EP0000028A1 - Process for the preparation of a water-insoluble enzyme composition - Google Patents

Abstract

The invention relates to a process for the preparation of water-insoluble enzyme preparations. According to the invention, inorganic carriers with different common pore diameters are first provided with functional groups capable of binding the enzyme to the carrier according to methods known per se, and enzyme preparations are produced from the functionalized carriers according to methods known per se by offering different amounts of enzyme to the different carriers . It is shown that the absolute activity of the preparations obtained, regardless of the amount of enzyme bound to them, depends on the most common pore diameter of the carrier and runs through a maximum for a certain most common pore diameter. Subsequently, enzyme preparations are made from the optimal carrier determined with regard to its most common pore diameter by offering this carrier different amounts of enzyme. It can be seen that, with a certain enzyme supply, the specific activity of the preparation approaches or reaches the specific activity of the enzyme in the free state. The amount of enzyme required for a preparation with maximum activity can thus be optimized. The invention also relates to enzyme preparations produced by the process and having amyloglucosidase or glucose isomerase activity.

Description

The invention relates to a process for the preparation of water-insoluble enzyme preparations and enzyme preparations produced thereafter.
It is known to immobilize enzymes by binding to organic or inorganic carriers, ie to render them water-insoluble, so that they are reusable and can be used in continuously operating processes.

Organic materials (e.g. cellulose, nylon, polyacrylamide) have considerable disadvantages as carriers because they do not have sufficient mechanical stability, can be attacked by the solvent, are sensitive to changing pH values and ionic strengths, and in some cases tend to attack microbes, causing the bond to Enzyme can be dissolved.

Therefore, inorganic substances have been proposed as carriers on which enzymes are bound by adsorption or covalently. The preferred type of binding depends on the type and conditions of use of the enzyme and the nature of the substrate. If, for example, the substrate is in a high salt concentration, the adsorption method cannot be used because desorption of the adsorbed enzyme molecules is possible. Therefore, the covalent binding of the enzyme to the carrier is preferred. The carrier surface must then contain specific functional groups that ensure binding of the enzyme. Since the carrier does not have these functional groups in most cases, the surface must be pretreated. For example, the coating of inorganic material with silanes is known, as a result of which the surface is given organic functional groups (for example alkylamine) which contain organic sub punch a covalent bond. The treatment of inorganic supports with sulfuryl chloride, thionyl chloride or cyanuric chloride has also been tried. In addition, it is possible to coat the support surface with a water-insoluble polymer which has free functional groups, such as polyacrolein, which has between 10 and 70% free aldehyde groups, based on the number of monomer molecules.

Aluminum oxide, nickel oxide, iron oxide, titanium oxide, zirconium oxide, hydroxylapatite, silicates and porous glass have been proposed as the material for inorganic carriers, the pore structure of which ensures the accessibility of the enzyme and the substrate to the inner surface, in addition to their other desirable properties, e.g. optimal pore distribution and surface size, however, there are very different information.

Regardless of the type of binding used, none of the carriers mentioned succeeded in immobilizing enzymes in such a way that their specific activity comes close to the specific activity in the free state. According to D.L. Latigue (Immobilized Enzymes for Industrial Reactors, London 1975, p. 127), even under the best immobilization conditions, only a maximum of 80% of the enzyme applied to the carrier is present in active form.

The invention is therefore based on the object of improving the known processes for the preparation of water-insoluble enzyme preparations, in which an inorganic carrier having functional groups for covalent bonding is brought into contact with an enzyme solution, so that a maximally active preparation is obtained with minimal enzyme expenditure.

This object is achieved according to the invention by selecting a carrier with such a most common pore diameter which, regardless of the amount of enzyme bound to it, results in a preparation with maximum activity, and by bringing the carrier selected in this way into contact with an enzyme solution which contains only enough enzyme that the specific activity of the preparation produced with it reaches or comes close to the specific activity of the enzyme in the free state.

In the teaching according to the invention, carriers with different common pore diameters are first provided with coupling agents according to known methods, which both adhere sufficiently firmly to the carrier, in particular form a covalent bond to the carrier, and are capable of covalent binding with the enzyme. In the past, silanization has become the most common method for inorganic carriers, but, as already mentioned, other coupling agents can also be used. The number of coupling links on the carrier must be sufficiently large and largely depends on the surface of the carrier.

The carriers pretreated in this way are then offered different amounts of enzyme by bringing them into contact with enzyme solutions of different concentrations and by covalent binding of the enzyme to the coupling member and thus to the carrier by known methods. To. Determination of the activity of the preparations thus obtained shows that the activity of the preparations depends on the most common pore diameter regardless of the amount of enzyme bound to them and runs through a maximum. It has also been shown that the grain size of the carrier is irrelevant to the position of the maximum and at best influences its absolute value. The grain size of the carrier has therefore only of secondary importance for the teaching according to the invention and largely depends on the intended use, for example the viscosity of the substrate, the process control and the like.

The optimal carrier determined in this way with regard to the most common pore diameter is then again offered different amounts of enzyme. It is shown that preparations are obtained at certain enzyme concentrations, the specific activity of which approaches or reaches the specific activity of the enzyme in the free state, i.e. the relative activity of the preparation reaches a value of 100%.

In the case of carriers which are produced from an Si0 ₂ gel, the optimum carrier can be obtained according to a further development of the invention if the gel, after setting an alkali content, calculated as Na ₂ 0 and based on dry substance, from 0.1 to 0, 5 wt .-%, and drying, 5 to 10 hours in a steam-containing air stream at 400 ⁰ C to 850 ° C, preferably 570 ⁰ C to 750 ° C, glows.

The drying is expediently carried out in water vapor-saturated air at 180 ° C. to 200 ° C. A water vapor-containing air stream with a relative humidity of 40 to 80% has proven to be advantageous for annealing.

The carrier thus produced has a most common pore diameter of 175 to 3,000 Å, preferably 250 to 600 Å, optimally about 340 Å.

The immobilization method according to the invention can be used for all technically and analytically important enzymes, for example for hydrolases (eg amylases, glycosidases, proteases), oxidoreductases (glucose oxidase, catalase), Isomerases (glucose isomerase), transferases (dextran sucrase).

In the event that the enzyme is amyloglucosidase, which has a specific activity of 10 to 15 units / mg in the free state, the optimal preparation is obtained when the optimal carrier is brought into contact with a solution which is 25 to 75 mg , preferably 50 mg, contains amyloglucosidase per gram of carrier.

If glucose isomerase is used as the enzyme, which has a specific activity of 50-70 units / mg in the free state, the optimal preparation is obtained if the optimal carrier is brought into contact with a solution containing 20-50 mg, preferably 25 mg, of glucose isomerase per Grams of carrier contains.

The following examples serve to explain the invention.

example 1

For the preparation of the carrier 1 a precipitated from sodium silicate solution with sulfuric acid was dried Si0 ₂ gel having a Na ₂ 0 content of 0.3 wt .-% at 180 ⁰ C in water vapor saturated air for three hours. 1 kg of this material was annealed for six hours at 730 ^° C. in an air stream of 2 l / min, which had a relative moisture content of 80%. After this treatment, the SiO _{2 had} a common pore diameter of 1400 Å. Carrier 1 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.5 mm.

150 g of this carrier fraction was refluxed for 8 hours with 4 l of a 10% solution of δ-aminopropyltriethoxysilane in benzene and, after cooling, washed three times with 1000 ml of benzene and with 1000 ml of acetone. After evaporating the solvent at room temperature in vacuo, the support was washed twice with 0.05 M phosphate buffer (pH 7) and three times with bidistilled. Washed water. Drying was carried out over P ₂ 0 ₅ in vacuo. Calculated on the basis of the mean value of the C and N determination, the support 1 contained 0.13 m eq of silane / g.

10 g of this carrier were suspended in 20 ml solution of 1 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7). The activity of the amyloglucosidase was 11.75 U / mg, the activity measure U being the formation of 1 _/ u mol of glucose / min at 25 ° C. The suspension was kept under vacuum for 20 minutes, re-vented and, after two hours, again evacuated for 20 minutes. After four hours, the support and solution were separated by filtration, followed by three washes with bidist. Water and finally a three washes with 0.01 m phosphate buffer (pH 5). The finished sample 1.1 was stored in phosphate buffer (pH 5) at 4 ° C. The CN analysis showed a protein content of 16.5 mg / g.

To prepare sample 1.2, 10 g of silanized carrier 1 were suspended in 20 ml solution of 0.5 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7). The further procedure corresponded to the preparation of sample 1.1. The C-N analysis of the finished sample 1.2 showed a protein content of 9.0 mg / g.

Example 2

For the preparation of the carrier 2 a precipitated from sodium silicate solution with sulfuric acid has been described Si0 ₂ gel having a Na ₂ 0 content of 0.3 wt .-% as in Example 1 dried. 1 kg of this material was annealed for 6 hours at 680 ° C in an air flow of 2 1 / min, which had a relative moisture content of 80%. After this treatment, the SiO _{2 has} a mode pore diameter of 340 Å had. The carrier 2 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.5 mm.

150 g of this carrier fraction was treated for 8 hours with 4 l of a 10% solution of δ-aminopropyltriethoxysilane in benzene in accordance with the procedure given in example 1.

Carrier 2, calculated on the basis of the mean value of the C and N determination, contained 0.19 m eq of silane / g.

10 g of this carrier were suspended in 20 ml solution of 1 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and prepared as described in Example 1. The finished sample 2.1 had a protein content of 30.8 mg / g after the C-N analysis.

Another 10 g of the silanized carrier 2 were suspended in 20 ml solution of 0.5 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and treated as described in Example 1 (sample 1.2).
The CN analysis of the finished sample 2.2 showed a protein content of 17.4 mg / g.

Example 3

For the preparation of the support 3 a precipitated from sodium silicate solution with sulfuric acid has been described Si0 ₂ gel having a Na ₂ 0 content of 0.3 wt .-% as in Example 1 dried. 1 kg of this material was annealed for six hours at 640 ° C in an air flow of 2 l / min, which had a relative moisture content of 60%. After this treatment, the Si0 _{2 had} a common pore diameter of 180 Å. The carrier 3 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.50 mm.

150 g of this carrier fraction was treated for 8 hours with 4 l of a 10% solution of y-aminopropyltriethoxysilane in benzene in accordance with the procedure given in example 1.

Carrier 3, calculated on the basis of the mean value of the C and N determination, contained 0.51 m eq of silane / g.

10 g of this carrier were suspended in 20 ml solution of 1 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and prepared as described in Example 1.

The finished sample 3.1 had a protein content of 26.2 mg / g after the C-N analysis. ,

A further 10 g of the silanized carrier 3 were suspended in 20 ml of a solution of 0.5 g of amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and treated as described in Example 1 (sample 1.2). The C-N analysis of the finished sample 3.2 showed a protein content of 12.7 mg / g.

Example 4

In order to demonstrate that the coupling agent has no effect on the activity of the preparation, the fraction 0.25-0.5 mm was sieved from the carrier 2 (most common pore diameter 340 Å) and 50 g thereof in 500 ml 12.5 % aqueous glutardialdehyde solution stirred for 5 minutes at room temperature. 500 ml of saturated NH ₄ C1 solution were then added. After four hours of stirring at room temperature, the sample was washed with water until free of chloride and dried over P ₂ 0 ₅ in vacuo. 10 g of this carrier were suspended in 20 ml solution of 1 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and prepared as described in Example 1.

The finished sample 4.1 had a protein content of 29.8 mg / g after the C-N analysis.

Another 10 g of carrier 2 were suspended in 20 ml solution of 0.5 g amyloglucosidase (Merek 1330) in 0.05 m phosphate buffer (pH 7) and treated as described in example 1 (sample 1.2).
The CN analysis of the finished sample 4.2 showed a protein content of 17.9 mg.

Example 5

10 g of carrier 1 (most common pore diameter 1400 Å) were in 40 ml of a 0.05 m phosphate buffer solution (pH 7), the 0.5 g glucose isomerase (Y.Takasaki, Agr.Biol.Chem.33, No. 11, 1527 -1534 (1969)), suspended.

The reaction time was 30 minutes at room temperature. After every 10 minutes, the reaction vessel was evacuated and, after the reaction had ended, the residual solution was suctioned off. Then 3 washes with water and 0.05 m phosphate buffer (pH 7).

The finished sample 5.1 had a protein content of 4.8 mg / g after the C-N analysis.

To prepare sample 5.2, a further 10 g of carrier 1 were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase. The further procedure corresponded to preparation 5.1.

The C-N analysis of the finished sample 5.2 showed a protein content of 2.0 mg / g.

Example 6

10 g of the carrier 2 (most common pore diameter 340 A) were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contained 0.5 g of glucose isomerase.

The further treatment corresponded to Example 5. The finished sample 6.1 had a protein content of 22.0 mg / g after the C-N analysis.

To prepare sample 6.2, a further 10 g of carrier 2 were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase. The further treatment corresponded to example 5. The C-N analysis of the finished sample 6.2 showed a protein content of 10.2 mg / g.

Example 7

10 g of the carrier 3 (most common pore diameter 180 i) were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contained 0.5 g glucose isomerase.

The further treatment corresponded to Example 5. The finished sample 7.1 had a protein content of 11.2 mg / g after the C-N analysis.

To prepare sample 7.2, a further 10 g of carrier 3 were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase. The further procedure corresponded to preparation 5.1.

The C-N analysis of the finished sample 7.2 showed a protein content of 5.1 mg / g.

Example 8

10 g of the carrier mentioned in Example 4 (most common pore diameter 340 Å, treated with aqueous glutardialdehyde solution) were suspended in 40 ml of a 0.05 M phosphate buffer solution (pH 7), which contained 0.5 g of glucose isomerase. The further treatment corresponded to Example 5. The finished sample 8.1 had a protein content of 21.3 mg / g after the C-N analysis.

To prepare sample 8.2, a further 10 g of the same carrier were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase. The further procedure corresponded to preparation 5.1. The C-N analysis of the finished sample 8.2 showed a protein content of 9.8 mg / g.

Example 9

The activity of the preparations 1.1, 1.2 described in Examples 1 to 4; 2.1, 2.2; 3.1, 3.2; 4.1, 4.2 and that of the enzyme used for fixation (amyloglucosidase, Merck 1330) was used according to the dinitrosalicylic acid method (see Rick, W., Stegbauer, HP in: Bergmeyer, HU "Meth. D. Enzymatic Analysis", Verlag Chemie 1970 S. 848 ff). One activity unit (U) corresponds to the amount of enzyme that releases 1µ equivalent reducing groups (calculated as glucose) per minute under incubation conditions.

Incubation conditions

2% substrate solution (Zulkowsky starch Merck 1257) in 0.1 m acetate buffer pH 5.0, 30 minutes, 25 ° C. Carrier-fixed preparations were suspended under the above conditions in a 40 ml reactor at a stirring speed of 600 min ¹ (product formation rate regardless of stirring speed.).

The protein content of the preparations was determined on the basis of the mean values of the C-N determination. The most common pore diameter of the carriers was determined from the pore distribution (measured with a high pressure porosimeter).

The results of samples 1-4 are summarized in Table 1 below. The parameters used are defined as follows:

Example 10

The activity of the preparations 5.1, 5.2 described in Examples 5-8; 6.1, 6.2; 7.1, 7.2; 8.1, 8.2 and that of the glucose isomerase used for fixation was determined using the Takasaki method (cf. Y.Takasaki: Agr.Biol.Chem. Vol.30, No. 12, 1247-1253, 1966 and Z.Dische uEBorenfreund: J Biol. Chem. 192, 583, 1951). One unit of activity (U) is defined as the amount of enzyme that forms 1 mg of fructose under incubation conditions.

Incubation conditions

Die trägerfixierten Präparate wurden wie im Beispiel 9 beschrieben unter Standardbedingungen im Rührreaktor suspendiert.

The preparations fixed to the support were suspended in the stirred reactor as described in Example 9 under standard conditions.

The protein content of the preparations was determined on the basis of the mean values of the C-N determination.

The results of samples 5-8 are summarized in Table 2 below.

The definition of the parameters used is given in Example 9.

The results show:

• 1. The activity U of the examined samples runs over a maximum which is dependent on the most common pore diameter of the carrier.
• 2. The specific activity U _s depends on the enzyme uptake and, at a specific enzyme concentration c _{E, reaches} the value of the activity of the enzyme in the free state ^U _SF , ^{so that} s ^s U _rel = 100. If this amount of enzyme is exceeded, the specific activity drops, the product of U _s and c _E remaining constant.
• 3. The amount of enzyme absorbed by the carrier is a function of the most common pore diameter. The enzyme / carrier system shows maximum activity with the smallest possible use of enzyme if the optimal carrier 2 (most common pore diameter 340 Å) has absorbed 17.4 mg amyloglucosidase / g or 10.2 mg glucose isomerase / g (samples 2.2 and 6.2).
• 4. Enzyme uptake and activity of the samples examined are independent of the coupling agent.

Since the enzyme costs are considerable and increase very strongly with the required degree of purity, i.e. they play a significant role for economic use, the method according to the invention has for the first time made it possible to use expensive enzymes technically, since the method allows the use of carrier according to the invention to optimize the amount of enzyme required for maximum activity.

Claims (11)

1. A process for the preparation of a water-insoluble enzyme preparation, in which an inorganic carrier with functional groups for covalent bonding is brought into contact with a solution of an enzyme, characterized in that one selects a carrier with such a common pore diameter, regardless of the bound to it Enzyme quantity results in a preparation with maximum activity, and that the carrier selected in this way is brought into contact with an enzyme solution which contains only enough enzyme that the specific activity of the preparation thus produced reaches or approaches the specific activity of the enzyme in the free state. 2. The method according to claim 1, characterized in that one selects a carrier from an Si0 ₂ gel, which after setting an alkali content, calculated as Na ₂ 0, to 0.1 to 0.5 wt .-% and drying 5- 10 hours in a water vapor-containing air stream at 400 ° C to 850 ^o C, preferably 570 ° C to 750 ^o C, is annealed. 3. The method according to claim 2, characterized in that the drying takes place in water vapor-saturated air at 180 ° C to 200 ° C. 4. The method according to any one of claims 2 or 3, characterized in that the annealing is carried out in a water vapor-containing air stream with a relative humidity of 40 to 80%. 5. The method according to any one of claims 1 to 4, characterized in that amyloglucosidase is used as the enzyme. 6. The method according to claim 5, characterized in that when using an amyloglucosidase with a specific activity in the free state of about 10 to 15 units / mg, the carrier is brought into contact with a solution which is 25 to 75 mg, preferably 50 mg, Contains amyloglucosidase per gram of carrier. 7. The method according to any one of claims 1 to 4, characterized in that glucose isomerase is used as the enzyme. 8. The method according to claim 7, characterized in that when using a glucose isomerase with a specific activity in the free state of about 50 to 70 units / mg, the carrier is brought into contact with a solution which is 20 to 50 mg, preferably 25 mg, Contains glucose isomerase per gram of carrier. 9. enzyme preparation prepared according to one of claims 1 to 8. 10. Enzyme preparation with amyloglucosidase activity, produced according to one of claims 1 to 6. 11. Enzyme preparation with glucose isomerase activity, produced according to one of claims 1 to 4, 7, 8.