GB2129809A

GB2129809A - Method for production of an immobilized enzyme preparation by means of a crosslinking agent

Info

Publication number: GB2129809A
Application number: GB08326640A
Authority: GB
Inventors: Shmuel Amotz
Original assignee: Novo Industri AS
Current assignee: Novo Nordisk AS
Priority date: 1982-10-06
Filing date: 1983-10-05
Publication date: 1984-05-23
Also published as: GB2128620A; GB8326641D0; GB2128620B; GB8326640D0; GB2129809B

Abstract

The following constituents are brought together in an aqueous medium: a) an enzyme preparation b) a crosslinking agent and c) a water-soluble salt in relatively high concentration. The salt renders the enzyme insoluble in the medium, but nevertheless the enzyme is fully available for the crosslinking agent. High enzyme recoveries can be obtained.

Description

1 GB 2 129 809A 1

SPECIFICATION

Method for production of an immobilized enzyme preparation by means of a crosslinking agent Enzymes immobilized by means of a crosslinking agent are some of the most wide-spread forms of immobilized enzymes. To produce such immobilized enzymes, a number of methods have been developed. In one such method, a carrier is first activated by the crosslinking agent, and then treated with the enzyme, which thus becomes firmly attached to the carrier. Such an activation may include an impregnation of the carrier with a polyamine and a subsequent 10 treatment with an excess of glutaraldehyde, as described in U.S. patent 4, 292,199, or in Biotechnology and Bioengineering, 22, pp. 271-287, 1980. Another activation of this kind may include a coating of a carrier with adsorption-promoting insoluble polymer, subsequent adsorption of the enzyme on the polymer, and a further immobilization by crosslinking in situ, as described in U.S. patent 3,705,084. Another activation of this kind is described in U.S. patent 15 4,069,106: A keratin-containing carrier is activated by reduction of the keratin and crosslinking of the enzyme thereto via S-S groups. Another activation of this kind is described in U.S.

patent 3,802,909: Glass is fractured in the presence of a protein, thus providing freshly produced active sites to bind the protein. Another activation of this kind is described in U.S.

patent 3,519,538: Glass is treated with a sequence of chemicals to produce the desired active 20 sites. Another method of production of an immobilized enzyme is described in Biochemical and Biophysical Research Communications Vol. 36, pp. 235-242, 1969: The enzyme is adsorbed on colloidal silica, without previous activation, and then further fixed by crosslinking with glutaraldehyde.

All the above mentioned methods are characterized by a thin layer of enzyme molecules being 25 directly attached to the active sites on a carrier and thus the amount of enzyme immobilized is determined by the number of the active sites.

A different approach altogether is to have a much thicker layer attached to the surface of the carrier whereby only a small fraction of the enzyme molecules is in direct contact with the carrier. In this way it is not the surface area of the carrier which determines the amount of enzyme immobilized, and this approach makes it possible to optimize the amount of bound enzyme, depending upon the process parameters during ultimate use. This approach, however, is more difficult to accomplish, as the relatively large amount of enzyme makes it difficult to maintain it in place during the immobilization. Therefore comparatively little has been published by way of this approach, despite its obvious advantages. One suggestion, described in Canadian 35 patents Nos. 1011671 and 1011672, is the use of an aqueous solution of a water-soluble organic solvent as the crosslinking medium, the organic solvent concentration being kept high enough to keep the enzyme insoluble, yet low enough not to interfere too much with the crosslinking reaction. The main draw-back in relation to this method is the requirement of relatively large amounts of organic solvents accompanied by explosion risks necessitating 40 elaborate safety provisions. Also, the product obtained in this way is not entirely satisfactory in terms of physical stability and activity recovery, probably due to the relatively high concentration of the organic solvent necessary to keep the enzyme out of solution during the immobilization.

Another method which pertains to this problem, is suggested in U.S. patent No. 4,116,771: In a limited volume of water the enzyme is treated with glutaraidehyde and an inert protein before 45 being quickly added to the carrier, then allowed to gel, then granulated and finally dried. The main draw-back of this method is the high concentration of the crosslinking agent, which is a consequence of the limited amount of water. Many enzymes are very sensitive to high concentrations of crosslinking agents, either because they are inactivated, or because they are rendered inaccessible during later use due to the extensive crosslinking, or both. This illustrates 50 the main difficulty when applying a large amount of enzyme to a carrier: in order to obtain a sufficiently low concentration of the crosslinking agent one needs relatively large amounts of medium, which makes it impossible to prevent the enzyme from dissolving in the medium, before the crosslinking becomes effective, whereas high concentration of crosslinking agent, which can keep the enzyme from dissolving, are undesirable, as explained above.

A completely different approach to the problem is avoiding the aqueous medium altogether, and use a gaseous phase instead; this is described in The Journal of Society of Chemical Industry, Vol 18, pp. 16-20 (1899), for the production of insoluble gelatine fibers, and in Japanese patent J 57002683 for immobilizing enzymes. However, this method would require a very elaborate ventilating system, and would, furthermore, require special means for preventing 60 the formation of insoluble deposits inside the ventilating, system.

Thus, an object of the invention is to provide a method for production of an immobilized enzyme preparation by means of a crosslinking agent which method should be simple to carry out, e.g. without the need of elaborate safety provisions, and by means of which the immobilized enzyme can be produced with-a high enzyme activity recovery and with good 65 2 GB 2 129 809A 2 physical stability.

Now, according to the invention it has been found that it is possible to achieve the above object by use of certain salts in a specified minimum concentration. Even though the prior art previously cited is related to immobilized enzyme preparations on carriers only, the invention is not restricted to such immobilized enzyme preparations but encompasses immobilized enzyme 5 preparations without a carrier as well.

Thus, the invention comprises a method for production of an immobilized enzyme preparation by means of a crosslinking agent, wherein the following components are brought together in.an aqueous medium:

a) an enzyme preparation in a solid or dissolved state, b) a crosslinking agent, and c) a water soluble salt which does not react with the crosslinking agent or inactivate the enzyme, in a concentration sufficient to prevent any essential part of the enzyme from dissolving in or mixing with the salt solution, while the crosslinking reaction is taking place, whereafter the immobilized enzyme is recovered.

In case the finished immobilized enzyme contains an excess of crosslinking agent, the latter can be removed by washing.

It is to be understood that the enzyme preparation can be in a solid or dissolved state; furthermore the enzyme preparation can be of high purity, or it can contain inert additives (e.g.

fillers, reinforcing agents, binders or granulating agents). Also, it is to be understood that the 20 sequence, in which the ingredients are brought together, is arbitrary except that in case the enzyme preparation and crosslinking agent are brought together before the addition of salt the enzyme yield will decrease if the salt addition is delayed unreasonably.

The above indicated category of salts (whereby a specific category of salts may be assigned to each combination of a specified enzyme and a specified crosslinking agent) usually include 25 cheap salts. It is to be understood that one or more enzyme preparations, one or more crosslinking agents and one or more salts may be used in the method according to the invention.

In a preferred embodiment of the method according to the invention the enzyme preparation comprises a carrier. Although the method according to the invention can be carried out without 30 a carrier (e.g. vide example 15), it is usually preferred to use a carrier, mainly because of the possibility offered by the carrier for producing a particle with superior flow properties useful for packed bed operations. No matter whether the enzyme is in a solid state or dissolved, it is in this case closely associated with the carrier: in a solid state the enzyme may be a coating layer on the carrier and in a dissolved state the enzyme solution may impregnate the (porous) carrier. 35 In a preferred embodiment of the method according to the invention the enzyme preparation is a carrier coated with a layer of solid enzyme. Hereby it is made possible to produce an enzyme preparation with any wanted enzyme loading inside wide limits by variation of the thickness of the enzyme layer.

In a preferred embodiment of the method according to the invention the enzyme preparation 40 is a porous carrier impregnated with an enzyme solution. Hereby a very simple method for production of the immobilized enzyme preparation according to the invention is provided.

In a preferred embodiment of the method according to the invention the crosslinking agent is glutaraidehyde. Glutaraidehyde is relatively cheap and not objected to by health authorities.

Also, in relation to many enzymes glutaraldehyde is a very effective and yet mild crosslinking 45 agent.

In a preferred embodiment of the method according to the invention the glutaraidehyde concentration in the aqueous medium is between 0.001 and 5% (w/v) preferably from 0.005 to 1 % (w/v). As it appears from the examples later in this specification the enzyme activity recovery depends on the concentration of glutaraldehyde, and usually the glutaraidehyde concentration corresponding to a.maximum enzyme activity recovery is to be found within the above indicated intervals. The percentage of glutaraidehyde (w/v) is calculated according to the formula w -1 weight of glutaraidehyde, g 55 volume of (salt solution and glutaraldehyde), mi X 100 In a preferred embodiment of the method according to the invention the salt is chosen from the group comprising a sulfate, phosphate, citrate, bicarbonate, carbonate, fluoride, acetate, 60 tartrate, polysulfate, polyphosphate, ferrocyanide, phenolsulfonate, sorbate, ethylsulfate, chlo ride, nitrate and succinate of secondary, tertiary or quaternary ammonium or one of the alkali metals, in particular sodium sulfate, sodium phosphate, potassium phosphate, and potassium citrate. Generally these salts are cheap and recoverable and open up the possibility for production of an immobilized enzyme preparation with a high enzyme activity recovery.

3 GB 2 129 809A 3 In a preferred embodiment of the method according to the invention the salt concentration is between 0. 1 M and saturation, in particular from about 0. 5 M to about 3 M. As appears from the examples later in this specification it is possible to choose a glutaraldehyde concentration and a salt molarity inside the above indicated intervals, which results in a very high enzyme 5 activity recovery.

In a preferred embodiment of the method according to the invention the enzyme is chosen from the group consisting of glucose isomerase, amylases, in particular amyloglucosidase, pullulanase, lactase, pectinases, naringinase, penicillin acylases, inulinases, lipases, and proteases.

Thus, the use of the above indicated salts, even in lower concentrations than needed for 10 precipitation of the enzymes, prevents the enzyme, for all practical purposes, from being dissolved in the crosslinking medium (salt solution), while keeping it fully accessible to the crosslinking agent, if the enzyme is in a solid state, or prevents the enzyme from being mixed with the salt solution, if the enzyme is dissolved. In this way, an optimum concentration of the crosslinking agent can be used which would cause only minimal damage to the enzyme, and yet 15 impart sufficiently good physical stability. Thus, it has been found, that to reduce the crosslinking agent concentration, one has to increase the salt concentration in order to maintain the enzyme activity recovery at the optimal level. According to the invention the use of salts which react with either the enzyme or the crosslinking agent such as silver or mercury salts, which may inactivate the enzyme, or ammonium salts or primary amine salts or sulfite salts, 20 which tend to react with a variety of crosslinking agents should be avoided. The salts which may be used for the purpose of this invention vary greatly with regard to their effectiveness, and, furthermore, this effectiveness would tend -to vary from one enzyme to another. Some broad guide-lines, however, with regard to the choice of salt, can be stated as follows: It is advisable to choose the more soluble salt when two salts are otherwise identical in regard to properties. 25 Thus Na2SO4 is usually preferred to K2S04, because of the much higher solubility. It has also been found that, in general, salts of multivalent anions, such as sulfates, phosphates, carbonates, citrates and the like, are more effective than the monovalent anions, such as chlorides and nitrates. The reverse, however, is usually true for the cations: The monovalent cations, such as Na+, K+ or tetra methylam mon iu m, are more effective than the multivalent 30 ones such as Mg + + or Ca + +. The preferred salts are, therefore, the alkali metal salts of sulfates, phosphates and citrates, and in particular sodium sulfate, sodium phosphate, potassium phosphate, potassium citrate and tetramethylammonium sulfate.

The salt concentration is usually kept to a minimum. This minimum is dependent on the one hand on the enzyme in question, as different enzymes often require different concentrations, and 35 on the other hand, on the crosslinking agent concentration: The higher the latter, the less salt is required, and vice versa. The crosslinking agent concentration is also kept to a minimum, as most enzymes are sensitive to crosslinking agents. The concentration, though, should be high enough to ensure an effective immobilisation. Care should be exercised, however, when establishing the optimal conditions for the crosslinking reaction at very high salt concentration, 40 especially with effective salts. Thus, it has been found that at high concentrations of Na,SO,, or potassium phosphate, for example, the activity yields are considerably reduced in comparison to those at moderate concentrations. This is presumably due to very tight cross-linking which render the enzyme molecules partially inaccessible.

Yet another advantage of using these salts in the crosslinking medium is the prevention of the 45 enzyme containing particles from aggregating during the immobilization reaction, if the enzyme is present in a solid state. Such aggregation is undesirable as it results in lower efficiencies and inferior flow properties during use. Also, in this regard salts differ widely.

The method according to the invention may be carried out in a number of steps, the order of which is not critical. Thus e.g. in a preferred embodiment of the invention, a carrier is first treated with an enzyme solution, dried, and then treated in a solution containing a crosslinking agent and salt, washed and optionally dried. In a variation of the same process, a part of the total amount of salt may be contained in the enzyme solution. In yet another variation the enzyme is first treated with the crosslinking agent and immediately afterwards with the salt. For certain purposes, for example when it is desired that the immobilized enzyme be light and fibrous the amount of the carrier may be very small, or the carrier may be avoided altogether, and the enzyme treated in a coagulating bath with the crosslinking agent and the salt. Thus the enzyme may be dissolved in a solution which is coagulated in a salt bath, the crosslinking agent being added to the enzyme solution or to the bath, either before or after coagulation. Also, the salt may be added as a dry powder, instead of a solution, when it is desired to limit the amount 60 of water. Thus, the order in which the different components are brought together/or their state, that is, whether in a solution or dry, is not critical for the method according to the invention.

The pH, temperature and duration of the crosslinking reaction may have a profound effect on the recovery of activity, depending on the enzyme and inert additives, if present. In general it has been found that a pH range from about 4 to about 9 is suitable. The temperature range 65 4 GB 2 129 809A suitable in most cases is from about 1 5'C to about 30C, though higher temperatures may be used with advantage in certain cases, e.g. when it is desired to shorter the duration of the crosslinking reaction, and lower temperatures may be used with advantage in case of very temperature sensitive enzymes. The duration of the crosslinking reaction may vary widely, from a few minutes to a few days depending on the type and concentration of the crosslinking agent, 5 the type and concentration of the salt, the enzyme, the pH and the temperature, and should therefore be determined in each individual case. For glutaraidehyde and most enzymes, however, the range from about 10 minutes to several hours at room temperature appears to be suitable.

In the following reference is made to different NOVO literature references. Copies of all these 10 references can be obtained from NOVO Industri A/S, Novo Alle, 2880 Bagsveerd, Denmark.

The method according to the invention will be illustrated by the following examples.

In the following part of the specification comprising the examples values of the pressure drop (physical strength) during column operation are indicated. This value is determined in accordance with AF 166/2, which is a description of a NOVO laboratory procedure. Some theoretical 15 considerations connected to this pressure drop determination are described in Starch/Stdrke 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 20 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

Portions of 20 9 of dry carrier particles coated with partially purified glucose isomerase 25 preparation to the extent of 28% w/w and produced according to the method described in example 8 in copending Danish application No. 4430/82 were suspended and gently stirred at room temperature in 500 mi solution containing 0.06 M sodium phosphate, 1. 4 M Na2SO, and different amounts of glutaraidehyde, adjusted to pH 7.0. After 1 hr, the particles were removed and suspended for 1 hr in 0.06 M sodium phosphate solution adjusted to pH 7.0. This washing 30 was repeated 3 times, and then the particles were left overnight in the phosphate solution, whereafter their activity was determined according to NOVO analyseforskrift AF 189/1. The results are presented in Fig. 1, which is a graph, where percentage of glutaraidehyde is plotted against enzyme activity recovery expressed in enzyme units/9, and which shows the sensitivity of the enzyme to glutaraidehyde.

Example 2 (comparison) Particles were prepared as in example 1, only no salt was added, apart from a minimal amount of phosphate to serve as a buffer (0.06 M sodium phosphate, pH 6.5). The percentage of glutaraldehyde was plotted against the enzyme activity recovery in units/g, vide Fig. 2, which 40 shows that the crosslinking agent has two opposite effects: On the one hand it increases the yield by preventing the enzyme from dissolving; on the other hand it reduces the yield by inactivating the enzyme. The optimum is established, in this case, at around 1 % glutaraldehyde.

From a comparison between Fig. 1 and Fig. 2 it appears that the optimum of the glutaraldehyde concentration is approximately 40 times as high as with 1.4 M sodium sulfate, and that the 45 yield in this case is only about 60% of the yield with the salt.

Example 3

Particles were prepared as in example 1, only in this case potassium phosphate was used as the salt, and the salt concentration was varied while keeping the glutaraldehyde concentration 50 constant on three levels an the pH at 6.5. The percentage of potassium phosphate was plotted against the enzyme activity recovery in units/g, vide Fig. 3, from which the beneficial effect of increasing the salt concentration clearly appears, especially at a low glutaraidehyde concentra tion.

Example 4

The experiment in example 3 was repeated, only here sodium sulfate was used instead of potassium phosphate. The molarity of Na2SO4 was plotted against the enzyme activity recovery in units/g vide Fig. 4, from which the beneficial effect of the salt clearly appears. As it also appears from Fig. 4, an optimum for the salt effect exists, beyond which the activity recovery 60 decreases, whereby this optimum depends on the glutaraldehyde concentration.

Example 5

The experiment described in example 3 is repeated, only the salt concentration was increased.

The molarity of potassium phosphate was plotted against the enzyme activity recovery in 65 GB2129809A 5 units/g, vide Fig. 5. A comparison between Fig. 4 and 5 shows that Na2SO, and potassium phosphate behave similarly.

Example 6

The experiment described in example 1 was repeated, only potassium citrate, pH 7.0, was 5 used instead of phosphate and sulfate in the crosslinking medium. In Fig. 6 the glutaraidehyde concentration is plotted against the activity in enzyme units/g, and the results clearly resemble those in example 1.

1 Example 7 g of dried carrier particles produced as in example 4 in copending Danish application No. 4430/82 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. 5 190/79) containing 80.1 U/g of thermophilic lactase from Bacillus sp. NRRL B-1 1.229 were sprayed onto the carrier 15 particles at 30-40C, and the coated particled were allowed to dry. The lactase activity unit is defined as that amount of lactase, which will split 1 jumol of lactose/minute under the following reaction conditions: Substrate concentration = 10% lactose, temperature = 60C, 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 Na2HPO41 1.4 M Na2SO4 and 0. 1 % 20 w/v glutaraidehyde 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 activity recovery in regard to the crosslinking step was 17.2%.

Example 8

24 g of dried carrier particles produced as in example 4 in copending Danish application No.

4430/82 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 NOVO Enzymes AMG, B 020 g-GB) 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 partially purified amyloglucosidase dry matter with 77.9% enzyme activity recovery.

g particles with 71.8% dry matter were then treated in 1600 ml of a solution of 0.06 M NaH2PO4, 1.4 M Na2SO, and 0.2% glutaraldehyde at pH = 4.5. After 1 hour the particles were 35 removed by filtration and washed with 0.06 M NaH2P04 at pH = 4.5.

The enzyme activity recovery in regard to the crosslinking step was 55.1 %.

Example 9

40 g carrier particles prepared as indicated in example 4 in copending Danish application No. 40 4430/82 and with a dry substance content of 98.8% and 24 g vacuum evaporated partially purified Bacillus coagulans glucose isomerase 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%.

18 9 portions of this preparation ( 14 g dry substance) were treated for 1 hour at room temperature with 375 mi of a solution containing in all cases 1.5 M sodium sulphate, 5% glucose, and 0.06 M sodium phosphate, adjusted to pH 7.5, and furthermore either 0.1 or 0.2 or 0.3% glutaraldehyde.

After this treatment the portions were washed five times with approx. 150 m] 1 % sodium 50 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 drained particles.

6 GB2129809A 6 % dry Immob.

substan- U/g U/g Yield, yield, ces wet dry % % 5 Enzyme concentrate 41.8 1415 3385 Enzyme concentrate - + carrier 79.2 463 585 86 - Immob. with 0. 1 % GA 32.5 158 486 72 83 10 Immob. with 0.2% GA 38.9 141 362 53 62 Immob. with 0.3% GA - 117 333 49 57 Portions equivalent to 5 9 dry substance were tested for pressure drop.

Glutaraldehyde concentration at Pressure drop immobilization 25 hours 50 hours 20.

Z 0.1% 3 5 0.2% 1 3 0.3% 2 3 Example 10

This example describes a method for producing an immobilized enzyme product based on activated carbon from Norit as a carrier.

Thus 20 g of Norit Rox 0.8 activated carbon, type A-3397 were soaked in 33 g solution of 30 39.2% w/w parily purified glucose isomerase from Bacillus coagulans (activity 3950 U/g dry matter, the activity unit being defined in Novo "Ana lyse-forskrift" AF 189/1).

Vacuum was applied for 20 h at 4'C except that vacuum was released 4 times during this period. The product thus obtained contained 35% by weight of partially purified glucose isomerase dry matter with 97% enzyme activity recovery.

98% of the above indicated, aggregate product was then immobilized in 890 ml of a solution of 0.06 M KH2PO41 14 M Na2SO4and 0. 18% glutaraldehyde, adjusted to pH 7.5 with 4 N NaOH. After 2 hours at room temperature with gentle agitation the particles were removed by filtration and washed thoroughly with 0.06 M KH2PO41 pH 8.0.

Recovery of activity in regard to the crosslinking step was 31 %.

Example 11 This example describes a method for producing an immobilized enzyme preparation with a carrier consisting of silica spheres with a diameter of approximately 2 mm. 45 A. 20 g of the silica spheres were fluidzed in a lab type fluid bed, and 40 g solution of 15% 45 w/w partly purified gluocose isomerase from Bacillus coagulans (activity 3306 U/g dry matter, the activity being defined in NOVO analyseforskrift AF 189/1) were sprayed onto the spheres at 25-30C, and the coated spheres were allowed to dry. The product thus obtained contained 23% by weight of partially purified glucose isomerase with 78% recovery of activity. 50 95% of the coated spheres was then treated in 500 mi solution containing 0.06 M K2HP04, 50 1.4 M Na2S04 and 0. 1 % w/v glutaraldehyde, adjusted to pH 7.5 with 4N NaOH; after one hour at room temperature the particles were removed and washed thoroughly with 0.06 M KH2P04 at pH 8.0. Then the activity was determined. The activity recovery in regard to the crosslinking step was 55%. 55 B. 20 9 of the silica spheres were soaked in 15 9 solution of 40% w/w partly purified glucose isomerase from Bacillus coagulans (activity 3270 U/g dry matter) with 0.56 M Na2S04 added. Vacuum was applied for 20 minutes except that vacuum was released 4 times during this period. The product thus obtained contained 20% by weight of partially purified gucose isomerase with 90% enzyme activity recovery. 95% of the coated spheres was then treated as described in part A of this example.

The activity recovery in regard to the crosslinking step was 45%.

In some cases where the enzyme is particularly difficult to crosslink, salts may become.not only advantageous but rather indispensible if the enzyme has to be immobilized in a pure state. A good example is thearnyloglucosidase, which is produced by NOVO (and sold e.g. under the trade mark AMG 200 L, vide the brochure NOVO enzymes AMG, B B 202 9-G13 2500 July 7 GB 2 129 809A 7 1982 which is practically impossible to immobilize with glutaraldehyde when in a pure state: it has been found impossible to insolubilize this amyloglucosidase even at as high a concentration as 50% w/w glutaraldehyde. However, by means of the salts used in the method according to the invention (though at rather high concentrations) it is possible to effectively insolubilize the enzyme even at as low a concentration as 0.05% w/v glutaraldehyde, as demonstrated by the 5 following examples 12-17. The thus obtained preparations, which have excellent filterability and yet can be easily floated, may be used with advantage in the production of low calory beer, for example.

Example 12

Commercial NOVO AMG 200 L diluted to a dry substance content of 30% w/v was mixed with Hyflo Celite and dried, whereby a preparation containing 21 % w/w dry matter originating from the AMG preparation was obtained. 1 g portions of the preparation were then added to a solution containing 2.4 M Na2SO4 0.06 M potassium phosphate at pH 6.5 and glutaraldehyde at different concentrations at 32C, and the entire mixture was left at that temperature for 20 15 hours. The particles were then filtered and rinsed three times with de- ionized water, and the activity yield measured. The results show the familiar pattern of the two opposite effects of glutaraldehyde with an optimum at 0.05%, vide Fig. 7.

Example 13

The same procedure as in example 12 was repeated, only here different concentrations of Na2SO4were used. The activity recovery with this enzyme is extremely sensitive to the amount of salt present, vide Fig. 8.

Example 14

The procedure in example 12 was repeated, except that here an AMG preparation containing only half as much enzyme was used. As a result a shift of the glutaraldehyde optimum to a lower concentration was clearly observed, vide Fig. 9.

Example 15

NOVO AMG 200 L was spray-dried, and the resulting powder was used as a starting material, and the procedure was varied somewhat: 0. 5 g of the AM G powder was added to 100 ml of a solution containing 2.5 M Na2SO41 0.1 M potassium phosphate, pH 6.5, and 1% w/v glutaraldehyde at 32C, and left at that temperature for 1 hour with occasional shaking. The insoluble particles thus formed were then filtered and incubated again in the same medium, only 35 without glutaraldehyde, and at the same temperature for further 20 hours. Then they were filtered and rinsed three times with de-ionized water. These easily-filtering micro-spheres had a diameter of about 10- 100 ILm, and retained 19% of the original activity.

Example 16

A procedure identical to that of example 15 was here followed except that the glutaraldehyde was added to the salt solution after the enzyme powder had been added to it. A similar product with 34% of the original activity was thus produced.

Example 17

Again the procedure in example 15 was followed, except that only 0.4% w/v glutaraidehyde was used and the total incubation was shortened to 3 hours. A product with 37% of the original activity was thus produced.

Example 18

This example illustrates the applicability of the method according to the invention with other crosslinking agents than glutaraldehyde, namely multivalent cations. In this case Sn.... was used as the crosslinking agent. Thus, 0.5 g of the Celite-AMG preparation from example 12, in the state before crosslinking was added slowly and with stirring to a 100 mi solution containing 2 M Na2S041 0,4 M potassium acetate (pH 4.35) and 0. 0086M SnC14, 5H20, and the mixture was left at room temperature for 5 minutes with occasional shaking. The particles thus formed were filtered and dispensed in 100 mi 0.2 M potassium acetate, pH 4.35, and the suspension stirred for 5 minutes. This procedure was repeated 3 times. The end product had an enzyme activity recovery of 43%.

Example 19

In this examle a product was made according to the method described in example 18 only the acetate and the stannic salt concentrations were doubled to 0.8 M and 0.017 M, respectively. A similar product was obtained, the enzyme activity recovery being 65%.

8 GB2129809A 8 Example 20

In this example also the procedure in example 18 was repeated only the acetate concentration was reduced to half, i.e. 0.2 M. Again a similar product was obtained, with an enzyme activity recovery of 48%.

Example 21

In this example the use of yet another cross-linking agent, namely benzoquinone, is described. Thus 0.5 g of a dried Celite-AMG preparation as in example 12 was added to 75 ml of a mixture containing 2.1 M Na2SO4and 0.05 M sodium acetate, and then 5 ml of a solution of 20% w/v of benzoquinone in pure ethanol was added, and the whole mixture was left alone at 10 room temperature for 80 minutes, with occasional stirring. The particles thus formed were then filtered, dispersed in 75 ml 0.1 M potassium acetate buffer, pH 4.4, stirred for 5 minutes and re- filtered. This procedure was repeated three times, and then the activity was determined. The enzyme activity recovery was 14%.

Claims

1. Method for production of an immobilized enzyme preparation by kneans of a crosslinking agent, wherein the following components are brought together in an aqueous medium:

a) an enzyme preparation in a solid or dissolved state b) a crosslinking agent, and c) a water soluble salt which does not react with the crosslinking agent or inactivate the enzyme, in a concentration sufficient to prevent any essential part of the enzyme from dissolving in or mixing with the salt solution while the crosslinking reaction is taking place, whereafter the immobilized enzyme is recovered.

i

2. Method according to claim 1, wherein the enzyme preparation comprises a carrier. 25

3. Method according to claim 2, wherein the enzyme preparation is a carrier coated with a layer of solid enzyme.

4. Method according to claim 2, wherein the enzyme preparation is a porous carrier impregnated with an enzyme solution.

5. Method according to claim 1 to 4, wherein the crosslinking agent is glutaraldehyde. 30

6. Method according to claim 5, wherein the glutaraldehyde concentration in the aqueous medium is between 0.001 and 5% (w/v), preferably from 0.005 to 1 % (w/v).

7. Method according to claims 1 to 6, wherein the salt is chosen from the group comprising a sulfate, phosphate, citrate, bicarbonate, carbonate, fluoride, acetate, tartrate, polysulfate, polyphosphate, ferrocyanide, phenoisuifonate, sorbate, ethyisulfate, chloride, nitrate and succinate of one of the alkali metals, in particular sodium sulfate, sodium phosphate, potassium phosphate, and potassium citrate.

8. Method according to claims 1 to 6, wherein the salt concentration is between 0.2 M and saturation, in particular from about 0.5 M to about 3 M.

9. Method according to claims 1 to 8, wherein the enzyme is chosen from the group 40 consisting of glucose isomerase, arnylases, in particular amyloglucosidase, pullulanase, lactase, pectinases, naringinase, penicillin acylases, inulinases, lipases, and proteases.

10. A method for production of an immobilized enzyme preparation by means of a crosslinking agent, substantially as described in any one of the foregoing Examples.

11. An immobilized enzyme preparation whenever prepared by the method of any one of the 45 preceding claims.

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

Printed for Her Majesty's Stationery Office by Burgess Et Son (Abingdon) Ltd.-1 984. Published at The Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.

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