JPS581916B2 - Carrier for immobilizing physiologically active substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a bioactive substance immobilization method comprising a new dihalotriazinylated resin represented by the general formula (1) (where R represents a support and X represents a halogen atom). This invention relates to a carrier for chemical conversion.

This carrier for immobilizing a physiologically active substance is a cyanuric halide and a resin having an amine group, a substituted amino group, an imino group, a hydroxyl group, or a sulfhydryl group (hereinafter abbreviated as R-resin).
Obtained by reacting with.

Furthermore, a resin composition obtained by reacting a halogenated S-cyanuric acid with an R-resin and a resin composition that exhibits improved activity without substantially containing inorganic chlorine when brought into contact with a buffer solution is obtained. It will be done.

Conventionally, carriers used for immobilizing physiologically active substances include:
As carriers for immobilizing physiologically active substances by adsorption methods, inorganic substances such as activated carbon, bentonite, and alumina, and ion exchangers such as ion exchange resins and ion exchange cellulose are widely used.

Also known are those in which the support is chemically modified with a specific compound to impart specific or non-specific affinity to the substance to be immobilized.

When immobilizing physiological ff3t9E substances using these carriers, the adsorbed amount is often small, the adsorbed physiologically active substance may be deactivated due to adsorption, and the adsorbed physiologically active substance may be easily eluted during use. It has various drawbacks such as irreversible adsorption, and although it can be applied to affinity chromatography, it cannot be used as a carrier for immobilizing physiologically active substances.

The present inventors previously made an invention regarding a carrier modified with an S-triazine derivative having an active halogen as a carrier for immobilizing a physiologically active substance (Japanese Patent Application Laid-Open No. 56770/1983).

Furthermore, the present inventors show general formula (If) modified with an S-triazine derivative having no active halogen.

(However, R', R'' represent H or an alkyl group having 1 to 6 carbon atoms, and R'' represents an amino acid residue.) It was found that it has a strong non-specific adsorption ability for physiologically active substances.

In addition, the present inventors have found that the carrier is composed of a conventional carrier and a support modified with an S-triazine derivative having an active halogen disclosed in the invention of JP-A No. 48-56770 in terms of adsorption power. They discovered that it could be applied to purposes such as enzyme immobilization because it showed unprecedented strength and could be regenerated and used repeatedly, and on August 19, 1975, A patent application was filed under the name "Carrier for Immobilization of Physiologically Active Substances."

After that, a more detailed study revealed that among the three halogens in the triazine skeleton, the first halogen reacts with the resin, and the active second and third halogen atoms remain; Surprisingly, a carrier for immobilizing a physiologically active substance consisting of a dihalotriazinylated resin composition represented by the general formula (1) having a third active halogen atom has the ability to bind enzyme proteins, etc. It has been found that the adhesion-bonded proteins are excellent in terms of stability, etc., and provide excellent immobilized enzymes.

That is, it was surprisingly found that even if the active secondary and tertiary halogens, which were thought to inhibit the activity expression of the physiologically active substance bound to the triazine ring, remained, they did not inhibit the activity expression of the physiologically active substance. .

Furthermore, we conducted a detailed study on the properties of the resin composition represented by the general formula (1) obtained by reacting the S-cyanuric halide with the R-resin. Even after washing the resulting resin composition with a sufficient amount of deionized water and confirming that no halogen was detected using silver nitrate from the washing solution, it was found that inorganic halogen remained and that this inorganic halogen was physiologically active. We have found that even when a substance is immobilized, it has a significant effect on the expression of its activity; in other words, although the activity of strongly acidic acid lactase and the like does not decrease much, the activity of some enzymes does decrease.

That is, as a result, we investigated a method for removing the remaining inorganic halogen, and by bringing the resin composition obtained by reacting the cyanuric halide and the R-resin into contact with a buffer solution, we obtained a method that substantially does not contain inorganic halogen. The present invention was completed by discovering a method for producing a resin composition.

The resin composition thus obtained exhibits improved properties in terms of expression of the activity of physiologically active substances, such as enzymes, as compared to resin compositions that are not treated with a buffer solution.

Next, to give a specific explanation of the present invention, the method for producing dihalotriazinyl resin is relatively easy, but the steps are (1) R-resin is washed alternately with a hydrochloric acid solution and a caustic soda solution in advance. After converting into an OH type, the process consists of (2) reacting the R1 resin sufficiently swollen with H20 with (3) cyanuric halide soaked in an organic solvent.

The reaction (3) is carried out by immersing the R-resin in a mixed solution of an organic solvent and water, adding cyanuric halide dissolved in the organic solvent, and stirring.

(The order of addition may be reversed.) After the reaction, add hydrochloric acid, sulfuric acid, acetic acid solution, etc. to complete the reaction, separate the reacted resin, thoroughly wash it with an organic solvent, and dry it. A halotriazinyl resin is obtained.

The resin used in the present invention may be a resin having an amine group, a substituted amino group, an imino group, a hydroxyl group, or a sulfhydryl group as a functional group. Polystyrene-based, aromatic amine-based, phenol-based, alkylene polyamine-based, aliphatic amine-based, polymers of melaminous anidine and formalin, aminothiazole-based resins, etc. have polystyrene, hydroxyl, and sulfhydryl groups as functional groups. Specifically, Duolite A2, Duolite A7 (both trade names, Chemical Process Co., Ltd.), Diaion 2OA, Diaion WA20, Diaion WA21 (all trade names, Mitsubishi Chemical Industries, Ltd.), Amberlight IR- 45, Amberlight IR-4B, Amberlight IRA-93 (all product names, Rohm & Haas), Imatsu A-17 (product name), Industry Remit Shirt Piece Activit N. V. etc. are preferably used.

As the cyanuric halide, cyanuric chloride, cyanuric bromide, and cyanuric fluoride can all be used.

Organic solvents used to dissolve cyanuric halides include acetone, acetonitrile, dioxane,
Examples include toluene.

Further, after the reaction is completed, the same organic solvent as mentioned above is used when the reacted resin is distributed and washed from house to house.

The reaction is usually carried out at 0° to 100°C, preferably at room temperature, but may be carried out under cooling in order to suppress heat generation.

The reaction time is usually completed within 5 minutes to 4 hours, but in order to avoid troubles due to heat generation when performing a large-scale reaction,
It is necessary to cool the reaction mixture, dilute it by increasing the amount of solvent, and control stirring especially at the beginning of the reaction.

The dihalotriazinyl resin obtained as above,
The method of bringing the buffer into contact may be as follows: After the reaction of the cyanuric halide with the R-resin, washing with an organic solvent, contacting with the buffer and drying as is, or again.
It may be replaced with water or an organic solvent and then dried.

Alternatively, a resin precursor obtained without contacting with a buffer may be brought into contact with a buffer prior to reaction with an enzyme.

The organic solvent used here for washing and substitution is also the same as that used to dissolve cyanuric chloride.

Further, the buffer used for contact after the reaction between the resin and the cyanuric halide preferably contains phosphoric acid or sodium acetate. For example, IM-phosphate buffer (pH
7-8), 0, IN-Sodium acetate solution W (pH approx. 8.8)
etc. are used.

Next, a method for producing a dihalotriazinyl resin will be described in detail with reference to Examples.

Example 1 Porous weakly basic anion exchange resin Duolite A-7 (
OH type) (trade name) (30 g) was treated with 500 ml of IN HCl, washed with water, treated with 500 ml of IN NaOH, and then washed with water until the washing solution became near neutral.

After adding 100 ml of a mixed solvent of acetone and water (volume ratio 1:1) and stirring for 15 minutes, a solution of 25 g of cyanuric chloride dissolved in 300 ml of acetone was added and stirred vigorously for 5 minutes at room temperature.

Then, 200 ml of 20% acetic acid aqueous solution was added and stirred for 10 minutes.

Quickly separate the resin and wash thoroughly with acetone.

After air drying, it is thoroughly washed with IM sodium acetate to remove inorganic chlorine, replaced with water and acetone again, and vacuum dried over phosphorus pentoxide at room temperature.

(By fractional and quantitative determination of halogen in the resin, 1.1-mmol/g of resin was found to have reacted with cyanuric chloride.

) Example 2 Alternatively, the reaction was carried out in the same manner as in Example 1, and after washing with acetone, it was immediately vacuum dried over phosphorus pentoxide at room temperature, and then,
Wash thoroughly with IN sodium monoacetate, replace with water and acetone, and vacuum dry over phosphorus pentoxide at room temperature.

(Similarly, 1.1 mmole/9 resin of cyanuric chloride was reacted.

) Example 3 Porous weakly basic anion exchange resin Amberlite IRA
-93 (Cl type) (trade name) 100g with 2NHCl50
After treatment with 0ml, wash with water and further add 2N NaOH 50
0 ml, and then washed with water until the washing liquid becomes approximately neutral.

Mixed solvent of acetonitrile and water (volume ratio 60:40) 5
After stirring for 15 minutes, add 70g of cyanuric chloride.
Add a solution of dissolved in 1000 ml of acetonitrile,
Stir vigorously for 10 minutes at room temperature.

Then, adjust the pH to 4 with INHCt and stir for 10 minutes.

Quickly wash each door with acetone, air dry, and add IM-phosphate buffer (pH 7-8) to remove inorganic chlorine.
), then replace with water and acetone again, and vacuum dry over phosphorus pentoxide at room temperature.

(By fractional and quantitative analysis of the halogen in the resin, 0.5 mmole/g of cyanuric chloride was found to have reacted with the resin.

) Example 4 A porous weakly basic anion exchange resin Diaion WA-21 (OH type (trade name) 5) was prepared by washing in the same manner as in Example 1.
0g of dioxane-water mixed solvent (volume ratio 30:70)
A solution of 40 g of cyanuric bromide dissolved in 500 wL of dioxane was added to the suspension, and the mixture was reacted at room temperature with vigorous stirring.

Thereafter, dibromotoriazinylated Diaion WA-21 was obtained by the same operation as in Example 1.

(The halogen content in this resin was determined to be 0.
48 mmole/g of resin of cyanuric bromide had completely reacted.

) Next, an experimental example will be shown in which the resin prepared according to Examples 1-3 was molded.

Experimental Example I 0.1 of each pH in which 200 mg of L-lysine hydrochloride was dissolved
To 40 ml of M buffer (acetic acid for pH 4-6, phosphoric acid for pH 6-8, boric acid for pH 118-10) was added 75 g of 4,6-dichloros-triazinyl-duolite A prepared in Example 1, The mixture was gently stirred at 5° C. for 15 hours.

After stirring, the mixture was filtered and thoroughly washed with INNaOH, and the amount of lysine contained in the washing liquid was measured to calculate the amount of lysine bound to the carrier.

The results are shown in Table 1.

Separately, as a control, Duolite A7 triazine derivative C (produced by the method described in Example 2 of JP-A No. 48-56770) was subjected to the same treatment, and the results are also appended.

Incidentally, even when 4,6-dichloros-triazinylruduolite A7 prepared in Example 2 was used, the same results as when using the one prepared in Example 1 were obtained.

Experimental Example 2 Table 2 shows the results of a reaction similar to Experimental Example 1 conducted at 30°C.

Experimental Example 3 750 mg of bovine serum albumin was dissolved in 0.00 mg of bovine serum albumin. 1M
25 g of 4,6-dichloros-triazinylruduolite A7 prepared in Example 1 was added to 200 ml of phosphate buffer (pH s.o), and the mixture was incubated at 5°C for 15 hours.
After stirring gently with
．． It was thoroughly washed with 1M phosphate buffer (pH 8.0) to obtain non-bathable albumin.

This contained 42 mg albumin/g resin.

Separately, the Duolite A7 triazine derivative used as a control in Example 1 and 4,6-dichloros-triazinylcellulose (produced by the method described in Example 1 of British Patent No. 1183258) were treated in the same manner. The immobilized albumin contained 13 mg/g resin and 8.5 mg albumin/g resin, respectively.

Experimental example 4 Aspergillus aminoacylase (manufactured by Amano Pharmaceutical Co., Ltd.) (specific activity 0.5
0.1M dissolved in International Units/mg protein) 2.5.9
25 g of 4,6-dichloro-s-triazinylruduolite A7 prepared in Example 1 was added to 200 ml of phosphate buffer (pH 8.0), and treated in the same manner as in Experimental Example 3 to fix aminoacylase. Approximately 25 g of compound was obtained.

This immobilization contained aminoacylase at 32 mg protein/g resin.

10 g of this immobilized material was placed into a diameter of 3 cm and a height of 1. Packed into an Ocm column, the temperature of the column was maintained at 37°C, and 0.0% of N-acetyl-DL-methionine was added. 2 mol/l and C
o 0.05M veronal buffer (pH 8.0) containing 0.2 mmole/t of Cl2 at SV (space velocity) 10
After 120 hours of passing through the tube, the liquid contained 75 mg/ml of L-methionine.

On the other hand, when the same treatment was carried out using the Duolite A7 triazine derivative used as a control in Experimental Example 1 and the 4,6-dichloro-s-triazinyl-cellulose used as a control in Experimental Example 3, the aminoacylase immobilized Aminoacylase is 11.1mg protein/
g resin and 2.1 mg protein/g resin, and the L-methionine in the tube fluid was 1.2 mg/ml and 0.1 mg/ml, respectively.

Experimental Example 5 4, prepared in Example 1, was added to 200 ml of 0.1 M phosphate buffer (pH 8.0) in which 1.5 g of yeast lactase (specific activity 119 international units/mg protein) and 2 g of lactose were dissolved. 25 g of 6-dichloros-triazinyl duolite A7 was added, stirred gently at 5°C for 15 hours, and then washed separately with the same buffer containing 5 M NaCl and 10 mg/ml lactose to remove insoluble lactase. Obtained.

10 g of this lactase immobilized product was packed into a column similar to that in Experimental Example 4, and city milk was passed through the column at 5° C. and sv10.

Glucose concentration in city milk after 20 hours in the bottle was 15.2 m
g/ml.

After that, the tube was continuously passed through the tube, and the glucose concentration after 4 days showed almost no change.

Separately, the glucose concentration in city milk after 20 hours of incubation of the lactase immobilized product obtained by performing the same treatment using the Duolite A7 triazine derivative used as a control in Experimental Example 1 was 16.3 mg/ml. However, the concentration after 5 days had decreased to 3.5 mg/ml.

Experimental Example 6 4,6-dichloros- prepared based on Example 1
Triazinyl Duolite A7 (However, inorganic chlorine was not removed using a phosphate buffer, and the product was thoroughly washed with water instead of a phosphate buffer.)

No more chloride ions were detected with silver nitrate in the cleaning solution.

), the same experiment as in Experimental Example 5 was conducted.

Glucose in the tube fluid was 2.5 mg/ml, but the concentration after 5 days was almost negligible.

Claims (1)

[Claims] 1. A carrier for immobilizing a physiologically active substance comprising a dihalotriazinylated resin represented by the general formula (1) (where R represents a support and X represents a halogen atom). 2. A carrier for immobilizing a physiologically active substance made of a resin represented by the general formula (1) (where R represents a support and X represents a halogen atom), which is characterized by reacting a support with a cyanuric halide. manufacturing method.