SI8112424A8 - Process for obtaining agglomerated fibrous ion-exchanging cellulose - Google Patents

Description

The field of technology in the invention is invention

Signed in! the invention falls within the field of preparing mixtures based on macromolecular compounds, even closer to the field of producing modified cellulose as well as to the application of an ion exchange process. According to the international classification, the invention could be classified in classes C 080 5 ^ 20. ' C 08B 15/00, C 08B 5/14. C 08B 11/145. ..., · ''

Technical problem

In the process of using an enzyme for industrial purposes, the enzyme is almost always (mobilized on a carrier during the process. Immobilization requires the enzyme to be physically bound to the carrier in some way. The more efficient the carrier holds the enzyme, the greater the efficiency of the process using the enzyme as a catalyst. Increasing the efficiency of the carrier binding capacity means that the carrier can hold more enzymes, thus facilitating the process.In some cases, however, the carrier may have adequate binding capacity but is not satisfactory in its ability to pump sufficient substrate flow through the carrier, and such carriers are often expensive. zaizruu,

A technical problem solved by the reported invention J® how to provide a process for obtaining agglomerated fibrous ion-exchange cellulose suitable for enzyme immobilization cell, which simultaneously exhibits executive ion-exchange properties as well as improved porosity characteristics to avoid flow problems while obtaining in a cost-effective manner from previously known methods.

BACKGROUND OF THE INVENTION The preparation and application of ion-exchange cellulose enzyme adsorbents is known from Peterson Sober's article, JAC.S.78, 751 (1956) Guthrie in Bullock, J / EC, 52,935 (1960) described methods for producing adsorptive cellulosic products that can be used to separate or purify enzymes and other proteins, tsmura et al. Nippon Shokuhin Kogyo Gakkaishi, 14 (12). (1967) demonstrate the binding of glucose isomerase to DEAE - Sephadex.

U.S. Pat. No. 3,708,397 to Sipos relates to a process for immobilizing glucose Isomerase to anion exchanger base cellulose. U.S. Pat. No. 3,823,133 to Hurst et al. refers to a process for the preparation of cationic cellulose ethers having a high adsorptive capacity for enzymes and other protein materials. 'American jaterite no. No. 3,838,007 to Velzen shows a process by which an enzyme preparation is obtained in the form of granules. U.S. Pat. No. 3,788,945 and 3,909,354 to both Thomson et al. Show a continuous process of converting glucose to fructose by transferring a solution containing glucose through fixed or floating strains containing glucose isomerase bound to various cellulose products. U.S. Pat. No. 3,947,325 to Dineliia et al. Relates to the production of enzymatic materials in the form of cellulose-containing beads. Cellulose is an emulsion containing aqueous enzyme solution and nitrocellulose. U.S. Pat. No. 3,956,065 to Idaszak et al. refers to a continuous glucose to fructose conversion process whereby a glucose solution is passed through a layer consisting of a cellulose derivative having glucose isomerase immobilized on it and non-porous or granulated polystyrene beads. . Granules prevent clogging and creasing when used in flow reactors. Sand and more. in an article titled ION-EXCHANGE1 granule cellulose derivatives., Dle Angewandto Macromolecules «Chemie, 53 p. 73-80 (1976), describes several cellulose derivatives obtained in the form of granules.

U.S. Patent Nos. 4,110,164 and 4,168250 to both Suthof et al., Relate to mixtures of agglomerated 'fibrous ion-exchange cellulose and processes for the preparation thereof. In these methods, the hydrophobic polymer is coupled to the -waxy polymer. cellulose previously derivatized as. would. obtain ion-exchange properties. Although these mixtures have been shown to be satisfactory in numerous applications, their ability to modify, ion and adsorb or bind glucose isomerase 'levels is as low as desired. In addition, the cost-effectiveness of these processes is such as to make finer production more expensive. ~; - - Description of the solution to these other problems:

Immobilized enzymes can be adapted for use in continuous conversion processes. In this way, enzyme utilization is achieved more efficiently and contact time between the enzyme and the substrate is reduced, whereby the lesulfate dabic ld improves the product quality. 'Although the description and embodiments are primarily directed to the use of agglomerating fibrous ion-exchange cellulose in the adsorption and immobilization of glucose isomerase. It is observed that the agglomerated material has the properties of adsorbing other enzymes, charged macro-molecules such as' proteins, nucleic acids and the like, except that it can. to regenerate said molecules from a range of substances such as food waste, e.g. regenerates whey proteins, meat and vegetables processing etc.

. In recent years, there have been developments in the production of fructose-containing solutions using glucose 'isomerase bound or immobilized on inert carrier materials. Such substances include various polymeric substances, such as cellulose derivatives, ion exchange resins and synthetic fibers, glass, insoluble organic and inorganic compounds, etc. Glucose isomerase was also encapsulated or shaped into beads in suitable materials. . '

In nature, cellulose occurs as a linear polymer consisting of anhydroglucose units coupled to each other by beta 41991

1.4 glucosidic bonds. Each unit of anhydroglucose contains three free hydroxyl groups that can react with the appropriate reagents to produce insoluble cellulose derivatives which, due to their relative inertia, large surfaces and open porous structures have a large adsorptive or ion-exchange capacity for protein molecules.

The invention relates to a process for the preparation of a mixture of agglomerated fibrous ion-exchange cellulose in which relatively large portions of cellulose are free to adsorb charged macromolecules. An agglomerate is obtained which contains woven cellulose and a hydrophobic polymer by coupling which the cellulose is derivatized to give ion-exchange properties.

The term "fibrous" is used in the present specification and claims when referring to cellulose originating from natural sources that is ground or converted into fibers by mechanical or chemical means and to include cellulose or derivatives thereof which have undergone chemical treatment.

'Fibrous, cellulose: -can be derivatized to da.bi. received a ion-softener material that has large charge capacities for tf- view, adsorption, or immobilization of macro-molecules. For this purpose, pulp. with / can ~ derivatize to obtain ion exchange material! having the properties of anion or cation exchange, depending on the charge present on the adsorbed material. When the adsorption material is glucose isomerase, cellulose is best derivatized in the form of an anion exchanger since in this form the filling capacity for this enzyme is large .. Typically, it would take the form of an anion exchanger to treat - agglomerated fibrous .cellulose suitable reagents - to obtain, inter alia, di-and three-letylaminoethip cellulose and cellulose derivatives epichlorohydrin and triethanolamine. Previous information and processes for the derivatization of cellulose are disclosed in U.S. Patent 3,923,133 to Hrust et al.

Due to the high loading capacities of fibrous ion-exchange cellulose preparations containing glucose isomerase when used in industrial applications, relatively small reactors can be used to convert large quantities of glucose to

- fructose.

In carrying out the present invention, a number of polymers may be used to agglomerate fibrous cellulose. Examples of these are melamine formaldehyde resins, epoxy resins, polystyrene and the like. The best polymer is polystyrene.

U.S. Patent Nos. 4,110, 164, and 4,168,250 show that agglomerated fibrous cellulose, which is derivatized to produce an ion-exchange substance !, with a hydrophobic polymer under suitable conditions, is light pulp having the properties to immobilize or bind glucose isomerase. The best process for preparing the mixtures involves treating the alkaline cellulose with a solution of diethylaminoethyl chloride hydrochloride and then agglomerating the resulting derivatized Jpno-measuring cellulose with polyester. Due to the solubility of polystyrene in the reaction mixture, it would be expected that the cellulose could not be effectively derivatized if the agglomerates were formed prior to the cellulose derivatization.

It has been found that fibrous cellulose can be effectively derivatized in the presence of a hydrophobic polymer by controlling the conditions of operation during derivatization to prevent the polymer from becoming dissolved in the derivatization solution. Thus, it was found that by adding a rate-controlled derivatization material to an aqueous suspension of agglomerates under alkaline conditions, the hydrophobic polymer component of the granular mixture does not become substantially soluble.

Another unexpected effect is that when the cellulose is derivatized followed by its agglomeration, the cellulose mixtures can be derivatized. in a larger roer and hence have a higher ion-exchange capacity than agglomerated cellulose mixtures produced by previously known processes in which the cellulose was derivatized prior to agglomeration. While the ion-exchange capacity of the fiber-cellulose mixtures according to the present invention may vary widely, typically the ion should be about 0.1 rriekv g-1 and preferably at least about 0j2 meq. g-1.

The mixtures of agglomerated fibrous ion-exchange cellulose according to the invention can also be regenerated, since the activity of the immobilized glucose isomerase is reduced to a certain extent - due to 'denaturation or other factors resulting from prolonged use, such that the soluble glucose isomerase solution can be brought into contact with layer iii column of the mixture so that its glucose isomerase activity again increases to the desired degree. Prior to regeneration, however, it is customary to treat the mixture with an alkaline solution to create ion-exchange sites of fibrous cellulose that are available for adsorption of isomerase.

When fibrous cellulose is derivatized prior to agglomeration, the materials used in the derivatization reaction may cause the cellulose to swell or become partially soluble and difficult to regenerate by filtration. The regeneration of the mixture according to the invention is simplified by the fact that such swelling as may occur does not pose a serious problem for liltration due to the granular nature of the derivatized product.

Depending on the specific weight of the substrate, mixtures of agglomerated fibrous ion-exchange cellulose may tend to flotate and hence, there may be some loss of the mixture through the inlet and outlet portions of the column-type reactor. Since problems may arise when initially filling a column with a mixture, in certain cases it is better to incorporate a thickening agent into the mixture of agglomerated fibrous ion-exchange cellulose to increase its density.

41991

Although many thickening agents can be used, they must, of course, be substantially inert to the substrate, and ¹ must also not deactivate glucose isomerase. Powdered metal oxides or silicates or mixtures thereof may be used as thickening agents.

In order to obtain a mixture of agglomerated fibrous-cellulose, the fibrous cellulose must be incorporated into the hydrophobic polymer in such a way that the cellulose is not completely complete. encapsulated or polymer coated. The larger the free surface of the pulp, the greater the adsorptive capacity of the mixture.

Although there are several ways to incorporate fibrous, cellulose into a hydrophobic polymer, two typical methods include: dissolving a hydrophobic polymer in an organic solvent and incorporating other materials into it, or warming the polymer to a plastic state and incorporating other materials. solvent evaporation. The resulting material can then be ground-by-milled UL similarly, the granules classified into - sieves of suitable dfriieriZi and agiomeriraria fibrous cellulose derivatized. The orariulometric composition of the granules may somewhat. widely varying The satisfactory results were obtained with the use of beads extending, with apertures of 0.84 mm, remaining. 'TL ⁷ _- To illustrate more clearly the process of the invention, specific embodiments will be provided. - 'EXAMPLES' This example illustrates the post post. ' ⁷ to obtain a mixture of agglomerated fibrous ion-exchange Geiui & ze wherein the cellulose component of the mixture is derivatized after agglomeration.

The agglomerate was obtained by mixing 25 parts of chemically pure cellulose (C-100 manufactured by International filter Corp., North Tonavwanda Ν.Υ.) with 25 parts of aluminum oxide and mixing the mixture with 50 parts of polystyrene on heated (180 - ^_ 200 ° C) mixing duo. within 10 minutes. After cooling, the fabricated mixture was ground and graded to a particle size of 0.42-0.149 mm.

220 g of the graded mixture was suspended in 616 ml of water containing 176 grams of Na ₂ SO ₄ and 26.4 grams of NaOH. The suspension was heated to 40 [deg.] C. after which it was quantitated into the suspension

- weighed - 57,2 gr 50% - - aqueous. of a solution of diethylaminoethyl chloride hydrochloride, stirring at a rate of 0.7 ml in a grinder for about 1 hour. Then 26.4 g NaOH dissolved in 26 ml water were added to the suspension, followed by an additional 57.2 diethylaminoethyl chloride hydrochloride solution at a rate of 0.7 m / min. ·

The temperature of the suspension was then raised to 60 ° C and maintained at the same temperature for 15 minutes. A volume of water approximately equal to the volume of the suspension was added and the mixture recovered at a sieve size of 0.250 mm. The mixture was washed with water in situ and resuspended in a volume of water similar to that previously added. This suspension was adjusted to pH about 7 with HO, washed on filter paper and dried.

The ion exchange capacity of the dried product was determined to be 0.84 meqv g-1 based on cellulose and 0.21 meqv g-1 based on the agglomerated mixture. ·

The ion exchange capacity of a mixture is determined by the following procedure:

Odmeriti 2O.g. dry matter derivatized agglomerated cellulose (5-10 g. cellulose base).

Suspend in water and adjust pH to -12.5 - 13.0 by aid 1. NNaOH. ' ⁷

Wash the suspension in the chromatographic column 1.

Add approximately 10 ml of IhLNaOH in coton j (MJstitf to bird in drops dd of plate level, wash the column with bottle; wash and dry to plate. · '

Wash with water volumes approximately equal to 6 volumes of corlstecl approximately 2 volumes of layers per rinse. Let it flow to the top surface of the plate with each rinse.

Add 25 ml of 1N HCl and the layers above isprati with about 10 ml of water ^- washing bottles. Start collecting the rising fluid, dropwise, at about 1-1.5 m / min. - Flush with the cadmium level washing bottle - the level reaches the plate.- r -. J

Wash with approx. 6 volume layers as in phase 5.

Titrate the outlet liquid to pH 7.0 with 1N NaOH. The ion exchange capacity was calculated as follows:

The capacity of I.J. (mEq.g-1, s.m.i = (mlHCIx N) - (ml NaOHx N gr.adsorbent s.m.

In Example 1 it can be calculated that the ratio of the derivatizing agent dlethylaminoethyl chloride hydrochloride to cellulose was 1.04 on a dry matter basis, whereas in the earlier process described in U.S. Patent 4,110,164, the cellulose was derivatized before agglomeration at a ratio of diethylaminoethylhydride hydrochloride. This value represents approximately the maximum amount to which non-agglomerated cellulose can be derivatized and regenerated.

EXAMPLE

This example illustrates a process for obtaining a mixture of agglomerated mixtures, as certain functional characteristics of the same

Table I.

41991 fibrous ion-exchange cellulose in which the cellulose is derivatized after agglomeration at a ratio of dlethylaminoethyl chloride hydrochloride to cellulose greater than 2.

The agglomerated mixture obtained as shown in Example 1 was ground and graded to a size of 0.177-0 ^ 12 mm. He suspended 100 g of the classified mixture in 280 ml of water in which 80 g of N ^ SO ^ and 24 g of NaOH were dissolved. With a suspension at 40 [deg.] C., 55 g of a 50% solution of diethylaminoethyl chloride hydrochloride were added quantitatively with stirring at a rate of 0.5 ml per minute over a period of about 1.5 hours. Additional NaOH (26 g of 50% solution) was then added in suspension as well as an additional 55 g of dlethylaminoethyl chloride hydrochloride solution with stirring at a rate of 0.5 ml per minute over a period of about 1.5 hours. Additional NaOH (26 grams of 50% solution) was then added to the suspension as well as an additional 55 grams of the solution - diethylaminoethyl chloride hydrochloride, quantitatively to the suspension as in the first addition. The reaction mixture was heated at 60 ° C for 30 min. At a temperature of 15 minutes / Water volume equal to the volume of the solution was added and diluted the suspension of the eye and washed in a 0.25-mm sieve. - The resulting slurry was 'resuspended' in water , adjusted to a pH of about 6.5-7.0 with HCl and washed in situ. \ - and the 'Dry Ion Change Capacity' in the average mode shown was 1.28 meqv.g-1 Ha base. cellulose and 0 ^ 32 meqv g-1 based on the mixture. Achieving comparable * ion exchange capacity by the earlier process in which derivatized diethylaminoethylcellulose was agglomerated with polystyrene would require such a degree of derivatization that the cellulose would become gelatinous and difficult to regenerate, filter and dry.

EXAMPLE! j '. ^: ~.

This example illustrates the adsorptive glucose isomerase capacity for the described mixtures of agglomerated fibrous ion-exchange cellulose and mixtures described in the prior art and provides a comparison of the characteristics and functional properties of said mixtures. _

Glucose isomerase derived from microorganisms of the Streptomyces species, of a strength of about 20 International Units of Glucose Isomerase (IGIU) per millimeter, is added to the same mass of agglomerated 'ion exchange cellulose mixtures obtained by the procedures described in (US Patent) US Patent 4, 110 ,. 164 and the above examples I ΐ II. The enzyme / mixture suspensions were adjusted to pH 7 and stirred for 5 hours at a temperature of 25 degrees. The mixtures were regenerated by filtration and the amount of enzyme adsorbed on them was measured by measuring the residual glucose isomerase activity. in the corresponding 'filtrates by the method described by N.E. Uoyd et al. in Gereal Chem. Vol, 49 (5), p. 544,1972.

Amount of glucose isomerase adsorbed by some? : ϋ ΐ I, χ i

(□ sa / azcz) iciM ion. ^k .n ** ncitct - (set. ¢ -1) ίΓ: ' ' ³ sl: EXAMPLE 1 1.04 ' 0.21 490 EXAMPLE 2.2 0.32 690 U.S. Patent 4,110,164 0.7 0.14 361

IGIU = international glucose isomerase units of meq.g-1 milliequivalents per gram = DEC = diethylaminoethyl chloride hydrochloride

EXAMPLE IV

This example illustrates the porosity characteristics of mixtures of agglomerated fibrous ion-exchange cellulose obtained by the present method and the comparison of the "characteristics" of the tendencies of said mixtures with those of mixtures according to the preceding one. state of the art.

Porosity characteristics determined by stf using the following materials:

Two non-agglomerated fiber pulp (a) and (b) Whatmah pulp manufactured by W-R Balaton Ltd. England, used for comparative purposes with products obtained according to the invention. "

Non-cross-linked diethylaminoethylcellulose (prepared as described in US 3,823,133. <

A mixture obtained by agglomerating fibrous ion-exchange cellulose and polystyrene (prepared as described in US 4,110,164).

A mixture of agglomerated fibrous ion-exchange cellulose obtained as described in Example i. 'A mixture of agglomerated fibrous ion-exchange cellulose obtained as described in Example 2.

The porosity constant was determined for each of the above materials by the following procedure:

- 75 grams of the dry matter of each of the comparative celluloses at points 1-5 were suspended in water and the suspension released from the air by stirring under a vacuum of 0.848 bar under atmospheric pressure for 15 minutes. A glass column (3.81 cm) in internal diameter and 40.64 cm in height, fitted with a porous glass plate and a tap at the bottom, is attached to a vacuum boco via a 'rubber stopper'. The bottle is in turn attached to a vacuum source / Suspended air is suspended in a column and a vacuum of 5 P · '' is included.

41991 {0,848 bar below atmospheric pressure) at the bottom of the column by opening the faucet, forming a layer of material on a porous glass plate. At the same time, water was released to the top of the column to replace the one removed by filtration so that about 12.7 cm of water was maintained above the layer at all times. When a total of 1000 ml of water was collected, the tap was closed, the bottle removed and the water drained from the bottle. The bottle was then reconnected to the column, vacuum recovered, open faucet 1 measured quantity (1000 - 3000 ml) of water (filtered through a thickened layer and collected. Chronometer 8 determined the time required to collect this water. The porosity constant was calculated according to the following equation :

K = (VH) / (TPA) where:

K = porosity constant (ml cm'g-1 min 1)

V = volume of water collected (ml)

H = height of thick layer (cm)

T = water collection time '(min)

P = pressure drop through the Layer (g per sq.cm.j A = cross-section of the layer (sq.cm.)

The results are shown in Table ll.

TABLES ·.

Material Porosity constant (1) (ml cm g-1 min-1) (a) '' · 0.21 (b) - 0.60

0.21 ³ ~ '· .. 4J

4. 2,6

3.6 (1) Minimum porosity constant of 1.5 ml cm g-1 min-1 is the minimum required to achieve efficient performance in a deep bed reactor.

As the best method for the industrial application of the present invention, the applicant provides the following example:

This example illustrates the procedure for. obtaining a mixture of agglomerated fibrous ion-exchange cellulose whereby the cellulose component of the mixture is derivatized after agglomeration.

The agglomerate is crushed by mixing 25 'parts of chemically pure cellulose (C-100 manufactured by International Filler Corp. North Tonawanda Ν.Υ., with 25 parts aluminum and mixing the mixture with 50 parts polystyrene on heated (180-200 C °) mixing mixers After 10 cooling hours, after cooling, the mixture is ground and graded with a grain size of 0.149 - 0.42 mm.

220 g of graded mixture suspended in 616 ml of water containing 176 grams of Na ₂ SO ₄ and 26.4 grams of NaOH. The suspension was heated to 40 ° C after which the suspension was quantitatively measured at 57.2 g of 50% aqueous solution of diethylaminoethyl chloride hydrochloride with stirring at a rate of 0.7 ml per minute for about 1 hour. A further 26.4 g of NaOH was dissolved in the suspension, dissolved in 26 ml of water, followed by an additional 57.2 g of diethylaminoethyl chloride hydrochloride solution at 0.7 ml per minute.

The temperature of the suspension was then raised to 60 ° C and held at this height for 15 minutes. The added volume of water is approximately equal to the volume of the suspension and the mixture recovered at a 0.25 mm aperture. The mixture was washed with water in situ and resuspended in a volume of water similar to that previously added. This suspension was adjusted to pH 7 with HCl, washed and dried on filter paper and dried.

The ion exchange capacity of the dried product was determined to be 0.84 meqv g-V per ~ 45azj of cellulose Ί 0.21 meqv-g - 1 based on the agglomerated mixture. '_ - -;

The capacity of the refined ion in deer is determined by the following procedure:. ^r '- r *

1. Weigh 20 g of dry matter of derivatized agglomerated cellulose (5-10 g of cellulose base).

2. Combine in water and adjust the pH to 12.5 - 13.0 with 1 N NaOH.

3. Wash the suspension in the chromatographic column and place the blank plate on top of the layer .. '.

4. Add approximately 10 ml of 1N NaOH to the column and let it drop to the level of the plate, wash the column with a washing bottle and dry to the plate.

5. Wash with water volumes of approximately 6 volume volumes using approximately 2 volume volumes per rinse. Let it leak to the top of the plate at each rinse.

6. Add 25 ml of 1N HCl above the shine and rinse with about 10 ml of water from the washing bottle. Start collecting the exit fluid, dropwise, at about 1-1.5 ml / min. Wash with bocorh for washing when upper level comes to plate.

7. Wash with approximately 6 volumes of coat as in f azi-5.

8. Titrate the outlet liquid to pH 7.0 with 1N NaOH. The ion exchange capacity was calculated as follows:

The capacity of I.J. (mEq. g-1, * m) = = (ml HCl x N) - (ml NaOHxNj gr adsorbent

41991

For example, it can be calculated that the ratio of the dlethylaminoethoxy chloride hydrochloride derivatization agent to the cellulose was 1.04 on a dry matter basis, whereas in the former process described in (U.S. Pat. No. 4,110,164), the cellulose was derivatized before agglomeration at a diethylaminoethyl chloride hydrochloride ratio.7 .

Claims (3)

PATENT REQUIREMENTS 1. A process for the preparation of agglomerated fibrous ion-exchange cellulose, which adsorbs ion-binding charged macromolecules, wherein the fibrous cellulose is agglomerated with a hydrophobic polymer, such as polystyrene, melamine formaldehyde or smokes, in an organic solvent such as diethylaminoethyl chloride I by incorporating it into cellulose, wherein the agglomerate is derivatized with diethylaminoethyl chloride hydrochloride, so that the ion-exchange capacity is at least 0.20 milliequivalents per gram of dried cellulose mixture. 2. The method according to claim 1, which is a macromolecule adsorbed by glucose isomerase. 3. The method of claim 1, characterized by t and m e, which is a fineness of the mixture to a particle size of 0.25 to 0.84 mm. Published and Printed by the Federal Patent Office, Belgrade, Uzun Mirkova l