JPS6283884A - Carrier and immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized enzyme having strong covalent bond by simple operation as ionic bond, by using a carrier prepared by reacting exchange groups of a water-insoluble weakly basic anion exchanger with a specific compound. CONSTITUTION:A water-insoluble weakly basic anion exchanger is reacted with a haloacetaldehyde or haloacetaldehyde dialkyl acetal in a state dispersed or dissolved in water. As a result, both above-mentioned compounds are reacted with exchange groups. In the case of the haloacetaldehyde dialkyl acetal, the reaction product is further hydrolyzed to introduce aldehyde groups into the weakly basic anion exchanger. The resultant carrier containing the aldehyde groups is reacted with an aqueous enzyme solution to readily afford the covalently bonded immobilized enzyme, having high enzymic activity, usable even in a high substrate concentration without elimination of the enzyme and further usable for a long period since the durability is very long.

Description

[Detailed Description of the Invention] (Industrial Application Field) Enzymes that catalyze various reactions at room temperature and normal pressure are used in the form of microscopic enzymes, enzymes found in living organisms, or extracted enzymes.

The present invention relates to a carrier particularly useful for immobilizing and effectively utilizing this enzyme, and an immobilized enzyme obtained by immobilizing the enzyme on the carrier.

(Prior art) Reactions using enzymes proceed at room temperature and pressure and are applied to a variety of reactions because they react only with substrates specific to the enzyme, but enzymes are generally water-soluble. After being used in one reaction, it is necessary to remove the enzyme from the target product, and the enzyme is deactivated in this process. Therefore, it has the disadvantage that an additional enzyme must be added in the first reaction. Various enzyme immobilization methods have been devised as a method for effectively using enzymes repeatedly rather than disposable, or as a method for continuously performing enzyme reactions. , covalent bonding or inclusion methods are known.

(Problems to be Solved by the Invention) Among these methods, in the case of the adsorption method in which enzymes are adsorbed and immobilized on a water-insoluble carrier, enzymes are held by relatively weak bonds (hydrogen bonds, hydrophobic bonds, ionic bonds), so that the enzyme is not immobilized. Alternatively, when reacting with a substrate, treatment with high temperature, strong acid, strong alkali, etc. must be avoided.

Immobilized enzymes obtained by immobilizing enzymes on water-insoluble carriers using these methods are affected by substrate concentration and ionic strength. When the substrate concentration or ionic strength increases, the enzyme tends to detach from the water-insoluble carrier and naturally decreases the substrate concentration or ionic strength. However, it has the drawbacks of short duration of activity and low stability. In addition, in order to compensate for these drawbacks, attempts have been made to prevent enzyme detachment by cross-linking the enzyme with a protein cross-linking reagent such as dialdehyde (Japanese Patent Application Laid-open No. 57-369A6), but the concentration of the reagent used If the concentration is too low, crosslinking will not be carried out sufficiently, which may prevent the enzyme from being desorbed, and if the concentration is too high, the enzyme will be severely deactivated, making it difficult to set the conditions for the crosslinking reaction. In addition, the adsorption method and the gel entrapment method, in which the enzyme is encased in the lattice of a polymeric carrier, have little deactivation during immobilization and high initial activity; Many have the disadvantage of poor stability.

In addition, as a carrier binding method, for example, a method of binding a carrier having a carboxyl group and an amine group of an enzyme using carbodiimides, and after activating the hydroxyl group using cyanogen bromide,
There are covalent bonding methods such as a method of reacting with an enzyme. In the covalent bonding method, the carrier and the enzyme are strongly bonded by covalent bonds, so the enzyme is less likely to be desorbed by highly concentrated substrate solutions or salt solutions, but compared to ionic bonding, the reaction conditions when bonding the enzyme are It is difficult to set up and complicated to operate, and in some cases, it has the disadvantage of causing a decrease in enzyme activity.

As described above, immobilized enzymes obtained by conventionally known methods have drawbacks in terms of their production method or in terms of their use.

(Means for Solving the Problems) As a result of extensive research in order to solve the above-mentioned drawbacks, the present inventors have discovered that aldehyde is produced by reacting haloacetaldehyde with the anion exchange group of a water-insoluble weakly basic anion exchanger. Immobilized enzymes can be obtained by a simple procedure of introducing an aldehyde group or by reacting and hydrolyzing I-roacetaldehyde dialkyl acetal and then applying an enzyme solution. It is combined.

The enzyme does not desorb even at high substrate concentrations and ionic strengths,
As a result, they discovered that the enzyme activity was stable over a long period of time, and completed the present invention.

That is, one aspect of the present invention is.

1. A carrier obtained by reacting an exchange group of a water-insoluble weakly basic anion exchanger with haloacetaldehyde or by reacting a haloacetaldehyde dialkyl acetal and hydrolyzing the reaction.

2. An immobilized enzyme obtained by immobilizing the enzyme on a carrier obtained by reacting haloacetaldehyde with the exchange group of a water-insoluble weakly basic anion exchanger or by reacting and hydrolyzing haloacetaldehyde dialkyl acetal.

It is related to.

The carrier and immobilized enzyme of the present invention can be easily produced, for example, as follows.

That is, by dispersing or dissolving haloacetaldehyde or haloacetaldehyde dialkyl acetal in water in a water-insoluble weakly basic anion exchanger, the aldehyde dialkyl acetal reacts with the exchange group, and in the case of haloacetaldehyde dialkyl acetal, it is further hydrolyzed to form an aldehyde group. is introduced into the weakly basic anion exchanger.

By allowing an aqueous enzyme solution to act on the aldehyde group-containing carrier thus obtained, an immobilized covalently bonded enzyme can be easily obtained.

Various types of water-insoluble weakly basic anion exchangers can be used in the present invention and are not particularly limited. for example,
Various ion exchangers having primary, secondary or tertiary amine groups as exchange groups can be used.

Examples of these are amino group; methylamino group.

ethylamino group, hydroxyalkylamine group.

Examples include secondary amine groups such as an aminoalkylamino group; and quaternary amine groups such as a dimethylamino group, a diethylamino group, and a bishydroxyethylamino group.

As the ion exchanger, various shapes, uses, and substances can be used as long as it is a water-insoluble, weakly basic anion exchanger. For example, (1) ion exchange resin, (2) ion exchange gel chromatography resin, (3) ion exchanger. Examples include exchange membranes or ion exchange fibers, (4) emulsions, (5) inorganic carriers, and (6) cationic natural polysaccharides.

More specifically, (1) ion-exchange resins (weakly basic anion-exchange resins) include, for example, the product names: AN-(-LiteE RA-35, -45, -6B, -95°-qa,
-qq (manufactured by Rohm & I.-S Co., Ltd.), product name DOWEX -66, -WGR-2 (manufactured by Dow Chemical Co., Ltd., product name Diamond Ion W A -10+-11, -20, -21
, -30 (Mitsubishi Chemical Industries, Ltd. yn>).

(2) Examples of the resin for ion exchange gel chromatography include resins for gel chromatography having dialkylamino groups such as diethylamino groups.

For example, the product name DEAI! : ) Yopal 65oM, (Toyo Sodasha 1!ri, product name DKAE Sephadex A-2
5, -50, DEAR-Sepharose CL-6B, DK
Examples include AK-Sephacel (manufactured by Pharmacia Fine Chemicals).

(3) The ion exchange membrane or ion exchange fiber may be any membrane or fiber having a weakly basic anion exchange group, such as DEAK-cellulose paper.

(4) Emulsions include 1, for example, polymers obtained by emulsion polymerization of dialkylaminoalkyl (meth)acrylate alone or by mixing dialkylaminoalkyl (meth)acrylate with an ethylenically unsaturated monomer copolymerizable with it in any proportion. It is usually obtained by a conventional radical polymerization method in an aqueous solvent in the presence of an emulsifier such as polyoxyethylene alkylphenyl ether, and the emulsifier can be removed by a treatment such as dialysis.

(5) Examples of inorganic carriers include porous glass or silica gel, and weakly basic anions such as amine groups can be formed by applying a silane coupling reagent having a primary, secondary, or tertiary amine group to the surface of the carrier. Examples include those in which an exchange group is introduced to form a weakly basic carrier.

(6) Examples of cationic natural polysaccharides include chitosan.

Heroacetaldehyde and I-roacetaldehyde dialkyl acetal used in the present invention.

Examples include chloracetaldehyde, bromoacetaldehyde, iodoacetaldehyde, chloracetaldehyde dimethyl (or ethyl) acetal, bromoacetaldehyde dimethyl (or ethyl) acetal, iodoacetaldehyde dimethyl (or ethyl) acetal, and the like.

The reaction of haloacetaldehyde or haloacetaldehyde dialkyl acetal with the exchange group of a weakly basic anion exchanger can be carried out by simply mixing the solution or dispersion containing the weakly basic anion exchanger tho-loacetaldehyde or...roacetaldehyde dialkyl acetal. Just that is enough. The reaction temperature is not particularly limited and may be any temperature that does not damage the slightly basic anion exchanger, but 0 to 50°C is particularly preferred.

The haloacetaldehyde or haloacetaldehyde dialkyl acetal reacted with a weakly basic anion exchanger has an ion exchange capacity (Meq/+
It is preferable to react 0.25 equivalent or more of (), especially 0.75 to 1.0 equivalent, but in one reaction -
Roacetaldehyde or...roacetaldehyde dialkyl acetal can be used in excess.

The haloacetaldehyde dialkyl acetal reacts with the exchange group of the weakly basic anion exchanger and is hydrolyzed, thereby introducing an aldehyde group into the weakly basic anion exchanger. As the hydrolysis conditions, an inorganic acid solution such as hydrochloric acid, sulfuric acid, etc. is used, and the concentration of the inorganic acid solution is not particularly limited, but a concentration of 5N or higher is particularly preferred. The reaction temperature is not particularly limited.

Particularly preferred is 0 to 70°C. After the reaction is completed, it is neutralized with an alkaline solution such as caustic soda or soda carbonate to return the pa to neutrality.

Unreacted haloacetaldehyde and I.roacetaldehyde dialkyl acetal or hydrolyzate thereof are removed by thorough washing with a large amount of water. In addition, a hydrophilic solvent such as methanol or acetone may be used. If a hydrophilic solvent is used, haloacetaldehyde and no-roacetaldehyde dialkyl acetal or its hydrolyzate can be removed by using a small amount, and by further washing with water. Hydrophilic solvents can also be easily removed.

The immobilized enzyme of the present invention can be produced, for example, by a batch method or a column method as described below.

That is, when using the batch method, the weakly basic anion exchanger into which aldehyde groups have been introduced as described above is suspended or immersed in water, an enzyme solution is added, and the mixture is shaken at an appropriate temperature, e.g., 4 to 50°C, for several hours. It is produced by continuous stirring and reaction.

Next, it is thoroughly washed with a suitable buffer solution (eg, phosphate buffer) to remove unreacted impurity proteins, dyes, etc.

Furthermore, washing by shaking several times with a buffer containing concentrated salts (for example, 1 molar salt) is repeated to remove enzymes adsorbed by weak bonds such as ionic bonds, and the immobilized enzyme is removed.
,-． 1. Purify.

In addition, when using the column method, a weakly basic anion exchanger into which an aldehyde group has been introduced is suspended or immersed in water, packed in a column, and the enzyme solution is allowed to flow down the column at an appropriate rate, and the enzyme solution is heated at an appropriate temperature (e.g. It is manufactured by reacting at 50°C). Next, the column is thoroughly washed with a suitable buffer solution, and a buffer solution containing concentrated salts is then connected to the top or bottom of the column to purify the immobilized enzyme.

The purified immobilized enzyme does not desorb the enzyme during the enzymatic reaction, and has high enzyme activity and can withstand long-term reactions. It has the characteristics that there is no need for this process.

In the immobilized enzyme of the present invention, the enzyme is not particularly limited, and various enzymes can be used.

For example, aminoacylase, pleomain inactivating enzyme, amylase, asparaginase, invertase, urease, urokinase, a7tylcholinesterase, trizucin, chymotrypsin, papain, pepsin,
Penicillin amidase.

Hydrolytic enzymes such as lipases and phosphatases.

Redox enzymes such as glucose oxidase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, catalase, amino acid oxidase, gluconate dehydrogenase.

Eliminating enzymes such as phenylalanine ammoniaase, curtamate decarboxyl 7ase, aspartase, glucose isomerase, isomerase such as glutamate racemase, glutamate-virupinoate aminotransferase, glutamate-oxaloacetate aminetransferase, loin aminobepte Examples include transferases such as pyruvate kinase, pyruvate kinase, and creatine phosphokinase.

(Example) After washing Amberlite RA-68 (a weakly basic anion exchange resin with a total exchange capacity of 1.6 Meq/gg or more, manufactured by Rohm & Co., Ltd.) of Example 110d with distilled water,
, bromoacetaldehyde dimethyl acetal of 694f was mixed with 20 scraps of water-containing methanol (methanol:water = 1+
1. A solution dissolved in water-containing methanol (hereinafter abbreviated as water-containing methanol) was added and shaken at 37°C for 15 hours. As a result of analysis by gas chromatography, no bromoacetaldehyde dimethyl acetal was detected in the water-containing methanol after the reaction.

30ffi/methanol was added to Amberlite IRA-6B which had reacted with the exchange groups of the weakly basic ion exchanger, it was shaken well and washed 3 times repeatedly.

Next, the same operation was repeated five times using distilled water to remove methanol.

Next, 30J of 6N hydrochloric acid! A hydrolysis reaction was carried out by shaking at 60° C. for 5 hours. Furthermore, 6N caustic powder 30
d was added to adjust the pH to 7.2. Distilled water (30 g) was added to Amberlite RA-68 into which an aldehyde group had been introduced, and the mixture was thoroughly shaken and washed 5 times. Next 600■
Aminoacylase (1s o o o u7y manufactured by Amano Pharmaceutical Co., Ltd.) was added to 20 ml of M/15 phosphate buffer (pH =
7.2) was added to this and shaken at 37°C for 4 hours to obtain an immobilized aminoacylase agent.

The obtained immobilized aminoacylase agent was packed into a column, and 5
Unreacted aminoacylase was removed by gravity flowing down 0 d of M/15 phosphate buffer (pH=7.2). 50 more
An M/15 phosphate buffer containing 111t of 2 M-NaCl was discharged in the same manner and purified by removing unreacted aminoacylase, dye, etc. adsorbed by ionic bonds.

Example 2 After washing 10at of Diaion WA-30 (styrenic weakly basic anion exchange resin with a total exchange capacity of 1-5 Meq 7Mg or more, manufactured by Mitsubishi Chemical Industries, Ltd.) with distilled water,
Add 2,945f of chloroacetaldehyde dissolved in aqueous methanol and heat to 37°C.

The time was displayed at 15:00.

Aldehyded Diamond Ion WA-5QIC30a
t of methanol was added, and the mixture was thoroughly shaken and washed 5 times. Next, the same operation was repeated five times using distilled water to remove methanol. Next, 600μ of aminoacylase (1e
o o o u7y manufactured by Amano Pharmaceutical Co., Ltd.) with M/1 of 20d
A solution dissolved in 5-phosphate buffer (pH near 4) was added thereto and treated in the same manner as in Example 1 to purify the immobilized aminoacylase agent.

Example 5 1.7ff DKAFi Sephadex A-
25 (total exchange capacity 5.5 Meq/9 or more weakly basic anion exchange carrier Pharmacia Fine Chemicals Co., Ltd.) was mixed with 50w1 M/15 phosphate buffer (pH near, 2
) was swollen, 1,574 f of bromoacetaldehyde dimethyl acetal was added, and the mixture was incubated at 37° C. for 15 hours.The immobilized aminoacylase agent was purified in the same manner as in Example 1.

Comparative Example 1110n1 Amberlite RA-68 was washed with distilled water, 600μ of aminoacylase dissolved in 2Qd M/15 phosphate buffer (pH = 7.2) was added, and the mixture was incubated at 37℃ for 4 hours. The immobilized aminoacylase agent was purified in the same manner as in Example 1 with time shaking.

After washing the Diaion WA-so of Comparative Example 210d with distilled water, the immobilized aminoacylase agent was purified in the same manner as in Comparative Example 1.

Comparative Example 3 DEAF of 1.77! Sephadex 8-2
5 to 30 mg M/15 phosphate buffer (pL=7.2
) and purified the immobilized aminoacylase agent in the same manner as in Comparative Example 1.

Test Example 1 1rJ of 2al immobilized aminoanlase agent
Collect 4 twl of 0.0 in a test tube with a rd cap.
A 5 mol N-acetyl-DL-phenylalanine aqueous solution (pH = 7.2, containing 5x10 mol CO++) was added and an enzymatic reaction was carried out with shaking at a reaction temperature of 37°C and a reaction time of 60 minutes to produce L-phenylalanine. was quantified to determine the reaction rate. In addition, the same reaction was repeated using the immobilized aminoase/lase agent for the third time.

The enzyme activity at the 30th, 60th, and 150th times was determined. The results of the immobilized aminoacylase agent of the present invention and the immobilized aminoanlase agent according to the prior art are as follows.

Test Example 2 2 kg of immobilized aminoacylase agent 1a, 1
0.05 mol N of 4d was collected in a test tube with a cap of 4d.
-Acetyl-DL-methionine aqueous solution (pH=7+2.
(containing s x 10 moles of co'++) was added, and an enzymatic reaction was carried out with shaking at a reaction temperature of 37° C. and a reaction time of 60 minutes, and the L-methionine produced was quantified to determine the reaction rate. The same reaction was repeated using the immobilized aminoacylase agent, and the enzyme activity at the 3rd, 50th, 60th, and 150th reactions was determined. The results of the immobilized aminoacylase agent of the present invention and the immobilized aminoacylase agent according to the conventional technology were as follows.

Test Example 32 The immobilized aminoacylase agent was collected in a 10 ml test tube with a cap, and 4 ml of 0.05 mol N was added.
-Acetate, II/ -D L -H IJ aqueous solution (pH
= 7.2.5 x 10 mol Co+) was added, and an enzymatic reaction was carried out with shaking at a reaction temperature of 57° C. and a reaction time of 60 minutes, and the L-valine produced was quantified to determine the reaction rate. Furthermore, the same reaction was repeated using the immobilized aminoacylase agent, and the enzyme activity at the 3rd, 30th, 60th, and 150th reactions was determined. The results of the immobilized aminoanlase agent of the present invention and the immobilized aminoacylase agent of the prior art were as follows.

Next, an example of immobilization of pleomycin inactivating enzyme extracted from bovine liver is shown.

Example 4 Fresh beef liver 207 was homogenated with 100 Rt 1/15 molar phosphate buffer (pH=7.2).

Centrifuge at 15.00 Orpm for 30 minutes, collect the supernatant and use it as a pleomycin inactivated enzyme solution. Example 2 was applied to IIIEAK Sephadex A-25 (manufactured by Pharmacia Fine Chemicals), which was separately equilibrated with the same buffer solution.
Chloracetaldehyde was reacted in the same manner as above, unreacted chloracetaldehyde was removed, 100 yr of pleomycin inactivated enzyme solution was added, and the mixture was slowly shaken at 37°C for 4 hours to immobilize. This was packed into a column with a heat insulation jacket, and 100 gJ of 1/15 molar phosphate buffer was added.
, 400 ml of the same buffer containing 2N-NaCl, and then 200 ml of the same buffer to wash out unreacted or adsorbed enzymes.

Warm water at 37° C. was flowed through the jacket, and a 1% solution of pleomycin B2 in the same buffer as a substrate was continuously flowed at a flow rate of 2.5 g/hr.

After 4 months of continuous operation, the hydrolysis rate of pleomycin B2 decreased to about 80%, and the average hydrolysis rate was 95.2%.
Met.

Comparative Example 4 The same procedure as in Example 4 was carried out except that chloracetaldehyde was not used in Example 4, and Pleomycin 7B was
The hydrolysis rate of 2 decreased to 80%.

Example 5 Porous glass CpG-TO (20-80 mesh, 5s
oX, manufactured by Electronucleonics) 82 to 10
%r-aminopropyltriethoxysilane KBK-90
5 (manufactured by Shin-Etsu Silicone Co., Ltd.) 160rtl was added, and 6N-
Adjust the pH to 5.5 with hydrochloric acid, shake at 75°C for 2 hours, separate with F, wash with water, and heat treat in a dryer at 120°C for 2 hours. The obtained aminopropyl glass was used in Example 4.
Similar results were obtained by reacting with chloracetaldehyde in the same manner as above to immobilize the pleomycin inactivating enzyme and performing an enzymatic reaction.

Comparative Example 5 The same procedure as in Example 5 was carried out except that chloracetaldehyde was not used in Example 5, and the hydrolysis rate of pleomycin B2 decreased to 80% in 10 days.

Example 6 An immobilized amino acrase agent was obtained in the same manner as in Example 1, except that chloroacetaldehyde dimethyl acetal was used instead of bromoacetaldehyde dimethyl acetal in Example 1, and using this, the same procedure as in Test Example 1 was carried out. When the enzymatic reaction was repeated, results similar to those obtained using the immobilized aminoacylase agent of Example 1 were obtained.

Example 7 An immobilized aminoacylase agent was obtained in the same manner as in Example 1 except that bromoacetaldehyde diethyl acetal was used instead of bromoacetaldehyde diethyl acetal in Example 1, and using this agent, an enzyme was prepared in the same manner as in Test Example 1. When the reaction was repeated, results similar to those obtained using the immobilized aminoacylase agent of Example 1 were obtained.

(Effects of the Invention) The present invention solves the drawbacks of conventional immobilized enzymes using ionic bonding methods and covalent bonding methods. By using a carrier.

It has become possible to covalently bond enzymes and carriers using a procedure similar to the ionic bonding method.

Therefore, according to the present invention, an immobilized enzyme with a strong covalent bond can be obtained from a water-insoluble weakly basic anion exchanger by a simple operation similar to the ion bonding method, and the enzyme has high enzyme activity and a high substrate concentration. It can be used even once, and since the # element does not come off and its durability is very long, it can be used for a long time.

Furthermore, according to the present invention, by using a weakly basic anion exchanger, the reaction with haloacetaldehyde and /S-roacetaldehyde dialkyl acetal proceeds extremely easily, and further by using a weakly basic anion exchanger, the enzyme can be immobilized. can be carried out very easily in a short period of time, and therefore the carrier and immobilized enzyme of the present invention can be produced by simple means.

Claims (2)

[Claims] (1) A carrier obtained by reacting an exchange group of a water-insoluble weakly basic anion exchanger with haloacetaldehyde or by reacting and hydrolyzing a haloacetaldehyde dialkyl acetal. (2) An immobilized enzyme obtained by immobilizing the enzyme on a carrier obtained by reacting haloacetaldehyde with the exchange group of a water-insoluble weakly basic anion exchanger or by reacting and hydrolyzing haloacetaldehyde dialkyl acetal. .