slurry was deaerated by stirring under reduced pressure for 15 min. A glass column (inner diameter 3.8 cm, height 45.7 cm) equipped with a porous glass plate and a bottom hook was attached to a vacuum flask with a rubber stopper. The bottle was connected to a suction pump. The airless slurry was poured onto the column, and a vacuum (0.83 atm below normal pressure) was adjusted to the bottom of the column by opening the bottom tap to form a layer of material on the porous glass plate. At the same time, water was added to the top of the column to replace the effluent by filtering so that there was about 13 cm of water on top of the bed at all times. After collecting a total of 1000 ml of water, the tap was closed, the flask was removed and the water was poured from the flask. The flask was then reconnected to 35 columns, the vacuum was adjusted, the tap was opened and a measured amount (1000-3000 ml) of water was filtered through condensed

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13,67560 layers and was collected. The time taken to collect the water was measured with a clock. The porosity constant was calculated using the following formula K— (VH) / (TPA) 5 where K = porosity loss (ml cm / g min) V = volume of water collected (ml) H = height of the compacted bed (cm) T = time taken to collect water (min ) 2 10 J? * pressure drop caused by the layer (g / cm) 2 A * cross-sectional area of the layer (cm)

The results are shown in Table II.

Table II

15 Material Porosity constant (1) _ (ml cm g ^ min ^) _ 1 (a) 0.21 1 (b) 0.60 2 0.01 20 3 4.7 4 2.6 5 3.6 (1) a porosity constant of at least 1.5 ml cm / g min is considered necessary for the satisfactory operation of equipment using thick layers.

Claims (10)

A process for the exchange of an agglomerated fibrous ion for the preparation of a cellulosic product capable of adsorbing or binding charged macromolecules, for example enzymes, by agglomerating fibrous cellulose with a hydrophobic polymer under aqueous conditions and converting ion exchange properties, whereby the hydrophobic polymer component of the granular product does not dissolve significantly and the product remains granular. Process according to Claim 1, characterized in that the modified cellulose formed is diethylaminoethylcellulose. Process according to Claim 1 or 2, characterized in that the hydrophobic polymer is polystyrene. Process according to one of Claims 1 to 3, characterized in that the product contains a density-increasing agent, such as powdered metal oxides, silicates or mixtures thereof. Process according to one of Claims 1 to 4, characterized in that the agglomerate is prepared by combining cellulose with a polymer which has been heated to a plastic state. Process according to one of Claims 1 to 4, characterized in that the agglomerate is prepared by dissolving the polymer in an organic solvent and adding cellulose to this solution. Process according to one of Claims 1 to 6, characterized in that the product is comminuted to a particle size such that the granules 35 formed pass through a 20-mesh sieve with a mesh width of 0.84 mm but stop at a 60-mesh sieve with a mesh width of 0.250 mm. Il 15 67560 Process according to any one of claims 1 to 7, characterized in that the product has an ion exchange capacity of at least about 0.10 meq / g of dry ion exchange cellulose product. Process according to any one of claims 1 to 7, characterized in that the product has an ion exchange capacity of at least about 0.20 meq / g of dried ion exchange cellulose product. Process according to one of Claims 1 to 9, characterized in that the fibrous cellulose is implanted in a hydrophobic polymer. Patent krav 6 V 5 6 O