JP2562085B2 - Enzyme stabilization method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing an enzyme capable of maintaining the stability of the enzyme in an aqueous solution, and a stabilized enzyme-bound product.

[0002]

2. Description of the Related Art Since an enzyme can carry out a catalytic reaction under mild conditions, the enzyme has been actively used in recent years in the fields of clinical examination, biochemistry, food industry and the like. When using the enzyme, it is convenient to use one that already exists as an enzyme solution, but generally the enzyme is unstable and the stability of the enzyme in the solution is particularly low. Therefore, various attempts have been made so far to stabilize the enzyme in a solution. For example, in the enzyme solution, amino acids or proteins such as sodium glutamate, albumin and skim milk, sugars such as sucrose and maltose, reducing agents such as glutathione and mercaptoethanol, polyhydric alcohols such as glycerol and sorbitol, and salts such as calcium salts and magnesium salts. Or a stabilizing method in which a water-soluble polymer such as dextran is oxidized and then converted into an aldehyde by a reducing agent, and an enzyme is covalently immobilized on the aldehyde, which is disclosed in JP-A-64-60380. .

[0003]

In the method of stabilizing a enzyme by adding a so-called stabilizer to the enzyme solution, the stable additive is generally different depending on the kind of the enzyme. Therefore, it was necessary to select a specific stabilizer.

A method of immobilizing an enzyme on a water-soluble polymer by a covalent bond method is excellent as a general-purpose stabilizing method for the enzyme, but this method has a low recovery rate of the enzyme when the enzyme is immobilized. There is a drawback that. For example, JP-A-64-60
The recovery rate of immobilized enzyme in No. 380 is 50% or less.
Thus, heretofore, there has been no universally applicable and efficient method for stabilizing enzymes in solution.

[0005]

[Means for Solving the Problems] Therefore, the present inventors diligently searched for a versatile and efficient method for stabilizing an enzyme in a solution. Then, it was found that the enzyme in the solution was dissociated into an anion type and then ion-bonded with the cation type water-soluble polymer to efficiently stabilize the enzyme, and this problem could be solved.

That is, according to the present invention, a method of stabilizing an enzyme is characterized in that the dissociation of an enzyme in a solution is made anionic, and then a cation-type water-soluble polymer is added to ionically bond the enzyme and the water-soluble polymer. , A water-soluble polymer ion-binding enzyme.

The enzyme used in the present invention may be derived from animals, plants and microorganisms. The type of enzyme may be any one of so-called hydrolases, oxidoreductases, transferases and the like. Specific examples include hydrolase glucoamylase, lipase, transglucosidase,
Examples include oxidoreductase glucose dehydrogenase, glucose oxidase, and transferase transglutaminase.

In the present invention, it is first necessary to dissociate the enzyme into an anion form in a solution. Enzymes are proteins,
It has a unique isoelectric point in solution. Therefore, in order for the enzyme to exhibit the anion type, the pH of the solution may be set to a pH higher than the isoelectric point of the enzyme by using an appropriate buffer solution. At this time, it is of course necessary to take care so that the enzyme is not deactivated.

Suitable buffers include, for example, phosphate buffer, Tris-HCl buffer, acetate buffer, Good's buffer, T
An EA / HCl (triethanolamine hydrochloric acid) buffer solution or the like.

The cationic water-soluble polymer used in the present invention is DEAE-dextran, polyethyleneimine and the like.

No special operation is required for the ionic bonding method of the anion-dissociated enzyme in solution and the cation-type water-soluble polymer. It is only necessary to add the enzyme to the water-soluble polymer and stir at room temperature for several minutes. As described above, it is a feature of the present invention that the enzyme is hardly deactivated at the time of ionic bond because it is extremely simple.

The present invention will be specifically described below with reference to examples.

Examples Example 1 Glucose dehydrogenase (hereinafter also referred to as GDH)
(Amano Pharmaceutical Co., pI 6.0) is dissolved in 50 mM phosphate buffer (pH 7.0) (enzyme concentration 0.1 u / ml),
Add 1000 to 15000 ppm of DEAE-dextran (Pharmacia) to this enzyme solution,
The time-dependent changes at 5 ° C. and 50 ° C. were examined, and the stability of the DEAE-dextran ion-binding enzyme was examined. The results are shown in Table 1. The method for measuring the activity of GDH is as follows.

D-glucose 5 mM, NAD 0.3
An enzyme solution was added to 50 mM of a phosphate buffer (pH 7.0) containing mM and reacted at 25 ° C. in a photometer cell.
This was done by examining the increase in absorbance at 0 nm. One unit was defined as the enzyme activity that produced 1 μmole of NADH in 1 minute of the reaction.

[0014]

[Table 1]

As is clear from Table 1, the solution stability of GDH at 50 ° C. was about 5 by the addition of DEAE-dextran.
It has increased by 0 times.

Example 2 Glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., pI
6.0) was dissolved in 50 mM triethanolamine hydrochloric acid buffer (pH 7.0) (enzyme concentration 0.001 mg / m
l), PEI is added to this enzyme solution at 246, 491, 966
Each was added so that the concentration would be ppm, and the temporal change at 50 ° C. was examined to examine the stability of the PEI ion-binding enzyme. The activity was measured according to Example 1 except that the buffer solution was changed to a phosphate buffer solution and a triethanolamine hydrochloric acid buffer solution was used. The results are shown in Table 2.

[0017]

[Table 2]

As is clear from Table 2, PEI 500p
By adding pm, the solution stability of GDH at 50 ° C. was increased by about 2470 times as compared with the case where no pm was added.

Example 3 Glucose oxidase (hereinafter also referred to as GO, manufactured by Amano Pharmaceutical Co., Ltd., pI 4.5) was dissolved in 100 mM Tris-HCl buffer (pH 7.7) (enzyme activity 20.2 u / m).
l), 5000ppm or 15000p in this enzyme solution
DEAE-dextran, 300 to reach pm concentration
Add PEI so that the concentration will be ppm and add 15 at 60 ° C.
After leaving for a minute, the residual activity was measured to examine the stability of DEAE-dextran and PEI ion-binding enzyme on the enzyme stabilization of GO. The results are shown in Table 3. In addition, GO
The activity measuring method is shown below.

0.1M phosphate buffer (pH 7.0) 10
After preparing a reagent containing 66 mg of o-dianisidine in 0 ml, 2.4 ml of the reagent was added to the cell, and then 0.5 ml of 10% glucose solution and peroxidase (500 u / 500 u /
(0.01 ml) and preheated at 25 ° C. for 5 minutes, 0.1 ml of enzyme solution (adjusted to 0.1 u / ml with 0.1 M phosphate buffer) was added, and the change in absorbance for 5 minutes was 436 n.
Measure in m. One unit is the amount of enzyme required to generate 1 μmole of hydrogen peroxide per minute.

[0021]

[Table 3]

However, in Table 3, * indicates DEAE-dextran and ** indicates PEI. As is clear from Table 3, the addition of the DEAE-dextran concentration of 15000 ppm improves the stability of GO by 4.1 times as compared with that without addition.

Example 4 Transglucosidase (manufactured by Amano Pharmaceutical Co., pI 4.5
~ 5.0) is dissolved in 10 mM acetate buffer (pH 6.0) (enzyme activity 1.746 u / ml), and D is added to this enzyme solution.
EAE-dextran at 5000 ppm or 15000
The residual activity after addition at a ppm concentration of 65 ° C. for 1 hour was examined, and the stability of DEAE-dextran ion-bound transglucosidase was examined. The results are shown in Table 4. The method for measuring transglucosidase activity is as follows.

Weigh 0.3 g of isomaltose and add 0.01
N acetate buffer (pH 5.0) was added and dissolved, 100 m
1 ml was used as a substrate, 2 ml of the substrate was placed in a test tube, preheated at 40 ° C for 5 minutes, and 0.5 ml of the enzyme solution was added, and 40 ° C.
React for 60 minutes. After the reaction, 0.3M Tris-HCl buffer (p
Add 2.5 ml of H8.0) and shake. This liquid 0.2
Transfer ml to a test tube and add 4-aminoantipyrine-
Add 3 ml of phenol glucose oxidase and peroxidase coloring reagent, shake well, leave at 40 ° C. for 20 minutes, and measure the absorbance at 500 nm. One unit is the enzymatic force that produces 1 mg of glucose in 60 minutes.

[0025]

[Table 4]

As is clear from Table 4, the stability of transglucosidase at 65 ° C. is increased about 17 times by adding DEAE-dextran concentration of 15000 ppm.

Example 5 Glucoamylase (manufactured by Amano Pharmaceutical Co., pI 3.8-4.
0) was dissolved in 10 mM acetate buffer (pH 5.0) (enzyme activity 0.983 u / ml), and DEAE was added to the enzyme solution.
-Dextran at 15,000 ppm or 45,000 pp
It was added so that the concentration would be m, and the residual activity after standing at 60 ° C. for 1 hour was examined to examine the stability of DEAE-dextran ion-bound glucoamylase. The results are shown in Table 5. The glucoamylase activity measuring method is as follows.

Weigh 2.381 g of soluble starch and weigh about 5
After dissolving in 0 ml of boiling water and making 100 ml with water as a substrate, 1 ml of the substrate and 1 ml of 0.02N acetate buffer (pH 5.0) were weighed into a test tube and left at 40 ° C. for 5 minutes or more, Add 0.5 ml of enzyme solution and shake. This is reacted at 40 ° C. for 60 minutes, put in a boiling water bath, heated for 5 minutes, and cooled in running water. Take 0.1 ml of this liquid in a test tube, and add 3 ml of 4-aminoantipyrine-phenol glucose oxidase and peroxidase coloring reagent.
, Shake well, leave at 40 ° C for 20 minutes, and
The absorbance at 00 nm is measured. 1 mg in 60 minutes
The enzyme power to produce glucose is defined as 1 unit.

[0029]

[Table 5]

As is clear from Table 5, the stability of glucoamylase at 60 ° C. is increased about 2.5 times by the addition of DEAE-dextran concentration of 45000 ppm.

Example 6 Transglutaminase (prepared by the method described in JP-A-1-27471, pI8.9) was dissolved in 0.1 M carbonate buffer (pH 10.0) (enzyme concentration 1.3 u / m
l), 250 ppm or 75 PEI in this enzyme solution
The residual activity was examined after 2 hours at 37 ° C. by adding 0 ppm, and the stability of PEI ion-binding transglutaminase was examined. The results are shown in Table 6. The method for measuring transglutaminase activity is as follows.

Reagent A 0.2 M Tris-HCl buffer (pH 6.0) 0.1 M hydroxylamine 0.01 M reduced glutathione 0.03 M benzyloxycarbonyl-L-glutaminylglycine Reagent B 3 N-hydrochloric acid 12% -trichloroacetic acid 5% FeCl ₃ .6H ₂ O (dissolved in 0.1N HCl) A reagent B is a 1: 1: 1 mixture of the above solutions. After adding 0.5 ml of reagent A to 0.05 ml of the enzyme solution and mixing and reacting at 37 ° C. for 10 minutes, reagent B is added to stop the reaction and form an Fe complex, and then the absorbance at 525 nm is measured. As a control, the absorbance of an enzyme solution preliminarily heat-inactivated and similarly reacted is measured to obtain the difference in absorbance with the enzyme solution. Separately, a calibration curve was prepared using L-glutamic acid γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the above-mentioned absorbance difference,
The enzyme activity for producing 1 μmol of hydroxamic acid per minute was defined as 1 unit.

[0033]

[Table 6]

As is clear from Table 6, PEI 750p
At pm concentration, the stability of transglutaminase at 37 ° C. is increased by about 4.3 times.

Example 7 Lipase (trade name Lipase F "Amano", manufactured by Amano Pharmaceutical Co., Ltd., 15000 u / g) was added to 0.05 M phosphate buffer (p.
H7.0) (enzyme concentration 150 u / ml) to dissociate to anionic form and then DEAE-dextran 5
It was added to 000 ppm, and the residual activity after 37 ° C. for 5 hours was examined to examine the stability of DEAE-dextran ion-bound lipase. The results are shown in Table 7. The method for measuring lipase activity is as follows.

1.0 ml of olive oil and 1.0 m of enzyme solution
1, Tris-hydrochloric acid buffer solution (pH 7.0) 5 ml,
The mixture was reacted at 37 ° C for 30 minutes (stirred at 600 rpm). Then, 20 ml of acetone-ethanol (1: 1) was added to stop the reaction, and 1% phenolphthalein solution was titrated with 0.1N KOH as an indicator. 1 μmol per minute
The amount of the enzyme that releases the fatty acid of e is 10 units.

[0037]

[Table 7]

As is clear from Table 7, inactivation of the lipase without DEAE-dextran was observed, whereas
When added, no deactivation was observed.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a versatile and efficient method for stabilizing an enzyme in a solution becomes possible, and it can be expected to expand the use of the enzyme in clinical tests, biochemistry, and food industry.

Claims (4)

(57) [Claims] 1. A buffer of an enzyme having a pH higher than the isoelectric point of the enzyme.
The dissociation of the enzyme in solution was added cationic water-soluble polymer After the anionic and dissolved in the liquid, the enzyme in solution and an enzyme and a water-soluble polymer, characterized in that to ionic bond
Stabilization method in. 2. The method for stabilizing an enzyme according to claim 1, wherein the cationic water-soluble polymer is DEAE-dextran or polyethyleneimine. 3. A buffer of an enzyme having a pH higher than the isoelectric point of the enzyme.
A water-soluble polymer ion-binding enzyme obtained by ionic binding of an enzyme that is dissolved in a liquid and anionized in the solution, and a cation-type water-soluble polymer. 4. The enzyme is glucose dehydrogenase, glucose oxidase, lipase, glucoamylase,
The water-soluble polymer ion-binding enzyme according to claim 3, which is one of transglucosidase and transglutaminase.