JPS635074B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme-immobilized membrane using a polymer membrane having maleic anhydride residues.

Maleic anhydride copolymers are known as reactive polymers because their maleic anhydride residues are highly reactive. However, this maleic anhydride copolymer is gradually hydrolyzed in an aqueous solution to become a water-soluble polymer with a high charge density, and even when crosslinked with a divalent crosslinking agent, it swells significantly in water and It was difficult to handle due to increased tackiness, making it difficult to use it as a practical membrane.

As a result of extensive research in order to develop a polymer membrane that can be used in practical use even in aqueous solutions from such maleic anhydride copolymers, the present inventor blended maleic anhydride copolymers with a supporting polymer, and further developed divalent polymer membranes. We have found that when partially crosslinked by treatment with a reactive crosslinking agent, the maleic anhydride copolymer becomes water insolubilized and exhibits only low swelling in water, and the resulting polymeric membrane is The present inventors have discovered that the membrane has excellent effects as an element-immobilizing membrane, and have completed the present invention.

That is, according to the present invention, maleic anhydride residues are composed of a blend of a maleic anhydride copolymer and a supporting polymer, and the blend is partially crosslinked with a divalent crosslinking agent. Provided is an enzyme-immobilized membrane characterized in that it uses a polymer membrane having the following as a base material, and has hexokinase bound to the base material.

Since the polymer membrane, which is the base material of the enzyme-immobilized membrane of the present invention, has maleic anhydride residues, hexokinase can be easily immobilized on the membrane using the maleic anhydride residues.

To prepare the polymer membrane that is the base material of the enzyme-immobilized membrane of the present invention, a maleic anhydride copolymer and a support polymer are mixed with acetone, tetrahydrofuran,
After dissolving in an organic solvent such as acetonitrile and thoroughly mixing (blending), add a divalent crosslinking agent dissolved in the same organic solvent, mix vigorously, and gently dry on a flat glass to form a film. in this case,
In the blend, the maleic anhydride copolymer is preferably present at less than 50% by weight, typically from 10 to 30%, and the supporting polymer is present at least 50%, typically from 70 to 90%. Examples of divalent crosslinking agents include divalent compounds that are reactive with maleic anhydride residues, such as divalent amines and divalent alcohols such as ethylene diamine, hexamethylene diamine, ethylene glycol, and propylene glycol. . The standard usage ratio of this divalent cross-linking agent is 1/10 equivalent of maleic anhydride residue, but generally it is 1/20 to 1/2 equivalent.
It is used in a range of 5 equivalents. That is, 5 to 20 mol% of the maleic anhydride residues contained in the blend.
It is preferable to crosslink it. In addition, in the blend body,
The amount of maleic anhydride copolymer is preferably 50% by weight or less, preferably 30% by weight or less; if it exceeds 50% by weight, the strength of the membrane tends to decrease.

Maleic anhydride copolymers are copolymers of maleic anhydride and olefins such as ethylene, propylene, and isobutene, and vinyl monomers such as vinyl acetate, allyl acetate, and isopropenyl acetate, and have a large molecular weight (usually 5 ml). 10,000 ~
500,000) is preferred. Maleic anhydride is known to form a 1:1 copolymer with a vinyl monomer, and the maleic anhydride residue in the copolymer is therefore about 50 mol%. As a supporting polymer,
Supports maleic anhydride copolymer as a film body, which has film-forming ability by itself, does not react with maleic anhydride copolymer, and even after drying, mixes uniformly with maleic anhydride copolymer and does not cause phase separation. For example, those that have the effect of
For maleic anhydride/acetic acid based polymers such as maleic anhydride/vinyl acetate copolymer and maleic anhydride/allyl acetate copolymer, alkyl methacrylate polymers such as polymethyl methacrylate are preferably applied. Further, as another example of this supporting polymer, ethylene-vinyl acetate copolymer and the like are known for maleic anhydride-styrene copolymer. (Koichi Kosai,
Tsuyoshi Higashino, Japan Adhesive Association Journal, 11(1), 2 (1975)) In the polymer film containing maleic anhydride residues, which is the base material of the present invention, the reactivity of the maleic anhydride residues is Taking advantage of the fact that amines are higher than alcohols, it is possible to react with an appropriate reaction reagent in an aqueous solution to obtain amphoteric membranes with amino groups introduced, or membranes with amino acids, acid azide groups, etc. introduced. .

The present invention takes advantage of the above-mentioned properties of a polymer membrane having maleic anhydride residues, uses it as a base material for enzyme immobilization, and binds an enzyme to this to form an enzyme immobilization membrane. It is something. In this case, hexokinase can be directly bound to maleic anhydride residues in the polymer membrane, but it is also possible to bind this type of oxygen derived from maleic anhydride residues using an appropriate spacer. Commonly used functional groups (acid azide, diazonium, imidoester, aldehyde, acid chloride, isocyanate,
isothiocyanate, etc.).

Next, the present invention will be explained in more detail with reference to Examples.

Reference example <Preparation of synthetic polymer membrane having maleic anhydride residues> Dissolve 5 g of maleic anhydride/vinyl acetate copolymer with a molecular weight of approximately 120,000 in acetone, and make the total amount 100 ml.
(Solution A). Dissolve 5 g of commercially available polymethyl methacrylate in acetone to make a total volume of 100 ml (solution B). Dissolve 0.5 g of hexamethylene diamine in acetone to make 100 ml (solution C). Solution A3
1 part and 7 parts of solution B were mixed, thoroughly stirred, sealed and left at room temperature overnight, and then 1.5 parts of solution C was gradually added dropwise while stirring vigorously. Spread 2 ml of this solution on a flat tray with a diameter of 6 cm, cover with a lid, and dry slowly on a flat surface at room temperature. A transparent film with a thickness of approximately 2.6×10 ^-3 cm is obtained.

Examples <Enzyme-immobilized membrane> (A) Acid azidation method The synthetic polymer membrane obtained in the reference example is soaked in a 5% hydrazine aqueous solution and left at room temperature for 12 hours.
After thorough washing with water, transfer to 30 ml of 2% aqueous hydrochloric acid solution (if you want to increase the pore size of the membrane, perform alkali treatment during this time). While stirring vigorously, add 4%
Add 8 ml of nitrous acid solution dropwise under ice cooling to obtain an acid azide film. A distinctive feature of this process is that it takes significantly less time than conventional acid azidation.

This membrane is immersed in a 0.02M phosphate buffer (PH7.4) containing 100 units of hexokinase (manufactured by Sigma, USA) per ml, reacted with stirring at 4°C for 12 hours, and then thoroughly washed. The enzyme activity was 1.9×10 ^-3 units/cm ³ and no enzyme detachment was observed (B) Direct immobilization method In the reference example, the polymer mixing ratio was 1:1.
The synthetic polymer membrane prepared in step (A) is soaked in a hexokinase solution and allowed to react for 12 hours. The enzyme activity obtained by this method is 3.9×10 ^-4 units/cm ³ . In this way, method (A) using a spacer is superior in immobilizing enzymes on the membrane surface.

Claims (1)

[Claims] 1 10 to 50% by weight of maleic anhydride copolymer;
An anhydrous product comprising a blend with 90 to 50% by weight of a support polymer consisting of an alkyl metharylate polymer or an ethylene-vinyl acetate copolymer, the blend being partially crosslinked with a diamine or diol. An enzyme-immobilized membrane comprising a polymer membrane having maleic acid residues as a base material, and hexokinase bound to the base material.