GB2128620A - Method for production of an immobilized enzyme preparation - Google Patents

Abstract

The method involves that a carrier comprising two phases, i.e. a) a continuous phase consisting of a binder and b) a discontinuous phase consisting of a multitude of separate, hard and inert particles, is contacted with an enzyme.

Description

SPECIFICATION Method for production of an immobilized enzyme preparation The invention comprises a method for production of a particulate, immobilized enzyme preparation consisting of a carrier, onto the surface of which the enzyme is attached. The surface of the carrier is here to be understood as the external surface as well as the internal surface in case of a porous carrier.

The field comprising immobilized enzymes and carriers for immobilizing enzymes is rapidly growing. Usually the carrier has the form of small particles, possibly weighted, in which the enzyme is embedded, or on the surface of which the enzyme is attached or fixed. Reference is made to U.S. patent 4 266 029, US patent 4 116 771, and Danish patent 133 380, which are only three examples of carriers (on or in which enzymes are fixed) known in the art.

The carrier utilized in relation to the method according to the invention belongs to the category of carriers, on the surface of which the enzyme is attached, in contradistinction to the catagory of carriers, in which the enzyme is distributed throughout the entire volume of the carrier.

A typical known carrier belonging to this just indicated category of carriers, on the surface of which the enzyme is attached, consists of granular gelatine and is described in Derwent 08061 C/s (J 5 4156-892). Particles of pure gelatine, however, are not hard enough for use in large scale fixed bed operations, where very high flow rates are required. Also, particles of pure gelatine are relatively expensive.

A similar carrier also belonging to this category, whereby an inert core is coated by gelatine, can be produced according to US patent No. 4 266 029. Though this carrier has good flow characteristics, it suffers from the disadvantage that the shape and size of the carrier particles can not be chosen in accordance with the criteria for best performance in a column, but are given with the particular sand fraction or other fraction of particulate dense material used as raw material. Furthermore, whereas it is easy to produce this carrier on a laboratory scale, it is difficult or perhaps impossible to manufacture this carrier on an industrial scale.

Another typical known carrier belonging to this category is described in Advances in Experimental Medicine and Biology, vol. 42, pp. 191-212, Immobilized Biochemicals and Affinity Chromatography.

This carrier consists of glass beads with a silane coupling agent. These beads have excellent flow properties and relatively high loading capacity. However, they are very expensive, and, like all inorganic carriers, to render them suitable for immobilizing enzymes, elaborate chemical treatment has to be performed, involving the use of undesirable materials.

Thus, an object of the invention is to provide a method for production of an immobilized enzyme comprising a carrier, onto the surface of which the enzyme is attached, whereby this carrier exhibits all the favourable properties listed above, i.e. sufficient hardness, ability to be produced on an industrial scale, and by means of a simple chemical treatment, and low production price.

Now, according to the invention a method for production of a particulate immobilized enzyme preparation comprising a carrier onto the surface of which the enzymes is attached has been found, wherein the carrier is formed by combination of two phases, i.e. a) a continuous phase of a water soluble binder at least partially dissolved or dispersed in an aqueous medium and b) a discontinuous phase of a multitude of discrete, hard, and inert particles, the size of which is small enough not to interfere with the shaping of the carrier, and by rendering the binder insoluble in the medium, in which the enzyme eventually will be utilized, if necessary, and wherein the carrier is contacted with an enzyme which is attached to the surface of the carrier.If the medium, in which the immobilized enzyme ultimately will have to be used, is an aqueous medium, it will be necessary to insolubilize the (originally) water soluble binder by a chemical or physical treatment, e.g. by crosslinking or heating. If the medium, in which the immobilized enzyme ultimately will have to be used, is a non-aqueous medium, the (originally) water soluble binder may be insoluble in this non-aqueous medium, and thus in this case it may not be necessary to insolubilize the binder. For the sake of completeness attention is drawn to the fact that the carrier may contain other components besides the above indicated imperative components a) and b), e.g. fillers and/or granulating aids.

Surprisingly, according to the invention it has been found that the method according to the invention fulfills the above indicated object of the invention. Also surprisingly it has been found that the carrier retains its excellent physical integrity even at very high proportions of the inert particles up to around 98 weight%, in relation to the weight of the carrier. This is very important, as the higher the proportion of the inert particles (up to the above indicated upper limit), the harder the carrier, and the lower its cost.

If the immobilized enzyme is a lipase, and the lipase is intended to interesterify lipids in a petroleum ether solution, then albumen, casein, soy protein, hydroxyethylcellulose, agar, alginate, polyvinylalcohols, starch, methylcellulose or carboxymethylcellulose may be used as the binder, as these materials are insoluble in petroleum ether. Only a loose attachment between the carrier and the enzyme is needed in this case. If, on the other hand the enzyme, with which the carrier eventually will be used, is amyloglucosidase, and the amyloglucosidase is intended to split dextrins in aqueous solution, then it will be necessary to insolubilize the (originally) water soluble binder. Usually the amyloglucosidase will be attached or fixed on the surface of the carrier by crosslinking with glutaraldehyde.

It is to be understood that not each and every combination of any enzyme and any of the two carrier phases a) and b) necessarily is operative. The skilled worker in the art will know that some enzymes may not tolerate certain cations which might be given off in small amounts from phase b), and also, if the immobilized enzyme is intended for use in the food industry, some of the phase a) constituants may be less desirable due to leakage of adverse constituents into the effluent from the enzyme reactor.

Immobilized glucose isomerase attached by ion exchange to a carrier consisting of fibrous, ion exchanging cellulose incorporated in a hydrophobic polymer and optionally containing a densification agent like powdered metal oxides is described in Danish patent application No.1592/78 (Standard Brands), corresponding to US 4,110,164 or BE 865,900. The carrier is produced by melting the hydrophobic polymer and mixing the cellulose and the densification agent into the melted polymer. This method, however, entails a number of disadvantages. In the first place the price of such hydrophobic polymers is rather high, and secondly it is much easier to operate with aqueous solutions or suspensions of binders according to the invention, than with melted hydrophobic polymers.Also, the method described in Danish patent application No.1592/78 is strictly limited to enzymes immobilized on ion exchange resins, i.e. with a monomolecular layer of enzyme, whereas our invention is adapted to all methods of attachments of enzymes to the carrier, and to monomolecular layers of enzymes as well as multimolecular layers of enzymes. Thus, in the case of glucose isomerase, for instance, due to this free choice of enzyme layer thickness, it is possible to obtain very high values of specific gravity in accordance with the invention, whereas the maximum value for specific activity of the immobilized glucose isomerase described in Danish patent application No.1592/78 is relatively low and definitely too low for industrial applications.

In a preferred method according to the invention the combination of the two phases is a mixing of the continuous phase and the discontinuous phase, whereafter the mixture subsequently is shaped to the particulate carrier, preferably spheronized. Hereby cheap and easily obtainable carrier particles are obtained.

Prior art methods for production of gelatine spheres or rounded gelatine particles are rather costly and tedious. Reference is made to Danish patent 1 33 380, in which it is described that such gelatine particles are produced by addition of an aqueous solution of gelatine (and enzyme) to n-butanol and separation of the formed, drop like particles. According to the invention it has been found, however, that spheres or rounded particles of a mixture of a binder and an inert material can be manufactured in a much cheaper way, e.g. by means of a Marumerizer (vide e.g. US patent 3 277 520) or by means of a special granulating device (vide e.g. US patent 4 106 991).Furthermore, the inclusion of a multitude of small hard particles imparts very high hardness to the carrier particles, thus substantially improving their flow properties, making them suitable for large scale column operations at very high flow.

In a preferred method according to the invention the shaping is a spheronizing treatment carried out in a Marumerizer according to US patent 3 2 i7 520. In this manner spheres suitable for column operation and with excellent physical properties can be produced.

In a preferred method according to the invention the shaping is a spheronizing treatment carried out in a granulating device as described in US patent 4 106 991. In this manner very cheap spheres with excellent physical properties can be produced.

In a preferred method according to the invention a gellable agent is added to the continuous phase before, during or after mixing with the discontinuous phase, whereafter the thus obtained mass is extruded or dripped into a gelling medium whereby a crosslinking agent can be added at any of these stages. Hereby particles with bery regular shapes can be obtained.

In a preferred method according to the invention the gellable agent is gelatine, alginate, carrageenan or chitosan. Hereby particles with very regular shapes and with excellent cohesion can be obtained.

In a preferred method according to the invention the gelling medium is a solution containing Ca++, Ba++, K+, polyphosphate or ferricyanide, or cold water, or a stream of cold air. Hereby particles with very regular shapes and with excellent cohesion can be obtained.

In a preferred method according to the invention the continuous phase is a water soluble protein, in particular gelatine, soy protein, casein, albumen, zein, gluten, or a protein hydrolysate, or a polysaccharide, in particular agar, alginate or other gums, flour, starch, or chitosan or a synthetic material, carboxy methyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinylalcohol, polyvinylpyrrolidon; or sodium silicate. The continuous phase may be any mixture of the binders usable in the invention, e.g. any mixture of the above indicated materials. With these binders it is possible to generate a carrier with excellent physical properties even in a very small proportion in relation to the discontinuous phase.

In a preferred method according to the invention the discontinuous phase is diatomaceous earth, crushed sand, brick dust, clay, a powder of nylon, insoluble metal oxides or insoluble metal salts, ground silica, aerosil, ground alumina, corundum, ground glass, ground flint, ground quartz, ground granite, aluminum phosphate, kaolin, bentonite, perlite, zeolites, calcium silicate, micro-cell filter-aid, crushed was added, the whole mixture was stirred for 10 minutes at 550C and pumped through a vibrating syringe to produce very fine droplets, which were allowed to drop into a 2% w/v CaCI2 2H2O solution, maintained at 50C. The sphere-like particles thus produced were stirred in the CaCI2 solution for a few minutes, then removed from the solution, washed with de-ionized water and allowed to dry for 2 days at room temperature.The particles were then gently stirred for 1 hour in 200 ml % w/v glutaraldehyde solution at pH 8.5, removed, washed in de-ionized water, and again allowed to dry. In this way spherical and extremely hard and cohesive particles with a diameter of approximately 2 mm and exhibiting excellent flow properties were obtained. The pressure drop was 2 g/cm2. The particles could be treated with citrate or phosphate, to remove the Ca++ and the alginate, if so desired, without any ill effect to the particles.

EXAMPLE 2 The procedure described in example 1 was repeated, except that half the amount of gelatine, that is 5 g, and twice as much Hyflo Celite, that is 20 g, was used, and the particles were not allowed to dry before being treated with glutaraldehyde. Essentially identical particles were thus obtained, with somewhat reduced hardenss, yet with almost the same excellent flow properties. The presure drop was 3 g/cm2.

EXAMPLE 3 The procedure described in example 2 was repeated, except that gelatine Bloom 200 was used, and the amount thereof reduced to 4 g. The concentration of glutaraldehyde was also reduced to 0.2% w/v. Particles with essentially identical properties were obtained in this manner.

EXAMPLE 4 In this example pilot-plant equipment was used. Thus, 0.7 kg cellulose fiber, type Arbocel BC 200, 2.8 kg Clarcel Celite (diatomaceous earth) and 4 kg 20% w/w gelatine Bloom 200 solution, all at 600C, were mixed in a plow-share mixer of the type Lödige FM 130 D, and the thick mass so obtained was extruded by an extruder equipped with a 1.5 mm screen and then spheronized in a Marumerizer, as described in US patent No 3 277 520. The extruder was of the twin screw type model EXDC-1 00, and the spheronizer model was Q-400. The particles thus obtained were dried in a fluid-bed tower (Glatt type WSG 1 5) and sieved, and the fraction 1.2-2.0 mm collected, with the residue recycled.A sample of 10 g was then treated for 3 hours at room temperature with 100 ml % w/v glutaraldehyde solution adjusted to pH 7.0, removed from the solution, and thoroughly washed with de-ionized water. Even without drying the thus obtained particles were extremely hard and cohesive and with excellent flow properties. The pressure drop was 2 g/cm2. In this example the discontinuous phase constituted 65% by weight of the particle.

EXAMPLE 5 In this example the same Marumerizer was used as in example 4, except that no extruder was used. Furthermore, the contents of the discontinuous phase was raised to 94% w/w of the total weight of the carrier. Thus, 1.5 kg Skamol clay particles with a particle size of 0.7-1.0 mm were loaded into the spheronizer, and 0.4 kg Hyflo Celite and 1.15 kg 10% w/w gelatine Bloom 80 solution at 600C were added alternately, so as to avoid formation of lumps. The particles thus obtained were treated as in example 4, whereby very hard particles with very good flow properties were generated. The pressure drop was 5 g/cm2.

EXAMPLE 6 In this example a granulator of a plow share type mixer, Lödige FM 50, equipped with a highspeed chopper as described in US patent 4 106 991 , was loaded with 5.0 kg Clarcel Celite, and 5.95 kg 16% w/w gelatine Bloom 200 was sprayed into it, whereby the treatment time and the rotation speeds of the mixer and the chopper were chosen in such a manner that a particle size of 0.7-1.5 mm was generated. These particles were treated as in example 4, whereby carrier particles with essentially the same properties as in example 4 were generated. The pressure drop was 4 g/cm2.

EXAMPLE 7 1 kg cellulose fiber, 4 kg Clarcel Celite and 10 kg of recirculated material produced according to this example were sprayed with 10.5 kg 10% w/w gelatine Bloom 200 in a plow share Lödige mixer type FM 1 30 D, whereby rounded particles of varying size were generated. During all the previously described operations both ingredients and equipment were maintained at 550C. The rounded particles were dried in a fluid-bed tower and sieved, and the fraction 0.5-0.7 mm comprising 32% was collected. The residue comprising coarser particles which were milled, and fines which were used directly, was recycled, as described in the beginning of this example.

EXAMPLE 8 The same mixer as in example 4, i.e. Lödige FM 130 D, was loaded with 3 kg cellulose fiber, magnesium silicate, talc, asbestos, abraded hornblende, titanium dioxide, stannic oxide, polishing powder, ground zirconium silicate, carbon black, active carbon, bone meal, fly ash or metal fines. The discontinuous phase may be any mixture of the discrete, hard, and inert particles usable in the invention, e.g. any mixture of the above indicated materials. These materials are cheap, and give rise to a carrier with good mechanical properties.

In a preferred method according to the invention the amount of the discontinuous phase is between about 10 and about 98 weight%, in relation to the total weight of the carrier, preferably between 50 and 95 weight%, in relation to the total weight of the carrier. In this manner a good combination of low cost and excellent physical properties of the carrier is obtained.

In a preferred method according to the invention the linear size of a single inert particle in the discontinuous phase, calculated as the diameter of the sphere with the same volume as the single inert particle, is less than 1/5 of the diameter of the carrier particle, preferably less than 1/20 of the diameter of the carrier particle. With such dimensions of the inert particles the shaping can be performed without difficulties.

In a preferred method according to the invention the continuous phase is insolubiiized by being crosslinked by means of a suitable crosslinking agent, preferably glutaraldehyde. In this way the mechanical integrity of the carrier particles is further improved.

In a preferred method according to the invention the shape of the carrier is spherical or rounded, and the average carrier diameter is between 0.1 and 5 mm, preferably between 0.2 and 2 mm, more preferably between 0.2 and 1 mm. Hereby a carrier with a good compromise between flow properties and surface area is provided.

In a preferred method according to the invention the carrier is treated with a solution of the enzyme and with a crosslinking agent. Hereby a splendid adhesion between carrier and enzyme is obtained and thus an immobilized enzyme, which has an extremely good physical stability.

In a preferred method according to the invention the carrier is introduced into a tower as a fluidized mass and the solution of soluble enzyme is introduced into the tower as a spray, whereafter the thus produced mass is removed from the tower and treated with the crosslinking agent. Hereby the immobilized enzyme according to the invention can be produced with a very high loading of the enzyme on the carrier.

In a preferred method according to the invention the carrier is introduced into a tower as a fluidized mass and a mixture of the solution of soluble enzyme and crosslinking agent is introduced into the tower as a spray, whereafter the thus produced mass is removed from the tower. Hereby the immobilized enzyme according to the invention can be produced with a very high loading of the enzyme on the carrier.

In relation to the preferred embodiments indicated in the two previous paragraphs it is to be taken into consideration that the carrier, crosslinking agent, enzyme and further agents, if present, may be brought together in any arbitrary sequence. Such further agents can be for instance granulating aids (e.g.

cellulose fibres or minute, hard and inert particles), soluble materials intended as porosity increasing agents (e.g. NaCI), or other proteins.

In a preferred method according to the invention the enzyme is a glucose insomerase, preferably originating from Bacillus coagulans. It has been found that the thus produced immobilized glucose isomerase can be provided with an excellent activity recovery and with superior characteristics in regard to continuous column operation.

The immobilized enzyme preparation according to the invention can be introduced as a layer in a column, whereafter a solution of a substrate for the enzyme is passed through said layer with a velocity permitting at least part of the substrate to be converted by the enzyme.

The following examples illustrate the method according to the invention. In some of the examples only the production of the carrier is described. For all these examples it is to be understood that the carrier is treated with the same enzyme solution as indicated in example 12 and further treated whereby an immobilized enzyme was produced.

In the following reference is made to different NOVO literature references. Copies of all these references can be obtained by NOVO Industri A/S, Novo All,2880 Bagsvaerd, Denmark.

In some of the examp les a value of the pressure drop (physical strength) during column operation is indicated. This value is determined in accordance with AF 166/2, which is a description of a NOVO laboratory procedure. Some theoretical considerations connected to this pressure drop determination are described in Starch/Stärke 31 (1979) No. 1, page 13-16. For comparison with known commercial products it may be mentioned that the best values of the pressure drop for the immobilized glucose isomerase preparations SWEETZYME is around 10 g/cm2. It appears from the examples that pressure drops with the immobilized enzyme preparations produced by means of the method according to the invention can be as small as 2 g/cm2 and that all values are considerably lower than 10 g/cm2, whereby the technical advantage of the invention is clearly demonstrated.

EXAMPLE 1 10 g gelatine Bloom 260 were dispersed in 60 ml H2O, 33 g of 6% w/v Na-alginate was added, the mixture was heated to 600C to dissolve the gelatine, then 10 g of Hyflo Celite (diatomaceous earth) 10.5 kg Clarcel Celite and 1.5 kg albumen at ambient temperature and sprayed with 15.2 kg water. The treatment time and rotation speeds of the mixer and the chopper was chosen in such manner, that particles of the preferred size were generated.The particles were dried in a fluid bed and a sieve analysis on the dried particles showed the following particle size distribution: > 1000,um 19.7% > > 850 m 35.6% > 707 m 58.8% > 600 cm 78.2% > 500 cm 92.1% < 420,um 1.1% EXAMPLE 9 The procedure in example 8 was repeated, except that 1.5 kg isoelectric soluble soy protein hydrolyzate was substituted for the albumen and that the amount of water was 14.8 kg. Sieve analysis on the dried particles showed the following particle size distribution: > 1000 m 24.2% > 850 Mm 43.2% > > 707 m 66.1% > 600,um 84.4% > > 500 m 95.2% < 420,um 1.0% EXAMPLE 10 The same mixer as in example 6, i.e.Lödige FM 50, was loaded with 11.3 kg ALTOS and 3.0 kg cellulose fiber and sprayed with 650 g Gelatine Bloom 200 in 4.55 kg water. The temperature was kept at 55 C. The particles formed by the rotation of the mixer and the chopper was dried in a fluid bed.

Sieve analysis showed the following particle size distribution: > 1000 tim 8.5% > > 850 m 16.1% > 707 tim 31.2% > 600 yam 46.2% > > 500 m 65.3% < < 420 m 15.6% EXAMPLE 11 The Lödige FM 130 D mixer was loaded with 2.1 kg cellulose fiber, 8.4 kg Clarcel Celite and 4.5 kg sodium chloride and sprayed with 11.0 kg 10% (w/w) gelatine Bloom 80. The temperature was 55 C. The particles were formed and dried.Sieve analysis showed the following particle size distribution: > 1000 tim 14.2% > > 850 m 23.7% > > 707 m 40.1% > 600 tim 59.1% > 500 tim 79.1% < 420 tim 5.8% EXAMPLE 12 This example describes a method for production of an immobilized enzyme preparation according to the invention.Thus, 4.5 kg of carrier particles produced as in example 4 treated with glutaraldehyde, washed and dried were fluidized in a pilot-plant fluid bed apparatus (Glatt type WSG 1 5), and 9.3 kg solution of 19% w/w partly purified glucose isomerase from Bacillus coagulans NRRL 5650 (activity 3240 units/g dry matter, the activity unit being defined in NOVO analyseforskrift AF 189/1), was sprayed onto the particles at 5O-550C, and the particles were allowed to dry. The product thus obtained contained 28% by weight of partly purified glucose isomerase, with 85% enzyme activity recovery. 20 g of these particles were then treated in 500 ml solution containing 0.06 M NaH2PO4.2H2O, 1.4 M Na2SO4, and 0.1% w/v glutaraldehyde, adjusted to pH 7.0 with 1 N NaOH.After 1 hour at room temperature the particles were removed and washed thoroughly with 0.06 M sodium phosphate of pH 7.0 and then superficially with de-ionized water, and part of them were allowed to dry. Both the wet and the dried particles showed the same excellent flow properties as the original carrier, and no leakage of activity could be detected. However, in the wet particles the enzyme activity recovery was 70%, while in the dried particles it was only* 48%.

EXAMPLE 13 20 g of dried carrier particles produced as in example 4 were fluidized in a Lab type fluid bed.

45.8 g of 11.0% w/w homogenized cell sludge (fermented as indicated in example 1 of Danish patent application No. 5190/79, sludge produced as indicated in example 4 of Danish patent application No.

5190/79) containing 80.1 U/g of thermophilic lactase from Bacillus sp. NRRL B-1 1.229 were sprayed onto the carrier particles at 30--400C, and the coated particles were allowed to dry The lactase activity unit as defined as that amount of lactase, which will split 1 timol of lactose/minute under the following reaction conditions: Substrate concentration = 10% lactose, temperature = 600 C, pH = 6.5 and reaction time = 30 minutes. The enzyme activity recovery was 79.8%. 10 g coated spheres were then treated in 250 ml solution containing 0.06 M Na2HPO4, 1.4MNa2SO4and0.1%w/v glutaraldehyde at pH 7.5. After 1 hour at room temperature the particles were removed and washed thoroughly with 0.06 M K2HPO4 at pH = 7.5.The enzyme activity recovery in regard to the crosslinking step was 17.2%.

EXAMPLE 14 24 g of dried carrier particles produced as in example 4 were soaked in 20.2 g solution of a 39.6% w/w partly purified amyloglucosidase from A. niger produced by ultrafiltration of the commercial product AMG 200 L (described in NOVO brochure AMG, B 020 g - GB 2500 July 1982) in order to remove low molecular constituents to a dry matter content of 39.6% w/w (activity 2610 IAG/g, the activity unit being defined in NOVO Analyseforskrift AF 159/2). Vacuum was applied for 1 hour. The product thus obtained contained 25% by weight of enzyme dry matter with 77.9% enzyme activity recovery.

20 g particles with 71.8% dry matter were then treated in 1 600 ml of a solution of 1% w/v NaH2PO4, 20% w/v Na2SO4 and 0.2% glutaraldehyde at pH = 4.5. After 1 hour the particles were removed by filtration and washed with 1% NaH2PO4 at pH = 4.5.

The enzyme activity recovery in regard to the cross-linking step was 55.1%.

EXAMPLE 1 5 40 g carrier particles prepared as indicated in example 4 and with a dry substance content of 98.8% and 24 g vacuum evaporated partially purified Bacillus coagulans glucose isomerase (NRRL 5650) concentrate with 5% glucose and 8% sodium sulphate added (dry substance 41.8%) was mixed and the liquid was allowed to displace the air in the pores of the particles by vacuum treatment. Weight after mixing was 63.22 g. Dry substance was 79.2%.

1 8 g portions of this preparation (--14 g dry substance) were treated for 1 hour at room temperature with 375 ml of a solution containing in all cases 22% sodium sulphate, 5% glucose, and 1% sodium phosphate, adjusted to pH 7.5, and furthermore either 0.1, or 0.2 or 0.3% glutaraldehyde.

After this treatment the preparations were washed five times with approx. 1 50 ml 1% sodium phosphate, pH 7.5.

The enzyme activity was determined according to AF 189/1 after draining of the liquid from the particles. Also dry substance was determined on the draining particles.

% dry Immob.

substance U/g wet U/g dry Yield, % yield, % Enzyme concentrate 41.8 1415 3385 - Enzyme concentrate + carrier 79.2 463 585 86 lmmob.with0.1%GA 32.5 158 486 72 83 Immob. with 0.2% GA 38.9 141 362 53 62 Immob. with 0.3% GA - 117 333 49 57 Portions equivalent to 5 g dry substance were tested for pressure drop.

Glutaraldehyde concentration at Pressure drop immobilization 25 hours 50 hours 0.1% 3 5 0.2% 1 3 0.3% 2 3 EXAMPLE 16 The same mixer as in Example 4, i.e. Lödige FM 130 D, was loaded with 3 kg cellulose fiber and 13.5 kg Clarcel Celite was sprayed with 1 5 kg 10% w/v polyethyleneimine PEI 1 5 T, Taihei Sangyo Kaisha Ltd., at ambient temperature, thereafter with 3.0 kg water and finally with 2 kg 50% w/v glutaraldehyde. The treatment time and rotation speeds of the mixer and the chopper was chosen in such manner, that particles of the preferred size were generated. The particles were dried in a fluid bed.

EXAMPLE 17 The same mixer as in Example 4, i.e. Lödige FM 130 D, was loaded with 3 kg cellulose fiber and 12.0 kg activated carbon Pecactif FGV (from the company Peca S.A., Levallois, Cedex, France) and sprayed with 20.0 kg 10% w/w gelatine Bloom 200 solution and finally with 4.5 kg water. The treatment time and rotation speeds of the mixer and the chopper was chosen in such manner, that particles of the preferred size were generated. The particles were dried in a fluid bed.

The utility of an immobilized enzyme produce produced according to the invention appears from the following application experiment. An immobilized glucose isomerase product was produced generally as in Example 12. The product contained 32.6% by weight of partially purified glucose isomerase and the crosslinking was performed as in Exmaple 1 2.

After the washing with 1% w/v sodium phosphate (pH = 7.0) 27.6 g moist particles, corresponding to 10 g particle dry matter, were filled into a water jacketed column with a diameter of 1.5 cm. The column was maintained at 650C and a 45% w/w glucose syrup at pH 7.8 (adjusted with Na2CO3) was continuously pumped through the enzyme bed at a rate that would allow a 40 42% conversion of glucose to fructose. The initial activity was 538 IGIC/g prep. dry matter (the activity being calculated according to NOVO Analyseforskrift AF 147/6) and the activity halflife was determined to 450 hours.

The outlet pH was 7.4-7.5.

For comparison it can be mentioned, that the widely used commercial immobilized glucose isomerase Sweetzyme has an activity of 225-300 lGlC/g and a similar activity half life, and in order to obtain an outlet pH of 7.4-7.5 with Sweetzyme at the same conditions as above indicated, i.e.

650C and 45% w/w glucose syrup, an inlet pH of 8.2 is necessary.

The word "Marumizer" is a Trade Mark, as are the words "Sweetzyme", "Bloom" and "Arbocel".

Claims (26)

1. Method for production of a particulate, immobilized enzyme preparation comprising a carrier, onto the surface of which the enzyme is attached, wherein the carrier is formed by combination of two phases, i.e. a) a continuous phase of a water soluble binder at least partially dissolved or dispersed in an aqueous medium and b) a discontinuous phase of a multitude of discrete, hard and inert particles, the size of which is small enough not to interfere with the shaping of the carrier, and by rendering the binder insoluble in the medium, in which the enzyme eventually will be utilized, if necessary, and wherein the carrier is contacted with an enzyme which is attached to the surface of the carrier.

2. Method according to claim 1 , wherein the combination of the two components is a mixing of the continuous phase and the discontinuous phase, and wherein the mixture subsequently is shaped to the particulate carrier, preferably spheronized.

3. Method according to claim 2, wherein the shaping is a spheronizing treatment carried out in a Marumerizer according to US patent 3 277 520.

4. Method according to claim 2, wherein the shaping is a spheronizing treatment carried out in a granulating device as described in US patent 4 106 991.

5. Method according to claim 2, wherein a gellable agent is added to the continuous phase before, during or after the mixing with the discontinuous phase, whereafter the thus obtained mass is extruded or dripped into a gelling medium, whereby a crosslinking agent can be added at any of these stages.

6. Method according to claim 5, wherein the gellable agent is gelatine, alginate, carrageenan or chitosan.

7. Method according to claim 5, wherein the gelling medium is a solution containing Ca++, Ba++, K+, polyphosphate or ferricyanide, or cold water, or a stream of cold air.

8. Method according to claims 1-7, wherein the continuous phase is a protein, in particular gelatine, soy protein, casein, albumen, zein, gluten, or a protein hydrolysate, or a polysaccharide, in particular agar, alginate or other gums, flour, starch, or chitosan or a synthetic material, such as carboxy methyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinylalcohol, polyvinylpyrrolidon; or sodium silicate.

9. Method according to claims 1 to 8, wherein the discontinuous phase is a diatomaceous earth, crushed sand, brick dust, clay, a powder of nylon, insoluble metal oxides or insoluble metal salts, ground silica, aerosil, ground alumina, corundum, ground glass, ground flint, ground quartz, ground granite, aluminum phosphate, kaolin, bentonite, perlite, zeolites, calcium silicate, micro-cell filter-aid, crushed magnesium silicate, talc, asbestos, abraded hornblende, titanium dioxide, stannic oxide, polishing powder, ground zirconium silicate, carbon black, active carbon, bone meal, fly ash or metal fines.

10. Method according to claims 1 to 9, wherein the amount of the discontinuous phase is between about 10 and about 98 weight%, in relation to the total weight of the carrier, preferably between 50 and 95 weight%, in relation to the total weight of the carrier.

11. Method according to claims 1 to 10, wherein the linear size of a single inert particle in the discontinuous phase, calculated as the diameter of the sphere with the same volume as the single inert particle, is less than 1/5 of the diameter of the carrier particle.

12. Method according to claims 1 to 11, wherein the continuous phase is insolubilized by being crosslinked by means of a suitable crosslinking agent, preferably glutaraldehyde.

1 3. Method according to claims 1 to 12, wherein the shape of the carrier is spherical or rounded, and wherein the average carrier diameter is between 0.1 and 5 mm, preferably between 0.2 and 2 mm, more preferably between 0.2 and 1 mm.

14. Method according to claims 1 to 13, wherein the carrier is treated with a solution of the enzyme and with a crosslinking agent.

15. Method according to claim 14, wherein the carrier is introduced into a tower as a fluidized mass and wherein the solution of soluble enzyme is introduced into the tower as a spray, whereafter the thus produced mass is removed from the tower and treated with the crosslinking agent.

16. Method according to Claim 14, wherein the carrier is introduced into a tower as a fluidized mass and a mixture of the solution of soluble enzyme and crosslinking agent is introduced into the tower as a spray, whereafter the thus produced mass is removed from the tower.

17. Method according to Claims 1 to 16, wherein the enzyme is a glucose isomerase.

1 8. Method according to Claim 1 7, wherein the enzyme is a glucose isomerase originating from Bacillus coagulans.

1 9. An immobilized enzyme granule adapted for fixed bed or fluidized bed continuous enzymatic reactions, which granule comprises a continuous phase hydrophilic binder material and a discontinuous phase particulate inert filler material, the binder and filler being insoluble in the enzymatic reaction medium, the granule also comprising enzyme immobilized to the binder material at the surface of said granule.

20. An immobilized enzyme granule adapted for employment in a aqueous medium, which granule comprises a continuous phase of hydrophilic binder material and a discontinuous phase of particulate inert filler material, the binder and filler being water insoluble, and enzyme crosslinked to the binder material at the surface of said granule.

21. An immobilized enzyme granule according to Claim 20, which further comprises a protein material binder water insolubilized by reaction with glutaraldehyde and enzyme crosslinked to the binder material by reaction with glutaraldehyde.

22. An immobilized enzyme granule according to Claim 21, which further comprises a gelatine binder and diatomaceous earth and cellulose fiber filler materials, the enzyme being glucose isomerase.

23. A method for the production of a particulate, immobilized enzyme preparation, substantially as described in any one of the foregoing Examples.

24. An immobilized enzyme preparation whenever produced by the method of any one of Claims 1 to 18 and 23.

25. An immobilized enzyme granule, substantially as described in any one of the foregoing Examples.

26. Any novel feature or combination of features described herein.