JPH0693837B2 - Immobilized enzyme and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immobilized enzyme and a method for producing the same, and more particularly to an immobilized enzyme having several kinds of enzymes immobilized in the same system and a method for producing the same.

[Conventional technology]

The enzyme reaction is used for production of various useful substances or removal of harmful substances because of its substrate specificity and high reactivity at room temperature and atmospheric pressure. In particular, due to recent advances in enzyme immobilization technology, it has become possible to reuse the enzyme effectively and to carry out the enzyme reaction continuously, even for some enzymes. Is becoming more and more popular.

As a method for immobilizing an enzyme, 1) carrier binding method and 2)
Cross-linking method, 3) comprehensive method, etc.

Of these, 1) is a method of binding to a water-insoluble carrier,
This is the oldest method for producing immobilized enzymes, and the number of reported cases is the largest. Depending on the binding mode, it can be further divided into three methods: a physical adsorption method, an ionic binding method, and a covalent binding method. The physical adsorption method is considered to be less likely to destroy the active center of the enzyme or change the higher-order structure as compared with the covalent bond method, etc. Therefore, it is an excellent method if a carrier suitable for the enzyme is found. However, since the interaction between the enzyme and the carrier is generally weak, there is a drawback that the enzyme is easily released. The ionic bond method is easier to operate than the covalent bond method, and the treatment is easy, so that an immobilized enzyme having a relatively high activity is often obtained. However, since the binding force between the carrier and the enzyme is weaker than the covalent bond, the enzyme is likely to be desorbed from the carrier when the reaction is carried out under the condition of high ionic strength because it is easily affected by the type of buffer solution or pH. is there. The covalent bond method is more difficult to set the reaction conditions than the physical adsorption method or the ionic bond method, the reaction procedure is complicated, and the immobilized enzyme having high activity cannot be obtained due to the relatively vigorous treatment. In some cases, there is a drawback that the enzymatic properties such as substrate specificity are changed. However, since oxygen is strongly bound to the carrier, it has the feature that it is not easily desorbed by a highly concentrated substrate solution or salt solution.

The cross-linking method 2) is a method of immobilizing an enzyme by cross-linking the enzyme with a reagent having two or more functional groups. As in the case of the covalent bond method of 1), this method performs the reaction under relatively violent conditions, and thus the activity of the obtained immobilized enzyme is often relatively low.

The encapsulation method of 3) includes a lattice type in which an enzyme is incorporated in a fine lattice of a polymer gel and a microcapsule type in which an enzyme is coated with a semipermeable polymer film. These methods are different from the carrier binding method of 1) and the crosslinking method of 2), and in principle, do not cause a binding reaction with the enzyme itself. Therefore, it can be applied to immobilization of many enzymes. However, when a chemical polymerization reaction is caused, the enzyme is easily deactivated.

As described above, the conventionally known enzyme immobilization methods have advantages and disadvantages and are not always satisfactory.

In view of the present situation, the present inventors have conducted various studies to establish an immobilization method having high enzyme activity and easy operation, which does not have the above-mentioned problems, and as a result, several types of enzymes have high activity. The present invention has been completed by finding a new immobilization method using a carrier binding method that can be easily immobilized in the same system.

Examples of this type of enzyme immobilization method include the methods described in JP-B-56-39879 and JP-A-54-41383.

[Problems to be solved by the invention]

The above-mentioned conventional techniques have advantages and disadvantages in terms of enzyme activity, operability, amount of immobilization, desorption of the enzyme from the carrier, etc., and are not necessarily satisfactory immobilization methods.

The purpose of this study is to provide a highly versatile immobilization method which has high enzyme activity, is easy to operate.

[Means for solving problems]

The above object is achieved by combining an enzyme with a physical adsorption method of a carrier binding method and an ionic binding method. The physical adsorption method is a method of physically adsorbing and immobilizing an enzyme on a water-insoluble carrier, and in the present invention, the enzyme is adsorbed on a water-insoluble carrier containing a hydrophobic group by hydrophobic interaction. . The ionic bond method is a method of binding an enzyme to a water-insoluble carrier containing an ion exchange group by utilizing the amino acid residue of the enzyme. The two types of carriers are mixed and the enzyme solution is brought into contact with the carriers to immobilize the enzymes. At that time, the pH and ionic strength of the enzyme solution have a great influence on the immobilized amount of the enzyme. Generally, by increasing the ionic strength, the adsorption force by the hydrophobic bond is strengthened, but the adsorption force by the ionic bond is weakened.
pH affects the dissociation of ionic groups of carriers and enzymes. Suitable immobilization conditions vary depending on the type of enzyme, conditions of enzyme reaction, etc. and are not specified, but for example, at a pH at which the enzyme is stable and the ionic groups involved in immobilization are maximally dissociated,
Considering the increase of the adsorptive power of the hydrophobic bond and the decrease of the adsorptive power of the ionic bond, the condition of ionic strength that maximizes the adsorptive power as a whole is preferable.

A first aspect of the invention is a mixture of a water-insoluble carrier containing a hydrophobic group and a water-insoluble carrier containing an ion-exchange group, which is immobilized by binding to a hydrophobic group and an ion-exchange group of the carrier, and the hydrophobic group is an alkyl group or an ion-exchange group. The group is an anion exchange group. The enzyme is characterized by containing acidic amino acid residues.

A second invention is characterized in that a water-insoluble carrier containing a hydrophobic group and a water-insoluble carrier containing an ion exchange group are mixed, and an enzyme solution is brought into contact with the mixed carrier to immobilize the enzyme. The immobilization is characterized in that it is carried out by physical adsorption by a hydrophobic bond and ionic binding by an ionic bond at a hydrophobic group portion and an ion exchange group portion of the mixed carrier packed in a column. .

Examples of the water-insoluble carrier applied to the present invention include agarose [for example, Sepharose (trade name of Pharmacia),
Biogel A (trade name of Bio-Rad Co.)], cross-linked dextran [for example, Cefadexx (trade name of Fuarasia Co.)], cellulose (for example, Serexus (trade name of Bio-Rad Co.), Azehiru (trade name of Asahi Kasei Co.)] , A synthetic polymer such as a polyacrylamide gel (for example, Biogel P (trade name of Bio-Rad)), a porous glass [for example, aminopropyl-CPG]
(Trade name of Electro Nucleonics Co., Ltd.)] and the like. The carrier is prepared by a known method (for example, AH Nishikawa, P. Bailon, J. Solid-Phase Biochem.,
1, 33-49 (1976)], an epoxidized product having an alkyl group introduced therein can be used as a water-insoluble carrier containing a hydrophobic group. Alternatively, an adsorption for a commercially available hydrophobic chromatograph containing a hydrophobic group, such as octyl-Sepharose CL-4B (trade name manufactured by Pharmacia), Phenyl-Sepharose CL-4B (trade name manufactured by Pharmacia). Agents can be used. In addition, a known method for the carrier (for example, J. Am. Chem. So
c., 78, 751 (1956)], a product having a diethylaminoethyl group introduced therein can be used as a water-insoluble carrier containing an anion exchange group. Alternatively, a commercially available ion exchange chromatography adsorbent containing a diethylaminoethyl group, such as DEAE-Sephadex (trade name, manufactured by Pharmacia) can be used.

The enzyme applied to the present invention is not particularly limited as long as it has an amino acid residue, that is, an electric charge. For example, the followings are preferable.

Redox enzymes: alanine dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, isocitrate dehydrogenase, formate dehydrogenase, glyceraldehyde phosphate dehydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate synthase, glutamate dehydrogenase, cystine reductase, serine dehydrogenase. Lactate dehydrogenase, malate dehydrogenase, leucine dehydrogenase, etc.

Transferases: Adenylate kinase, glycogen synthase, glcerate kinase, glycerol kinase, glucokinase, glutamate kinase, choline kinase, acetate kinase, histamine methyl tranferase, pyruvate kinase, hexokinase, etc.

Synthase: Asparagine synthetase, acetyl-CoA synthetase, carbamoyl phosphate synthetase, glutathione synthetase, glutamine synthetase, dihydrofolate synthetase, biotin carboxylase, pyruvate synthetase, etc.

[Action]

In the present invention, since the enzyme is immobilized by the combination of the physical adsorption method and the ionic bond method, the operation is simple,
An immobilized enzyme having a relatively high enzymatic activity can be obtained. Moreover,
Since it is more strongly adsorbed than the physical adsorption method or the ionic bond method alone, the influence of pH and ionic strength is reduced, more enzymes can be immobilized, and the enzyme can be desorbed from the carrier. Few. Since it is not necessary to limit the carrier and the enzyme in particular, this immobilization method is highly versatile, and several kinds of enzymes can be immobilized in the same system.

According to the enzyme immobilization method of the present invention, the type of enzyme desired to be immobilized (the enzyme of the present invention is originally an enzyme that is immobilized by both physical adsorption method and adsorption method by ionic bond, Therefore, there are those more suitable for physical adsorption and those more suitable for adsorption by ionic bond).
Simply by changing the mixing ratio of the carrier containing the hydrophobic group and the carrier containing the ion-exchange group, the enzyme selects the carrier with the stronger binding force and fixes it, so that the enzyme is efficiently and firmly fixed. Can be converted.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to this.

[Example 1] 8 ml of distilled water, 30 ml of dioxane, 12 ml of 1N NaOH aqueous solution, and epibromohydrin were used against Sepharose 4B (trade name of Pharmacia) (still volume 50 ml) washed with distilled water.
4 ml was added, and the mixture was stirred at 30 ° C. for 4 hours. After the reaction,
The gel was thoroughly washed with distilled water (500 ml or more) collected by filtration. The epoxidized Sepharose 4B thus obtained was immediately used in the next reaction. Epoxidized Cepharose 4B (Static volume 50m
l) in 50 ml of 60% aqueous dioxane solution,
l-N-octylamine was added, and the mixture was stirred at room temperature for 8 hours. After standing at room temperature for 2 days, the gel is collected by filtration,
It was washed with a 40% aqueous dioxane solution. A small amount of an aqueous solution of 2,4,6-trinitrobenzenesulfonic acid was added to the washing solution, and the washing was continued until the color reaction did not occur, and then thoroughly washed with distilled water. The obtained n-octylated cepharose 4B is
Stored in distilled water at 4 ° C. DEAE Sephadex 50 (trade name, manufactured by Pharmacia) is swelled in 100 times or more of distilled water for 2 days (room temperature), then repeatedly washed with distilled water while sucking with a Buchner funnel, and then degassed for 1 hour. It was The obtained DEAE Sephadex A50 was stored in distilled water at 4 ° C.

n-Occlusion Cefarrows 4B and DEAE Sephadex A5
Each column was filled with 0 ml of 0 and mixed. After washing with 50 mmol / l triethanolamine buffer (pH 7), it was cooled at 4 ° C, and the enzyme solution dissolved in the same buffer had a space velocity SV = 1.5 h ^-1.
And then leave it all day and night. After the completion of the immobilization reaction, wash with 15 mmol or more of 50 mmol / l triethanolamine buffer (pH 7) of the gel, and confirm that the enzyme does not desorb from the carrier and does not leak out. (Bradford method), confirmed. As the enzyme, formate dehydrogenase (abbreviated as FDH) was used. Table 1 shows the relationship between the amount added and the amount immobilized, and the presence or absence of desorption of the enzyme from the carrier.

[Comparative Example 1] Table 1 shows the results of immobilizing FDH in the same manner as in Example 1 using 2 ml of n-octylated cepharose 4B prepared by the same method as in Example 1.

It can be seen that the examples of the present invention in Table 1 are immobilized enzymes in which the enzyme is firmly adsorbed on the carrier without desorption from the carrier.

Comparative Example 2 DEAE-Sephadex prepared in the same manner as in Example 1
Table 1 shows the results of immobilizing FDH using A502 ml in the same manner as in Example 1.

[Example 2] A 50 mmol / l triethanolamine buffer solution (pH 7) containing 1 mmol / l NAD and 100 mmol / l sodium formate was added to an immobilized FDH column prepared by the same method as in Example 1.
Table 2 shows the results of conducting NADH regeneration reaction by conducting at different flow rates (room temperature). The concentration of NADH in the effluent was 260 nm (ε = 15 cm ² / μmol) and 340 nm (ε = 6.2
cm ² / μmol), NAD concentration is 260 nm absorbance (ε = 18 cm ² /
μmol) was measured. The amount of FDH immobilized is 20.35 mg in 1 ml protein gel.

According to Table 2, the conversion rate of the substrate (substance that assists the immobilization of the enzyme) is close to 100%, which is favorable for reuse for the immobilization of the enzyme.

[Example 3] A column prepared by the same method as in Example 1 was mixed and filled with n-octylated cepharose 4B and DEAE Sephadex A50 and washed with 50 mmol / l triethanolamine buffer (pH 7). Lactate dehydrogenase (abbreviated as LDH) solution containing about 25 mg of FDH (about 4.3 mg of protein)
1 ml) 0.8 ml was immobilized by the same method as in Example 1, and FDH and LSH could be immobilized quantitatively at the same time.

Example 4 Various concentrations of formic acid and N were measured by the following enzyme activity measuring method.
The initial reaction rate against AD, pyruvate, NADH was measured and Km
(Michaelis constant) was calculated. The results are shown in Table 3.

According to Table 3, the obtained immobilized enzyme retains the enzymatic activity, and it is found that the enzymatic properties are not changed even by the immobilization.

The enzyme activity of FDH was measured as follows. Into a UV cell with an optical path length of 1 cm, 400 mmol / l sodium formate aqueous solution, 40 mmol / l NAD aqueous solution and 50 mmol / l triethanol aqueous solution (pH 7) are put in appropriate amounts, and the total volume is 3 ml.
Add 0.01 ml of FDH solution to this, mix quickly, and
The initial reaction rate was determined by the time change of the absorbance at 340 nm at ° C. In the case of immobilized FDH, 0.01 ml of a suspension of immobilized FDH (same volume of 50 mmol / l triethanolamine, pH 7) was added instead of the enzyme solution, and the mixture was stirred.
The time change of the absorbance at 340 nm was measured at 25 ° C to determine the initial reaction rate.

For LDH as well as FDH, the time change of the absorbance at 340 nm at 25 ° C. was measured. At that time, sodium formate aqueous solution and NAD
Instead of the aqueous solution, 50 mmol / l sodium pyruvate aqueous solution and 5 mmol / l NADH aqueous solution are used. LDH solution is 0.001 ml
Added. In the case of immobilized LDH as well as immobilized FDH,
The initial rate of reaction was determined.

The immobilized enzyme is a mixture of 25.9 ml of n-octylated cepharose 4B and DEAE sephadex A50 prepared by the same method as in Example 1 with FDH and LDH immobilized by 15.9 mg and 1.53 mg, respectively.

[Effect of the invention] According to the present invention, a large amount of an enzyme can be easily immobilized depending on the type of the enzyme, and moreover, it is strongly adsorbed as compared with the case of only the hydrophobic adsorption method or the ionic bond method. It also has the effect of preventing the enzyme from desorbing from the immobilized enzyme due to changes in ionic strength.

Claims (7)

[Claims] 1. An immobilized enzyme which is immobilized by binding to a hydrophobic group and an ion exchange group of a mixed carrier of a water insoluble carrier containing a hydrophobic group and a water insoluble carrier containing an ion exchange group. 2. The immobilized enzyme according to claim 1, wherein the hydrophobic group is an alkyl group. 3. The immobilized enzyme according to claim 1 or 2, wherein the ion exchange group is an anion exchange group. 4. The immobilized enzyme according to any one of claims 1 to 3, wherein the enzyme contains an acidic amino acid residue. 5. An immobilization characterized in that an enzyme is immobilized by mixing a water-insoluble carrier containing a hydrophobic group and a water-insoluble carrier containing an ion exchange group, and bringing the enzyme solution into contact with the mixed carrier. Method of oxidase. 6. The immobilized enzyme according to claim 5, wherein the immobilization is performed at a hydrophobic group portion and an ion exchange group portion of the mixed carrier packed in a column. Manufacturing method. 7. The method according to claim 5, wherein the immobilization is carried out by physical adsorption by a hydrophobic bond and ionic binding by an ionic bond.
The method for producing an immobilized enzyme according to the item.