CS204402B1 - Process of immobilisation of beta-fructosidase on insoluble carrier - Google Patents

Description

The invention relates to a method of immobilizing β-structosidase to an insoluble carrier comprising suitable reactive groups bound to a solid backbone capable of reacting with complementary enzyme groups that are not part of the active center, i. There is no significant decrease in immobilized enzyme activity.

One aspect of the choice of immobilization method is the choice of coupling the carrier to the enzyme. There are currently four basic ways to use it:

1. Physical adsorption, which, however, in many cases does not meet the requirement of a strong bond between the enzyme and the carrier, and when the external conditions (pH, ionic strength of the environment, etc.) change, the enzyme may be washed out of the carrier.

2. Capture and closing of the enzyme within the gel matrix in the copolymerization in _the ongoing přítomnosti.enzymu. However, there is usually a significant loss of enzyme activity, since the initiation reaction as well as the growth of the chains proceed under unfavorable enzymes conditions (high energy, temperatures, presence of free radicals, etc.).

3. Encapsulation of enzyme solutions in the form of tiny drops with a permeable membrane of natural or synthetic polymers. This method of immobilizing enzymes also has numerous disadvantages. In case of mechanical breakage of the membrane during handling, contamination of the treated material may occur

204 402

204 402 medium by free enzyme. Furthermore, the mechanical strength of these particles, with a sufficiently thin membrane to allow rapid diffusion, is low.

4. Covalent binding of the enzyme to the carrier through the reactive groups of the carrier and from the corresponding groups of the enzyme that are not part of its active center. Nowadays, there is already a large selection of reactions or activations which make it possible to carry out the process under very fine conditions, in which the enzyme is not inactivated. On the other hand, from the very beginning, the carrier has the desired mechanical and physical properties and the enzyme is bound so tightly that the elution into the medium to be treated does not change by external conditions. Particular amino acid side chain groups such as the amino group of the lysine residue, the carboxylic acid group of aspartic acid or glutamic acid, the hydroxyl group of tyrosine or lilac, and the like are particularly useful in the binding of the enzyme to the carrier. Depending on the type of groups indicated in the previous sentence, the method of activation or the functional group of the carrier is then chosen.

Regardless of the functional groups, a number of requirements are placed on the carrier, postulating the characteristics of an ideal carrier:

1. Good mechanical, physical and hydrodynamic properties.

2. Reactive groups bonded to a sufficiently long side chain that is flexible.

3. Hydrophilicity.

4. Chemical stability.

5 »Sufficient capacity.

6. Appropriate reactivity without complex activation.

7. Minimal nonspecific eorpee.

8. Sufficiently large surface available for reaction.

One of the few carriers meeting most of these requirements is, for example, a polymeric material containing reactive epoxy groups according to U.S. Pat. No. 175112, which provides a number of possibilities for enzyme binding. One possibility is, for example, the direct binding of ampholytic substances, described in U.S. Pat. Certificate No. 185717.

This carrier has many other properties which can be advantageously used in the field of enzyme immobilization. In a simple manner, it is possible to modify the epoxy group contained therein to obtain a polymeric carrier with groups significantly different. For example, reaction with a diamine as described in U.S. Pat. No. 162535, results in a product containing free primary amino groups, while retaining the original properties of the epoxy starting polymer. These free amino groups are then capable of binding the following reaction to the next reaction. a carbonyl group to form a so-called Sehiff base.

204 40

However, this reaction is uncommon in terms of enzyme binding, as the enzyme itself generally does not contain sufficient reactive carbonyl groups. Preferably, however, this reaction can be used when dialdehyde is used as the activating agent, thereby forming a Schiff base (iminomethylene bond) from one of its carbonyl groups, while the remaining second group is available for binding the enzyme via its amino groups. Another possibility is chemical modification of the enzyme in such a way that it will eventually contain an aldehyde group,

-Fructoidase (invertesis, aacharase, -fructofuranoside-fructohydrolase, E.C.

3.2.1.26) is an enzyme of the group of hydrolases, ie. hydrolyzes sucrose to fructose and glucose.

-Fructoidase is by its chemical nature a glycoprotein with a molecular weight of 260,000, of which the protein component has a mol. weight 135,000. 50% is mannan and 3% glucosamine. Each molecule contains a cluster of bound mannan at 20 to 30 sites.

In principle, the literature provides three types of carriers for immobilizing $ -fructosidase:

(a) inorganic carriers, such as glass, betonite, brick grit, which are activated, for example, by thionyl chloride, cyanuric chloride or -aminopropyltriethoxysilane, that is to say all substances difficult to handle;

(b) natural polymers such as cellulose derivatives, collagen, gelatin but which are attacked by micro-organisms and whose mechanical properties do not meet the necessary technological requirements for operational application;

c) synthetic polymers such as polyacrylamide gel, poly (acrylonitrile), aminostyrene gel, phenol formaldehyde resins, copolymers of 2-methoxy-4-formylphenyl methacrylate, poly (vinyl alcohol).

Using all of the above carriers, the enzyme is immobilized by all of the previously mentioned methods.

A common feature of the prior art that overcomes the method of the invention is the relatively low conserved relative activity (i.e., activity of bound amount of enzyme relative to the same amount of native enzyme), ranging from 0.5% (polyaminostyrene) to 80% (polyacrilamide gel). and at the same time disadvantageous mechanical and hydrodynamic properties.

SUMMARY OF THE INVENTION The present invention provides a process for immobilizing? -Fructosidase to an insoluble carrier, wherein the carrier based on glycidyl esters containing primary amino groups is a reaction product of a chemical reaction of crosslinked polyglycidyl acrylate or polyglycidyl methacrylate with ammonia or primary diamine; at 0-80 [deg.] C. is mixed with the [beta] -fructosidase solution and allowed to react for 0.1 to 30 hours at a pH of the mixture of 4 to 8.

The immobilized [beta] -fructpsidase can then be used in the production of invert sugar, i.e. a mixture of fructose and glucose from sucrose, which is immediately useful for food purposes. The commonly used method of inverting sucrose with a solution of sulfuric acid usually does not give this product because invert sugar contains trace amounts of metals in the food industry

204,402 inadmissible such as arsenic. The ion exchange method has the disadvantage of low ductility. Also, the use of the native enzyme is not very advantageous since the enzyme has only a single use and the protein remains present in the final product after the reaction, which is not desirable or has to be laboriously removed.

In the following, the invention is illustrated in more detail by means of examples without limiting them.

Example 1 g of glycidyl methacrylate-ethylenedimethacrylate copolymer (specific surface 60 m ² / g, epoxide content 3.05 mmol / g) was heated in an Erlenmeyer flask for 3 hours with 5 ml of ethylenediamine at 80 ° C with shaking occasionally. The solid phase was filtered off and washed on a glass frit with 100 ml of 0.5 N HCl each time, water, 0.5 N NaOH and again water. The dried product contained 1.8 mmol / g of primary amino groups.

The resulting carrier was suspended in 100 ml of water for 3 hours and washed with 200 ml of phosphate buffer pH ³ 6.9 after suction. It was then resuspended in 20 ml of the same buffer and, together with 8 ml of glutardialdehyde (25% in water), shaken at 37 ° C for 18 hours, washed with 400 ml of pH ³ buffer 6.9 and 200 ml of water.

To 0.2 g of activated carrier, washed with 100 ml of pH ³ 6.9 buffer, was added 15 ml of the same buffer and 10 ml conc. enzyme solution (about 200 mg protein per g carrier). The reaction was run at 4 ° C for 15 hours and 2.5 hours at 18 ° C. The solid phase was washed with β NaCl buffer (500 ml each). Specific activity of immobilized enzyme is 17,971 and relative activity 71% ·

Example 2

Immobilization was performed in a manner similar to Example 1, except that after 18 hours of reaction, 10 ml of the? -Fructosidase solution was added to the suspension of glutaraldehyde in pH = 5 buffer solution and the reaction was continued at 4 ° C for 20 hours. The specific activity of the preparation was 15.0 in cat and relative activity 60% ·

Example 3 g of glybidylacrylate-ethylenedimethacrylate copolymer (35 nm / g specific surface area, epoxide content 3.05 mmol / g) was suspended in 10 ml of a 25% aqueous ammonia solution and refluxed at 90 ° C for 5 hours. . After solid-phase separation and washing, as in Example 1, 1.7 mmol / g of primary amino groups were found in the product.

The sample of the carrier was suspended for 2 hours in 10 ml of phosphate buffer pH 7 and then added 5 ml of a 25% glutardialdehyde solution and shaken at 25 ° C for 1 hour, then washed with 100 ml of the same buffer, 100 ml of water and 100 ml of acetate buffer. 4.

After aspiration, 10 ml of a concentrated N-fructosidase solution in 15 ml of acetate buffer pH = 4 was added to the support and shaken at 37 ° C for 30 hours. The preparation was separated and washed as in Example 1. The specific activity was 16.3 µm and the relative activity retained was 65%.

Example 4

Glycidyl methacrylate-2-hydroxypropylene dimethacrylate copolymer (specific surface area m / g, epoxide content 3.1 mmol / g) (1 g) was reacted with ethylenediamine (5 mL) at 80 ° C for 3 hours. It was then washed as in Example 1, dried and found to contain 1.2 mmol / g of bound diamine. The reaction with glutardialdehyde proceeded as in Example 3. After washing, the activated carrier was suspended in 10 ml of phosphate buffer pH 8 and 10 ml of concentrated enzyme solution was added.

Immobilization was then carried out at 80 ° C for 6 minutes. The product was aspirated on a glass frit and washed with 500 ml each of a 1% surfactant solution (Tween 20), phosphate buffer pH-8 and 0.5 N NaCl in the same buffer. The specific activity was 3.8 µm.

Example 5 g of glycidyl methacrylate-divinylbenzene copolymers (surface area 45 m / g, epoxide content 2.6 mmol / g) were cooled to -70 ° C in an autoclave and overlaid with 5 ml of liquid ammonia. After closing, the autoclave was heated at 100 ° C for 3 hours, cooled again to 20 ° C and opened. The sample was transferred to a petri dish and kept in a desiccator at 20 ° C for 1 hour under a water pump vacuum. The dry product contained 2.1 mmol / g of primary amino groups.

The above support was shaken for 3 hours with 50 ml of phosphate buffer pH = 7, 10 ml of a 20% solution of glyoxal in water was added and shaken for a further 3 hours at 25 ° C. Without washing, concentrated enzyme solution (20 mL) was added to the suspension and the mixture was shaken at 0 ° C for 10 hours. Excess reagents were removed in the same manner as in Example 4. The immobilized preparation exhibited 4.1kat activity and the relative activity captured was 26%.

Claims (1)

SUBJECT OF THE INVENTION A process for immobilizing? -Fructosidesis to an insoluble carrier, characterized in that the glicidyl ester carrier containing primary amino groups and the reaction product formed by the chemical reaction of a crosslinked polyglycidyl acrylate or polyglycidyl methacrylate with ammonia or primary diamine is activated at a temperature of 0 to glutardialdehyde. 80 ° C is mixed with the solution of N -fructosidesis and allowed to react for 0.1 to 30 hours at a pH of the mixture of 4 to 8.