DK148513B - Process for obtaining an immobilized enzyme preparation, using a cross-linking agent - Google Patents

Description

1 148513

Enzymes inunobilized by a crosslinking agent are some of the most widespread embodiments of ununobilized enzymes. In order to produce such ununobilized enzymes, a number of methods have been developed. In a method of this kind, a carrier is first activated with the cross-linking agent and then treated with the enzyme, which thus becomes firmly attached to the carrier. Such activation may include impregnating the carrier with a polyamine and subsequent treatment with an excess of glutaraldehyde, as described in U.S. Pat.

No. 4,292,199, or in Biotechnology and Bioengineering 22, pp. 271 - 287, 1980. Another activation of this kind may include coating a carrier with adsorption-insoluble polymer, subsequent adsorption of the enzyme onto the polymer, and further immobilization by in situ crosslinking, as described in U.S. Patent No. 3,705,084. Another activation of this kind is described in U.S. Patent No. 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 No. 3,802,909: Glass breaks down in the presence of a protein, thereby producing freshly made, active centers for binding the protein. Another activation of this kind is described in U.S. Patent No. 3,519,538: Glass is treated with chemicals in a specific order to produce the desired active centers. Another method of preparing an immobilized enzyme is described in Biochemical and Biophysical Research Communications, Vol. 36, pages 235 - 242, 1969: The enzyme is adsorbed on colloidal silica, without prior activation, and then further stabilized by cross-linking with glutaraldehyde.

All of the above methods are distinguished by the fact that a thin layer of enzyme molecules is directly associated with the active centers of a carrier and that the amount of immobilized enzyme is thus determined by the number of the active centers.

A completely different problem approach involves providing a much thicker layer attached to the surface of the support, whereby only a small fraction of the enzyme molecules are in direct contact with the support. In this way, it is not the surface area of the carrier that determines the amount of immobilized enzyme and this problem approximation allows one to optimize the amount of bound enzyme, depending on the process parameters of the final application. However, this problem approximation is more difficult to implement because the relatively large amount of enzyme makes it difficult to hold the enzyme in place during immobilization. Therefore, relatively little has been published in connection with this approach, despite its clear advantages. One suggestion described in Canadian Patent Nos. 1,011,671 and 1,011,672 is the use of an aqueous solution of a water-soluble organic solvent as the crosslinking medium, whereby the concentration of the organic solvent is kept at one. value sufficiently high to keep the enzyme in undissolved state, yet small enough not to interfere too much with the crosslinking reaction. The main disadvantage of this method is the requirement of relatively large quantities of organic solvents, which is accompanied by explosion risks which necessitate extensive safety measures. The product thus obtained is also not completely satisfactory in terms of physical stability and yield, probably due to the relatively high concentration of the organic solvent necessary to keep the enzyme out of solution during immobilization. Another method related to this problem is proposed in U.S. Patent No. 4,116,771: The enzyme is treated with glutaraldehyde and an inert protein in a limited volume of water before being rapidly added to the carrier, followed by 3,88513 the mixture gels, and then granulated and finally dried. The major disadvantage of this process is the high concentration of crosslinking agent, which is a consequence of the limited amount of water. Many enzymes are very sensitive to high concentrations of cross-linking agents, either because they become inactivated or because they become inaccessible in later use because of the extended cross-linking, or because of both of these conditions. This illustrates the greatest difficulty that occurs when using a large amount of enzyme relative to a carrier:

To obtain a sufficiently low concentration of the cross-linking agent, relatively large amounts of medium are required which make it impossible to prevent the enzyme from dissolving in the medium before the cross-linking becomes effective, whereas high concentration of the cross-linking agent which can prevent the enzyme from being dissolved. is undesirable, as explained above.

A completely different problem approach is the complete avoidance of the aqueous medium and the use of a gas phase instead; this is disclosed in The Journal of the Society of Chemical Industry, vol. However, this method will require a highly implemented ventilation system, and it will further require special means to prevent the formation of insoluble deposits within the ventilation system.

It is thus the object of the invention to provide a process for preparing an immobilized enzyme preparation by means of a crosslinking agent, which method should be simple to carry out, e.g. without the need for cumbersome safety precautions, and with the help of which the immobilized enzyme can be prepared with high enzyme yield and with good physical stability.

According to the invention, it has now been found that it is possible to fulfill the object of the invention by means of certain salts at a specified minimum concentration. Although the aforementioned prior art relates exclusively to immobilized enzyme preparations on carriers, immobilized enzyme preparations without any carrier can also be produced by the process of the invention.

The method according to the invention, which is of the kind specified in the preamble of claim 1, is thus characterized by the characterizing part of claim 1.

In case the final immobilized enzyme contains an excess of glutaraldehyde, this can be removed by washing.

It is to be understood that the enzyme preparation may be a solid or dissolved enzyme; in addition, the enzyme preparation may be in high purity, or it may contain inert additives (eg fillers, reinforcing agents, binders or granulating agents). It is also to be understood that the order in which the constituents are brought together is arbitrary, with the proviso that the enzyme yield will decrease,. if the salt addition is delayed excessively, in the event that the enzyme preparation and cross-linking agent are brought together before the salt addition.

The above-mentioned category of salts (whereby a specific category of salts can be assigned to each combination of a specific enzyme and a specific crosslinking agent) usually comprises cheap salts. It is to be understood that one or more enzymes, one or more crosslinking agents and one or more salts can be used in the process of the invention.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 2. Although the process according to the invention can be carried out without a carrier, cf. Example 15, it is usually preferable to use a carrier, mainly because of the possibility presented by the carrier with respect to the preparation of a particle having superior flow properties which is advantageous in the use of packed masses. Regardless of whether the enzyme is in solid or dissolved state, in this case it is firmly bound to the carrier; in the solid state, the enzyme may be a coating layer on the support, and in the dissolved state, the enzyme solution may impregnate the (porous) carrier.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 3. This makes it possible to produce an enzyme preparation with any desired enzyme charge within wide limits by varying the thickness of the enzyme layer.

A preferred embodiment of the method according to the invention is characterized by that described in the characterizing part of claim 4. This provides a very simple process for preparing the immobilized enzyme preparation of the invention.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 5. Glutaraldehyde is relatively inexpensive and health authorities do not object to glutaraldehyde. In addition, glutaraldehyde to many enzymes is a very effective yet mild crosslinking agent.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 6. As will be seen from the examples later in this specification, the enzyme activity yield depends on the concentration of glutaraldehyde, and usually the glutaraldehyde concentration corresponding to a maximum enzyme activity yield will be found within the above ranges. The percentage content of glutaraldehyde (w / v) is calculated according to formula 6 148513 weight of glutaraldehyde, g x 100 volume of (saline and glutaraldehyde), ml

A preferred embodiment of the method according to the invention is characterized in that according to the characterized part of claim 7. Usually these salts are inexpensive and recyclable, and they offer the possibility of producing an immobilized enzyme preparation with high enzyme activity yielding tea.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 8. As will be seen from the examples later in this specification, it is possible to select a glutaraldehyde concentration and a salt molarity within the ranges indicated above which correspond to a very high enzyme activity yield.

Thus, even if the salts listed above are used at lower concentrations than necessary for precipitation of the enzymes, the enzyme will in practice prevent the enzyme from dissolving in the cross-linking medium (the saline solution) while being fully accessible to the cross-linking agent. if the enzyme is in solid state or the enzyme will be prevented from mixing with the saline solution if the enzyme is dissolved. In this way one can use one optimal concentration of the cross-linking agent, which will only damage the enzyme to a minimal extent, and yet provide a sufficiently good physical stability. Thus, it has been found that in order to reduce the concentration of cross-linking agent, the salt concentration must be increased in order to keep the enzyme activity yield at the optimum level. According to the invention, the use of salts which react with either the enzyme or crosslinking agent such as silver or mercury salts which can inactivate the enzyme or ammonium salts or primary amine salts or sulfite salts which tend to react with many different crosslinking agents should be avoided. The salts which can be used for the invention vary greatly in their efficiency, and in addition, this efficiency tends to vary from one enzyme to another. However, some general guidelines regarding the choice of salt can be drawn up, namely the following. It is advisable to choose the most soluble salt when two salts are otherwise identical in their properties.

Thus, Na2 SO4 is usually preferred over I2 SO4, because the first mentioned salt has much higher solubility.

It has also been found that it is usually the case that salts of multivalent anions such as sulfates, phosphates, carbonates, citrates and the like are more effective than salts of the monovalent anions such as chlorides and nitrates. The opposite usually applies to the cations: The monovalent cations, such as Na +, K + or tetramethylammonium, are more effective than the multivalent cations such as Mg ++ or Ca ++. Therefore, the preferred salts are the alkali metal salts of sulfates, phosphates and citrates, especially sodium sulfate, sodium phosphate, potassium phosphate, potassium citrate and tetramethyl ammonium sulfate.

The salt concentration is usually kept as low as possible. This minimum depends on one side of the enzyme in question because different enzymes often require different concentrations, and on the other hand the concentration of the cross-linking agent: the higher the latter, the less salt is required, and vice versa. The concentration of crosslinking agent is also kept at a minimum, since most enzymes are sensitive to crosslinking agents. However, the concentration should be high enough to ensure effective immobilisation. However, special care should be taken when establishing the optimal conditions for the crosslinking reaction at very high salt concentrations, especially with effective salts. Thus, it has been found that the activity yields at high concentrations of e.g.

Na SO or potassium phosphate is significantly reduced in weight with the activity yields at moderate concentrations. This is probably due to the very solid crosslinking which makes the enzyme molecules partially inaccessible.

A further advantage of using these salts in the crosslinking medium is that the enzyme-containing particles are prevented from aggregating during the immobilization reaction if the enzyme is in the solid state. Such aggregation is undesirable because it results in lower efficiencies and inferior flow characteristics during use. It has also been found that salts differ widely in this respect.

The process of the invention 3.an is carried out in a number of steps, the order of which is not critical. Thus, in a preferred embodiment of the invention, a carrier is first treated with an enzyme solution which is dried, and then treated in a solution containing a crosslinking agent and salt, which is then washed and optionally dried. By varying the same process, a portion of the total amount of salt may be present in the enzyme solution. In another variation, the enzyme is first treated with the crosslinking agent and immediately thereafter with the salt. For certain applications, e.g. when it is desirable that the immobilized enzyme be light and fibrous, the amount of the carrier may be very small or the carrier can be completely avoided and the enzyme treated in a coagulation bath with the crosslinking agent and salt. Thus, the enzyme can be dissolved in a solution which is coagulated in a salt bath, whereby the cross-linking agent is added to the enzyme solution or to the bath either before or after coagulation. The salt can also be added as a dry powder, instead of a solution, when it is desired to limit the amount of water. The order in which the various components are brought together, or their state, i.e. thus, whether in a solution or in a dry state is not critical to the process of the invention.

The pH, temperature and duration of the crosslinking reaction can have a profound effect on the activity exchange, depending on the enzyme and inert additives if 9 U8S13 are present. Usually, it has been found that a pH range of about 4 to approx. 9 is suitable. The temperature range which is most suitable in most cases is between approx. 15 ° C and approx. 30 ° C, although higher temperatures may advantageously be used in some cases, e.g. when it is desired to shorten the duration of the crosslinking reaction, and lower temperatures may advantageously be used in the case of highly temperature sensitive enzymes. The duration of the crosslinking reaction can vary widely, from a few minutes to a few days, depending on the type and concentration of the crosslinking agent, the type and concentration of the salt, enzyme, pH and temperature, and it should therefore be determined in each individual case. However, for glutaraldehyde and most enzymes, the range seems to be from ca. 10 minutes to several hours at room temperature to be suitable.

Various NOVO references are referred to below. Copies of all these references can be obtained from NOVO Industri A / S, Novo Alle, 2880 Bagswaard,

Denmark.

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

In the following part of the description comprising the examples, values of the pressure drop (physical strength) are indicated during column operation. This value is determined in accordance with AF 166/2, which is a description of a NOVO laboratory method. Some theoretical considerations in relation to this pressure drop determination are described in Starch / St'årke 31 (1979) Nos. 1, pages 13 - 16. For comparison with known commercial products, it may be stated that the best values for the pressure drop for the immobilized glucose isomerase preparations SWEETZYME is approx. 10 g / cm 2. It can be seen from the Examples that pressure drops with the immobilized enzyme preparations produced by the method of the invention can be as small as 2 g / cm 2 and that all values are significantly lower than 10 g / cm 2, the technical advantage of the invention being clearly demonstrated.

10 U8513

Example 1

20g portions of dry carrier particles coated with a partially purified glucose isomerase composition to an extent equal to 28% w / w and prepared according to the method described in Example 12 of the co-pending Danish Application No. 4427 / 83, was suspended and kept under moderate stirring at room temperature in 500 ml of solution containing 0.06 M sodium phosphate, 1.4 M Na 2 SO 4 and various amounts of glutaraldehyde adjusted to pH 7.0. After 1 hour, the particles were removed and suspended for 1 hour in 0.06 M sodium phosphate solution adjusted to pH 7.0. This washing was repeated 3 times and then the particles were left to themselves overnight in the phosphate solution, after which their activity was determined according to NOVO assay specification AF 189/1. The results are shown in FIG. 1, which is a plot in which the percent amount of glutaraldehyde is plotted against the enzyme activity yield expressed as enzyme units / g and showing the sensitivity of the enzyme to glutaraldehyde.

Example 2 (Comparison)

Particles were prepared as in Example 1, but no salt was added, except for a minimal amount of phosphate which served as a buffer (0.06 M sodium phosphate, pH 6.5). The percentage amount of glutaraldehyde was plotted against the enzyme activity yield in units / g, cf. FIG. 2, which shows that the crosslinking agent has two opposite effects: on the one hand, it increases the yield by preventing the enzyme from dissolving, and on the other hand it reduces the yield by inactivating the enzyme. In this case, the optimum value is at approx. 1% glutaraldehyde. By comparing FIG. 1 with FIG. 2 it appears that the optimum value for the glutaraldehyde concentration is approx.

40 times as high as with 1.4 M sodium sulfate, and the yield in this case is only approx. 60% of the yield with the salt.

11 148513

Example 3

Particles were prepared as in Example 1, but in this case potassium phosphate was used as the salt and the salt concentration was varied while keeping the glutaraldehyde concentration constant at three levels and maintaining the pH of 6.5. The percent amount of potassium phosphate was plotted against the enzyme activity yield in units / g, cf. FIG. 3, which clearly shows the beneficial effect of increasing the salt concentration, especially at a low glutaraldehyde concentration.

Example 4

The experiment in Example 3 was repeated, except that sodium sulfate was used instead of potassium phosphate. The molarity of Na 2 SO 4 was plotted against the enzyme activity yield in units / g, cf. FIG. 4, which shows the beneficial effect of the salt. As can also be seen from FIG. 4, there is an optimal value for the salt effect, beyond which the activity yield is reduced, whereby this optimum depends on the glutaraldehyde concentration.

Example 5

The experiment described in Example 3 is repeated, except that the salt concentration is increased. The molarity of potassium phosphate was plotted against the enzyme activity yield in units / g, cf. FIG. 5. A comparison of FIG. 4 and FIG. 5 shows that Na2 SO4 and potassium phosphate behave similarly.

Example 6

The experiment described in Example 1 is repeated except that potassium citrate, pH 7.0, is used instead of phosphate and sulfate in the crosslinking medium. In FIG.

6, the concentration of glutaraldehyde is plotted against the activity in enzyme units / g and it is evident that the results are similar to those of Example 1.

Example 7 20 g of dried carrier particles prepared as in Example 4 of the co-pending Danish patent application No. 4427/83 were fluidized in a laboratory type fluidized mass. 45.8 g 11.0% w / w homogenized cell sludge (fermented as 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 units / g thermophilic lactase from Bacillus sp. NRRL B-11,229 was sprayed onto the carrier particles at 30-40 ° C and the coated particles were allowed to dry. The lactase activity unit is defined as the amount of lactase that will cleave 1 μπιοί lactose / minute under the following reaction conditions: substrate concentration = 10% lactose, temperature = 60 ° C, pH = 6.5 and reaction time = 30 minutes. The enzyme activity yield was 79.8%. 10 g of coated beads were then treated in 250 ml of solution containing 0.06 M Na 2 HPO 4, 1.4 M Na 2 SO 4 and 0.1% w / v glutaraldehyde at pH 7.5. After 1 hour at room temperature, the particles were removed and washed thoroughly with 0.06 M K 2 HPC> 4 at pH = 7.5. The activity yield during the cross-linking step was 17.2%.

Example 8 24 g of dried carrier particles prepared as in Example 4 of co-pending Danish Patent Application No. 4427/83 were soaked in 20.2 g of a 39.6% w / w partially purified A. niger amyloglucosidase solution prepared by ultrafiltration of it. commercial product AMG 200 L (described in NOVO brochure NOVO ENZYMES AMG, B020 g-GB) to remove low molecular weight constituents to a solids content of 39.6% w / w (activity 2610 IAG / g, whereby the activity unit is defined in NOVO Assay Specification AF 159/2). Vacuum was used for 1 hour. The product thus obtained 13AA8513 contained 25 wt% solids of partially purified amyloglucosidase with an enzyme activity yield of 77.9%.

20 g of particles with 71.8% solids were then treated in 1600 ml of a solution of 0.06 M NaH4 PO4, 1.4 M Na2SC> 4 and 0.2% glutaraldehyde at pH = 4.5. After 1 hour, the particles were removed by filtration and washed with 0.06 M NaH 2 PO 4 at pH = 4.5.

The enzyme activity yield during the cross-linking step was 55.1%.

Example 9 40 g carrier particles prepared as set forth in Example 4 in co-pending Danish Patent Application No. 4427/83 and having a solids content of 98.8% and 24 g under vacuum treated partially purified glucose isomerase concentrate from Bacillus coagulans to which 5% glucose was added and 8% sodium sulfate (dry matter 41.8%) were 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. The solids content was 79.2.

18 g portions of this preparation (14 g dry matter) were treated for 1 hour at room temperature with 375 ml of a solution containing in all cases 1.5 M sodium sulfate, 5% glucose and 0.06 M sodium phosphate adjusted to pH 7 , 5, and which further contained either 0.1 or 0.2 or 0.3% glutaraldehyde.

After this treatment, the portions were washed 5 times with ca. 150 ml of 1% sodium phosphate, pH 7.5.

The enzyme activity was determined according to AF 189/1 after the liquid had run out of the particles. Dry matter was also determined on the drained particles.

14 148513 Solids, Unit Yield, Irmtob.

% dsr / g there / g% yield,% wet dry

Enzyme Concentrate 41.8 1415 3385 -

Enzyme Concentrate 79.2 463 585 86 + Carrier

Inniobilized m. 32.5 158 486 72 83

0.1% GA

Inniobilized m. 38.9 141 362 53 62 0.2% (¾

Inniobilized m. - 117 333 49 57 0.3%

Portions corresponding to 5 g of dry matter were examined for pressure drop.

Glutaraldehyde - Pressure drop concentration 25 hours 50 hours during immobilization OTTI 3 5 0.2% 1 3 0.3% 2 3

Example 10

This example describes a process for the preparation of an immobilized active carbon based carrier from Norit as a carrier.

Thus, 20 g of Norit Rox 0.8 activated carbon of type 33-3397 was soaked in 33 g of solution of 39.2% w / w partially purified glucose isomerase from Bacillus coagulans (activity 3950 units / g dry substance, whereby the activity unit is defined in NOVO " Analysis Specification "AF 189/1).

Vacuum was treated for 20 hours at 4 ° C, however during this period the vacuum was interrupted 4 times. The product thus obtained contained 35 wt% partially purified glucose isomerase solids with 97% enzyme activity yield.

98% of the above product as an aggregate present was then immobilized in 890 ml of a solution of 0.06 M KH 2 PO 4, 1.4 M Na 2 SO 4 and 0.18% glutaraldehyde adjusted to pH 7.5 with 4 N NaOH. After 2 hours at room temperature with gentle stirring, the particles were removed by filtration and washed thoroughly with 0.06 M KH 2 PO 4, pH 8.0.

The activity yield during the cross-linking step was 31%.

Example 11

This example describes a process for preparing an immobilized enzyme preparation with a carrier consisting of silica spheres having a diameter of approx. 2 mm.

A. 20 g of the silica beads were fluidized in a laboratory-type fluidized mass, and 40 g of solution of 15% w / w partially purified glucose isomerase from Bacillus coagulans (activity 3306 units / g of dry matter, the activity being defined in NOVO assay specification AF 189/1) was sprayed onto the balls at 25-30 ° C and the coated balls were allowed to dry. The product thus obtained contained 23 wt% partially purified glucose isomerase with 78% activity yield.

95% of the coated beads were then treated in 500 ml of solution containing 0.06 MF ^ HPO 2, 1.4 M Na 2 SO 2 and 0.1% w / v glutaraldehyde adjusted to pH 7.5 with 4 N NaOH ; after one hour at room temperature, the particles were removed and washed thoroughly with 0.06 M KH 2 PO 4 at pH 8.0.

Then the activity was determined. The activity yield during the crosslinking step was 55%.

B. 20 g of the silica beads were soaked in 15 g solution of 40% w / w partially purified glucose isomerase from Bacillus coagulans (activity 3270 units / g dry matter), with 0.56 M Na 2 SO added. ^. Vacuum was treated for 20 minutes, but the vacuum was interrupted four times during this time. The product thus obtained contained 20 wt% partially purified glucose isomerase with 90% enzyme activity yield. 75% of the coated balls were then treated as described in Part A of this example. The activity yield during the cross-linking step was 45%.

In some cases where the enzyme is particularly difficult to cross-link, the presence of salts is not only advantageous but rather imperative if the enzyme is to be cross-linked in its pure state. A good example is the amyloglucosidase produced by NOVO (and sold, for example, under the trademark AMG 200 L, cf. the NOVO ENZYMES AMG brochure, B 020 g-GB 2500 July, 1982), which is practically impossible to immobilize with glutaraldehyde when in pure state: it has been found impossible to insolubilize this amyloglucosidase even at as high a glutaraldehyde concentration as 50% w / w. However, with the salts used in the process of the invention (albeit at fairly high concentrations), it is possible to efficiently insolubilize the enzyme, even at a glutaraldehyde concentration as low as 0.05% w / v , as shown in the following Examples 12 - 17. The compositions thus obtained which have excellent filterability but which can still be easily suspended can advantageously be used in the preparation of e.g. low calorie beer.

17 148513

Example 12

Commercial NOVO AMG 200 L diluted to a solids content of 30% w / v was mixed with Hiflo Celite and dried to obtain a composition containing 21% w / w solids derived from the AMG preparation. 1 g portions of the preparation were then added to a solution containing 2.4 M Na 2 SO 4, 0.06 M potassium phosphate at pH 6.5 and glutaraldehyde at various concentrations at 32 ° C and the whole mixture was left to itself at this temperature for 20 hours. The particles were then filtered and rinsed three times with deionized water, and the activity yield was measured. The results show the usual pattern comprising the two opposite effects of glutaraldehyde with an optimum of 0.05%, cf. FIG. 7th

Example 13

The same method was repeated as in Example 12, but different concentrations of Na 2 SO 4 were used. The activity yield with this enzyme is highly sensitive to the amount of salt present, cf. FIG. 8th

Example 14

The procedure of Example 12 was repeated except that an AMG preparation containing only half as much enzyme was used here. As a result, one could clearly observe a shift of the glutaraldehyde optimum to a lower concentration, cf. FIG. 9th

Example 15 NOVO AMG 200 L was spray dried and the resulting powder was used as starting material, varying the method to a certain extent: 0.5 g of AMG powder was added to 100 ml of a solution containing 2.5 M Na 2 SO 4, 0.1 M potassium phosphate, pH 6.5, and 1% w / v glutaraldehyde at 32 ° C, and the mixture was left to itself at this temperature for 1 hour with occasional shaking. The insoluble particles thus formed were then filtered and incubated again in the same medium, simply without glutaraldehyde, and at the same temperature for a further 20 hours.

Then it was filtered and rinsed three times with deionized water. These lightly filtered microspheres had a diameter of approx. 10 - 100 μιίι and retained 19% of the original activity.

Example 16

A procedure identical to that of Example 15 was followed except that the glutaraldehyde was added to the saline solution after the enzyme powder was added thereto. In this way, a similar product was obtained with 34% of the original activity.

Example 17

Also in this case, the method of Example 15 was followed except that only 0.4% w / v glutaraldehyde was used and that the total incubation was shortened to 3 hours. In this way, a product was prepared with 37% of the original activity.

Example 18

This example illustrates the utility of the process of the invention in connection with cross-linking agents other than glutaraldehyde, namely, multivalent cations. In this case, Sn ++++ was used as the crosslinking agent. Thus, 0.5 g of the Celite-AMG preparation of Example 12, in the state in which it was pre-crosslinking, was slowly added with stirring to a 100 ml solution containing 2 M Na 2 SO 4, 0.4 M potassium acetate (pH 4.35 ) and 0.086 M SnCl4, 5 H2 O, and the mixture was left to itself at room temperature for 5 minutes with occasional shaking. The particles thus formed were filtered and fed to 100 ml of 0.2 M potassium acetate, pH 4.35. and the suspension was stirred for 5 minutes. This procedure was repeated 3 times. The final product showed an enzyme activity yield of 43%.

Example 19 In this example, a product was prepared according to the method described in Example 18 except that the concentrations of acetate and stannic salt were doubled to 0.8 M and 0.017 M. A similar product was obtained, whereby the enzyme activity yield was 65%.

Example 20

Also in this example, method 18 from Example 18 was repeated; but here the acetate concentration was reduced to half, i.e. 0.2 M. Also in this case a similar product was obtained with an enzyme activity yield of 48%.

Example 21 This example describes the use of a further crosslinking agent, namely benzoquinone. Thus, 0.5 g of a dried Celite-AMG preparation was added as in Example 12 to 75 ml of a mixture containing 2.1 M Na 2 SO 4 and 0.05 M sodium acetate and then 5 ml of a 20% solution was added. w / v benzoquinone in pure ethanol and the whole mixture was left to itself at room temperature for 80 minutes with occasional stirring. The particles thus mixed were then filtered, introduced into 75 ml of 0.1 M potassium acetate buffer, pH 4.4, kept under stirring for 5 minutes and again filtered. This procedure was repeated 3 times and then the activity was determined. The enzyme activity yield was 14%.

Claims (4)

20 148513 A process for preparing an immobilized enzyme preparation by means of a crosslinking agent, characterized in that the following components are brought together in an aqueous medium: a) a solid or dissolved enzyme preparation, b) a crosslinking agent and c) a water-soluble salt which does not react with the cross-linking agent or inactivate the enzyme, at a concentration sufficient to prevent any substantial part of the enzyme from dissolving in or mixing with the saline solution while the cross-linking reaction takes place, upon which the immobilized enzyme preparation is recovered. Process according to claim 1, characterized in that the enzyme preparation comprises a carrier. Process according to claim 2, characterized in that the enzyme preparation is a carrier coated with a layer of solid enzyme. Process according to claim 2, characterized in that the enzyme preparation is a porous carrier impregnated with an enzyme solution.