JPS6137913B2 - - Google Patents

Description

[Detailed description of the invention]

As a method for immobilizing an enzyme using a polyamino acid as a carrier, a method is known in which the carboxyl group of the acidic amino acid unit of the polyamino acid is converted into an acid azide and peptide bonded to the amino group of the enzyme protein.
51−19031). This method is an excellent method in which the enzyme activity of the immobilized product is high and this high activity can be maintained for a long period of time. However, in the case of glycoprotein enzymes such as glucose oxidase, basic amino acid residues such as urease and uricase Since it is only a fraction of the amount, it has the disadvantage that the enzyme activity per unit weight of the immobilized product is low. Therefore, the inventor focused on the sugar chain moiety of the glycoprotein enzyme, and by treating the glycoprotein enzyme with periodic acid or a periodate salt, an aldehyde group was formed in the sugar chain moiety, and this was converted into an acidic amino acid unit. By reacting with a hydrazide of a polyamino acid, we succeeded in obtaining an immobilized glycoprotein enzyme with high enzyme activity per unit weight and increased stability. Glycoprotein enzymes are complex protein enzymes containing sugars or derivatives thereof, such as glucose oxidase, peroxidase, ribonuclease, α-
Includes galactosidase, glucoamylase A, lipase, hyaluronidase, etc. The cation that forms periodic acid or periodate salt is not particularly limited as long as it does not inhibit enzyme activity. For example, ions such as sodium, potassium, calcium, and magnesium may be used. A method for treating enzymes with periodic acid or periodate salts is the method of PK Nakane (J.
Histochem.Cytochem., 16 , 9 (1968)) is known, and other enzymes may be used in accordance with this method.
Specific conditions for individual enzymes, i.e. pH;
Regarding temperature, time, concentration, etc., it is necessary to consider both the progress of the reaction and the inactivation of the enzyme, and since the optimum conditions differ depending on the enzyme, it is best to determine them by conducting tests in advance. Usually, the pH is within the stable range of the enzyme, the temperature is about 0°C to room temperature, and the time is about 5 minutes to 2 hours. Preferably, the reaction is carried out in a known buffer, such as an aqueous solution of a salt such as bicarbonate, phosphoric acid, boric acid, acetic acid, or the like. Although the reaction may be stopped by limiting the amount of periodic acid itself, it is usually accomplished by adding a diol such as ethylene glycol or a reducing agent such as ascorbic acid. In order to increase the enzyme activity per unit weight of the immobilized product, it is preferable to remove reaction reagents and the like from the periodate-treated enzyme solution and concentrate it before the immobilization reaction. For this purpose, known means such as dialysis, ultrafiltration, gel filtration, etc. may be selected as appropriate. The enzyme treated with periodic acid or periodate salt is reacted with a polyamino acid containing an acidic amino acid unit (hereinafter referred to as polyamino acid hydrazide) in which the carboxyl group at the α- or ω-position has been converted to an acid hydrazide group. . Acidic amino acids are monoaminodicarboxylic acids such as glutamic acid, aspartic acid, and α-aminoadipic acid. Other constituent units of the acidic amino acid unit in which the carboxyl group at the α- or ω-position of a polyamino acid is an acid hydrazide group include acidic amino acids, their lower alkyl esters, acid amides, and metal salts. In addition, as necessary, neutral amino acids such as leucine, alanine, and phenylalanine, oxyamino acids such as serine and threonine and their O-substituted forms, sulfur-containing amino acids and their S-substituted forms such as cysteine and methionine, lysine,
It can contain one or more of basic amino acids such as arginine and histidine and their lower acyl forms. These amino acids and derivative units thereof may be optically active, racemic, or a mixture thereof. Furthermore, when these polymers are produced by the hydrazine method described below, hydrazine acts between the ester residues of the two polymers to form a -CONHNHCO- type crosslinking bond. Such polyamino acid hydrazide is produced by reacting a molded or liquid product of a homopolymer or copolymer containing acidic amino acid lower alkyl ester units with hydrazine, and then, if necessary, removing part or all of the unreacted ester groups. can be obtained by converting into a carboxyl group (hydrazine method). A method for obtaining a polyamino acid hydrazide as a molded product is to hydrazide a molded product of polyacidic amino acid lower alkyl ester obtained by a known method, or to obtain a polyacidic amino acid hydrazide obtained by a known method. A molded article having a desired shape may be formed from a solution by a known dry method or wet coagulation method. In addition to the hydrazine method described above, polyamino acid hydrazide can be produced by any method capable of producing a hydrazide, such as by reacting hydrazine with polyamino acid chloride. Since polymers of acidic amino acid derivatives are soluble in organic solvents, they can be easily molded into various shapes or coated on solid surfaces, and can be used in any desired shape for use with immobilized enzymes. It has characteristics. A method for immobilizing enzymes treated with periodic acid or periodate salts is to immobilize the enzyme in a known buffer solution with a stable pH range, such as an aqueous solution of salts such as bicarbonate, phosphoric acid, boric acid, etc., and add polyamino acids to this solution. If necessary, suspend the hydrazide in water, the above buffer solution, etc., and then mix.
The contact may be carried out for about 1 hour to 48 hours at 40°C or lower, preferably 25°C or lower. The immobilized product is separated from the reaction solution by filtration, centrifugation, etc., if necessary, and washed with a buffer before use. Next, the results of measuring the relationship between enzyme activity and PH for the enzyme immobilized product of the present invention and the raw enzyme obtained in Example 1 are shown in FIG. For measurement, 10U of glucose oxidase was added to 0.1M phosphoric acid, 0.1M citric acid,
Suspend or dissolve in 5.0 ml of a solution containing 2mM MEDTA and 2mM NaN ₃ adjusted to the specified pH, and 20mM containing 1mg/ml benzoic acid as a substrate.
0.1 ml of glucose was added and enzymatic reaction was carried out for 1 minute while stirring with a magnetic stirrer at 26°C, and the rate of decrease in dissolved oxygen concentration was determined by measuring with a dissolved oxygen electrode. In the figure, the solid line indicates the enzyme immobilized product of the present invention, and the dotted line indicates the raw enzyme. The thermostability of the enzyme is shown in Figure 2. In the figure, the solid line is the enzyme immobilized product of the present invention obtained in Example 1, the chain line is the oxygen immobilized product obtained by the acid azide method, also obtained in Example 1, and the dashed line is the amino derivative immobilized product obtained in Example 2. The dotted line indicates the raw enzyme.
The measurement method was to leave each enzyme in a 0.1M phosphate buffer solution of PH6.5 at each temperature for 30 minutes, and then measure the residual activity. Glucose oxidase activity in the examples was measured using an oxygen electrode cell (5.0ml) containing a small magnetic stirrer.
0.1M phosphate buffer PH containing 200mM glucose, 2mM MEDTA, 2mM sodium azide.
6.5, add 0.1 ml of glucose oxidase solution or immobilized glucose oxidase suspension while stirring (500 r.pm) at 25°C, measure the maximum oxygen reduction rate, and correct for the case where no enzyme is added. Activity was calculated. 1U refers to the activity that consumes 1 μmol of dissolved oxygen per minute. Example 1 4 mg of glucose oxidase (210 U/mg, manufactured by Boehringer) was dissolved in 0.4 ml of 0.3M NaHCO ₃ ,
After adding 0.4 ml of 0.06M NaIO ₄ and reacting at room temperature for 30 minutes, the reaction was stopped by adding 0.4 ml of 0.1M ascorbic acid with a pH of 7.0. This reaction solution was ultrafiltered at 5° C. under nitrogen gas pressure using Diaflow PM-10 manufactured by Amicon and concentrated to 0.4 ml. cold
Adding 1.6 ml of 0.1M NaHCO ₃ and concentrating to 0.4 ml by ultrafiltration was performed three times to obtain 1.0 ml of an activated glucose oxidase solution with a concentration of 530 U/ml. Poly-η-methyl-D- with intrinsic viscosity [η] = 1.2
50 g of a solution prepared by dissolving 6 parts by weight of glutamate, 3 parts by weight of polyurethane diisocyanate, and 1 part by weight of epichlorohydrin in 300 parts by weight of a solvent consisting of 9 parts by volume of dichloroethane and 1 part by volume of toluene to give a concentration of 3% by weight is made into 80 to 100 meshes. After stirring thoroughly, the solvent was removed using an evaporator and the mixture was dried at 100°C for 5 hours. The obtained dried product was added to 50 ml of a solution of 10 parts by volume of 80% foamy hydrazine and 1 part by volume of pyridine, and heated at 50°C.
After reacting for 3 hours, the mixture was thoroughly washed with water to obtain a glass bead-coated PMG hydrazide. Add 1.5 ml of cold 0.1 M NaHCO ₃ to 0.5 g of the hydrazide, stir, let stand, and remove the supernatant. Immediately add 1.0 ml of the above activated glucose oxidase solution and gently shake at 5°C for 2 days. and reacted. Next, after leaving it at room temperature for 1 day, the reaction supernatant was removed and washed five times with 0.1M phosphate buffer PH7.0 containing 0.5M NaCl and 2mM EDTA to obtain an immobilized glucose oxidase (enzyme activity 428U). Ta. The enzyme activity in the washing solution was 33U. When this immobilized product was repeatedly measured 200 times over 2 months under the measurement conditions of PH7 shown in Figure 1, the activity at the 200th measurement was 94% of the first measurement. Next, 0.5 g of the glass bead-coated PMG hydrazide obtained above was added with ice-cooled 0.1N hydrochloric acid 5
ml for 15 seconds, add 0.75 ml of 3% by weight aqueous sodium nitrite solution, stir gently for 20 minutes to react, then filter the glass beads, add ice-cold water and then add 0.05 M phosphoric acid at pH 8.5. The azide was obtained by washing with a buffer solution. This azide was added to a solution in which 4 mg of the same glucose oxidase was dissolved in 4 ml of 0.3M NaHCO ₃ , and the reaction and washing were carried out in the same manner as above, whereby an immobilized glucose oxidase with an enzyme activity of 140 U was obtained. The enzyme activity in the washing solution was 688U. Example 2 The same glucose oxidase as in Example 1 was treated in the same manner as in Example 1 to obtain an activated glucose oxidase solution. Glass bead coating obtained in Example 1
1.0 g of PMG hydrazide was treated with 1260 U of the above activated glucose oxidase in the same manner as in Example 1 to obtain 1100 U of immobilized glucose oxidase. 10% of 10g of the same porous glass beads as in Example 1
The beads were added to 80 ml of a toluene solution of γ-aminopropanetriethoxysilane, refluxed for 5 hours, and then washed with ethanol to obtain glass beads bound with γ-aminopropanetriethoxysilane. 1.4 g of the glass beads were treated with 1760 U of the above activated glucose oxidase in the same manner as in Example 1,
900 U of glucose oxidase immobilization was obtained. The residual activity of these two immobilized products was measured after treatment under the conditions shown in the table below, and the results are shown in the table below.

[Table] Example 3 1.2 mg of 250 U/mg peroxidase (manufactured by Boehringer) was dissolved in 0.25 ml of 0.3M NaHCO ₃ .
0.2 ml of 0.06M NaIO ₄ was added and the mixture was reacted in the same manner as in Example 1, and then treated in the same manner as in Example 1 to obtain 0.5 ml of an activated peroxidase solution of 510 U/ml. Add 0.5ml of this activated peroxidase solution
1 ml of 0.1M NaHCO ₃ and 0.1 g of the glass bead-coated PMG hydrazide obtained in Example 1 were added, and the mixture was gently shaken and reacted at 4° C. for 2 days. After washing with 2 ml of 0.1M phosphate buffer, pH 7.0, add 1 ml of the same buffer, add 0.2 mg of NaBH ₄ and react at 4°C for 5 hours, and then add 0.1M phosphate buffer, pH 7.0 five times. After washing, 104 mg of immobilized peroxidase with 2200 U/g was obtained. Enzyme activity was determined by measuring color development with phenol antipyrine using 0.01% H ₂ O ₂ as a substrate.

[Brief explanation of the drawing]

Figure 1 shows the relationship between PH and enzyme activity for the immobilized glucose oxidase obtained in the present invention and the raw enzyme used for immobilization, and Figure 2 shows the results of measuring the thermostability of the enzyme. It shows.

Claims (1)

[Claims] 1. A glycoprotein enzyme is treated with periodic acid or a periodate salt to generate an aldehyde group in the enzyme, and then the carboxyl group at the α-position or the ω-position is converted to an acid hydrazide group. 1. A method for immobilizing a glycoprotein enzyme, which comprises reacting with a polyamino acid containing an acidic amino acid unit. 2. A polyamino acid containing an acidic amino acid unit in which the carboxyl group at the α- or ω-position has been converted to an acid hydrazide group is obtained by reacting poly-γ-methylglutamate with hydrazine. The method described in Scope 1.