JPH0269180A - Polymerized enzyme and production thereof - Google Patents

Abstract

PURPOSE:To polymerize a protein without deteriorating the activity and water- solubility of the protein by treating a protein with a specific water-soluble polysaccharide (derivative) and an aliphatic dialdehyde. CONSTITUTION:The objective polymerized enzyme can be produced by dissolving (A) 0.05-1.5% of a protein, especially an enzyme (e.g., amylase), (B) 0.1-10% of a water-soluble polysaccharide (derivative) containing >=0.02 units of 2- aminoethyl group per one monosaccharide unit and having a molecular weight of 5,000-500,000 (e.g., dextran) and (C) 0.05-1.0% of an aliphatic dialdehyde (e.g., 1,3-dipropanal) in (D) a buffer liquid of pH 6-8 and capable of stably dissolving protein (e.g., phosphoric acid buffer) and reacting the components at 10-40 deg.C for 1-10hr. The obtained polymerized enzyme contains >=0.02 units of 2- aminoethyl group per one monosaccharide unit and contains a water-soluble polysaccharide (derivative) having a molecular weight of 5,000-500,000 and bonded at the 2-aminoethyl group through the aliphatic dialdehyde to the amino group of the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to proteins, particularly enzymes, which are polymerized without impairing water solubility, and a method for producing the same.

[Conventional technology]

Developments in biotechnology have made it possible to utilize biological proteins such as enzymes, antibodies, lymphokines, and blood factors, as well as physiologically active substances such as hormones, peptides, and pharmaceuticals, to a greater extent for humankind. Attempts have begun to chemically modify these biochemical substances or physiologically active substances to enhance their functions and to provide them with new functions.

In the production of substances using enzymes, immobilized enzymes are well known in which the enzyme is immobilized on a water-insoluble carrier to facilitate separation from the product. Although immobilized enzymes are extremely effective for certain enzymes and are used industrially, immobilization of some enzymes may result in an extremely reduced activity.

For such enzymes, membrane reactors that use membranes to separate enzymes and products are being investigated. In this method, only the product is separated from the reaction mixture through a membrane, and the enzyme is confined within the membrane and reused. However, if the size of the product is not significantly different from the enzyme used, the enzyme may It often leaks from the membrane and separation from the product is not sufficient. For example, when trying to obtain peptides and oligosaccharides by hydrolyzing proteins and carbohydrates with enzymes, etc. In such cases, it may be possible to make the enzyme into a polymer to eliminate the porosity from the membrane. Further, in order to increase the membrane permeability of the product, it is preferable to use a membrane with a large pore size, but in this case as well, it is preferable to use a polymerized enzyme in order to prevent leakage of the enzyme.

[Problem to be solved by the invention]

Attempts have been made to react enzymes and polymeric compounds to form polymers. For example, methods include introducing a functional group to the end of polyethylene glycol and bonding it to an enzyme, condensing polysaccharides containing carboxyl groups with enzymes using a condensing agent such as carbodiimide, and oxidizing polysaccharides with periodic acid. A known method is to introduce an aldehyde group and bind it to an enzyme. However, in many cases, the water solubility of the enzyme is impaired, the reaction is complicated, and the enzyme activity is decreased, making it impractical. Therefore, a polymerized enzyme without loss of water solubility or enzyme activity has been desired. On the other hand, 1 is a functional group that is relatively easy to react with and can react under mild conditions with little risk of damaging enzyme activity.
However, until now, no polymer compounds containing an appropriate amount of amino groups have been known, and only polymer compounds with very low or high amine group contents have been used. It wasn't possible and it wasn't practical.

From this point of view, the present inventors conducted extensive studies and succeeded in introducing an appropriate amount of 2-aminoethyl groups into a water-soluble polysaccharide or a derivative of a water-soluble polysaccharide. They have now invented a polymerizing enzyme in which a polysaccharide or a polysaccharide derivative containing polysaccharide or a polysaccharide derivative and an enzyme are bonded via an aliphatic dialdehyde.

[Means to solve the problem]

That is, the present inventors simply and efficiently activated the enzyme by reacting the enzyme with a water-soluble polysaccharide or a water-soluble polysaccharide derivative containing a 2-aminoethyl group using an aliphatic dialdehyde. It was discovered that a polymerized enzyme could be obtained without losing its water solubility.

The polymerizing enzyme according to the present invention is polymerized without impairing the enzyme's inherent water solubility and enzyme activity, and can be used as an enzyme for membrane reactors. In addition, such polymerized enzymes may no longer exhibit antigenicity and may be used as enzyme preparations, making them extremely useful.

Enzymes that can be used in the present invention are not particularly limited, and any enzyme can be polymerized. for example,
amylase, protease, lipase, oxidase,
Hydrogenase, cellulase, etc.

In addition, examples of proteins other than enzymes include chlorophyll protein.

The 2-aminoethyl polysaccharide or polysaccharide derivative used in the present invention must have an average content of 2-aminoethyl groups of 0.02 or more per ton unit;
The preferred average degree of substitution of the aminoethyl group is 0.02 to 1.
0/- monosaccharide unit. If it is 0.02 or less, there are too few active sites to polymerize the enzyme, and a sufficient effect cannot be obtained. On the other hand, if it is 1.0 or more, the water-soluble polymer has many active sites, so too many enzymes bind to each other, and the enzyme activity inhibits each other, resulting in deactivation or reduction of enzyme activity, which is not preferable.

The polysaccharide or polysaccharide derivative is not particularly limited as long as it is a water-soluble neutral polysaccharide, but examples include dextran, amylose, pullulan, curdlan, mannan, hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, methylcellulose, ethylcellulose, Examples include hydroxyethyl methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxypropylethylcellulose. Its molecular weight is 5,000~oooo,ooo,
Practically speaking, it is preferably 10.000 to 200.000.

In the present invention, an aliphatic dialdehyde is used to bind the enzyme and 2-ethylaminopolysaccharide or polysaccharide derivative (hereinafter abbreviated as aminoethylated water-soluble polymer). The aliphatic dialdehyde used in the reaction is not particularly limited, but includes 1,3-dibrobanal, 1,4-dibutanal, 1,5-dipentanal, 1,6-dihexanal/be-1,7-dioctanal, and 1,8-dibutanal. Aliphatic dialdehydes having 2 to 10 carbon atoms such as dioctanal are preferred.

In order to actually produce a polymerizing enzyme, reaction conditions must be selected so that the enzyme to be polymerized does not become inactivated, and although it differs slightly depending on the individual enzyme, in general, the reaction conditions are Add enzyme to 0.05-1% of the total amount of reaction solution.
．． 5%, and the amine ethylated water-soluble polymer serving as a carrier was 0.
1 to 10%, preferably 0.1 to 5%, and aliphatic dialdehyde to 0.05 to 1.0%, dissolved in a buffer solution in which the enzyme can be stably dissolved, at pH 6 to 8, preferably pH 7. , by reacting at 10 to 40°C, preferably at room temperature, for 1 to 10 hours. The structure and properties of the obtained polymerizing enzyme are as follows.

Structure〉 (WSP)-C112['H2NH2+OHC6CH2
h"CHO+HJ (ENZ) (WSP) CH2
CH-N = [:HgoCH,hiCH=N (BNZ)
WSP + Approximate date of water-soluble polymer NZ: Approximately 2-aminoethylation of enzyme Molecular weight of water-soluble polymer 5.000 ~
500,000.2-aminoethyl group content 0.02 or more/-monosaccharide unit Enzyme molecular weight 10.000-100.000 <Physical properties> Viscosity 1% aqueous solution 20-15,000 cps (B-type viscometer, 25° C., 60 rpm) [Examples] Examples are shown below, but the present invention is not limited to these Examples.

Method for producing aminoethylated dextran The aminoethylated dextran used in the present invention may be obtained by any method, but the following method has the highest reaction efficiency and is suitable for the present invention. can be obtained.

That is, the amount of dextran per glycol unit is 1.0 to 8.
0 mol, preferably 3.0 to 6.5 mol of hydroxide)
It can be easily obtained by dissolving it in water containing IJum, then adding the desired amount of β-aminoethyl sulfate in a nitrogen stream, and reacting at 90 to 110°C for 1 to 4 hours in an oil bath etc. . The aminoethylated dextran thus obtained was used in the present invention.

Method for producing aminoethylated hydroxyethylcellulose The aminoethylated hydroxyethylcellulose used in the present invention may be produced by any method, but aminoethylated hydroxyethylcellulose obtained by the same method as the aminoethylated dextran shown above can be used. Hydroxyethylcellulose is effective. The manufacturing method is the same as for dextran.

Example 1 4g of aminoethylated and droxyethylcellulose (hereinafter abbreviated as AHEC, nitrogen content 0.25%, average molecule of hydroxyethylcellulose 120,000) was added to 50mM,
pH 7.0, phosphate buffer y -80mj! An enzyme solution prepared by dissolving 20 rnl of glucoamylase agent Gluczyme (manufactured by Ohno Pharmaceutical Co., Ltd.) Ig in the same buffer was added to the solution and mixed.

Thereafter, while stirring the mixed solution, 2 ml of a 5% concentration glutaraldehyde aqueous solution was added, and the mixture was reacted at 25° C. for 2 hours.

The reaction solution was purified using 5ephadex G-75 manufactured by Pharmacia.
The AHEC-bound enzyme and unreacted enzyme were separated by fractionation through a gel filtration column, and the AHEC-bound enzyme fraction was concentrated under reduced pressure to obtain 30 ml of enzyme solution.

The glucoamylase activity of this solution was 1161j/d, and the activity yield was 58%.

In addition, 1 unit is 40℃ using soluble starch as a substrate.
, the activity of producing lomg of glucose in 30 minutes.

The attached drawings show gel filtration column chromatography. Figure 1 shows the gluczyme stock solution, Figure 2 shows the solution immediately after reaction at 25°C for 2 hours, and Soot 3 is 5ephadex G-75.
The results of gel filtration column chromatography of each solution after fractionation separation using the column are shown, and it is clear from the results that polymerization of glucoamylase has occurred.

In addition, gel filtration is performed using 5uperose1 manufactured by Pharmacia.
A 2■ column was used.

The solid line indicates protein based on absorbance at 280 nm, and the dotted line indicates glucoamylase activity.

Example 2 A reaction was carried out using the same reaction solution composition as in Example 1 and using glucose oxidase (manufactured by Boehringer) in place of glucoamylase.

FIG. 4 is a diagram showing the chromatography results of gel filtration before the reaction, and Soot 5 shows the chromatography results of the gel filtration after the reaction.

As is clear from the results of this analysis, it is clear that glucose oxidase is polymerized by AHEC.

[Brief explanation of the drawing]

1 to 5 are diagrams showing the results of gel filtration column chromatography in Examples.

Claims (1)

[Claims] 1.0.02 2-aminoethyl groups per monosaccharide unit
Contains 5,000 or more and has a molecular weight of 5,000 or more and 500,000
2- of the following water-soluble polysaccharides or water-soluble polysaccharide derivatives:
A polymerizing enzyme in which an aminoethyl group is bonded to the amino group of the enzyme via an aliphatic dialdehyde. 2 protein, (a) a water-soluble polysaccharide or water-soluble polysaccharide derivative containing 0.02 or more 2-aminoethyl groups per monosaccharide unit and having a molecular weight of 5,000 to 500,000; (b) A method for modifying proteins, which comprises treating with an aliphatic dialdehyde. 3. The method according to claim 2, wherein the protein is an enzyme.