JPH0728741B2 - Stable β-galactosidase aqueous composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Object of the Invention <Industrial field of application> The present invention relates to a stable β-galactosidase aqueous composition originating from Aspergillus oryzae.

β-galactosidase is an enzyme that hydrolyzes lactose to produce glucose and galactose, and is used as a lactose-degrading enzyme in the pharmaceutical or food manufacturing industry.

As a drug, it is widely used for the treatment of lactose intolerance (lactose-degrading enzyme deficiency) and diarrhea of infants.

In the food manufacturing industry, lactose-free milk is produced by whey (solid content 6.3%, lactose 4.5%) and whey powder (solid content 9%).
It is used to produce glucose and galactose by hydrolyzing wastes containing a large amount of lactose (4.6%, lactose 72.3%).

The β-galactosidase actually used in these industrial fields is mainly Aspergillus oryzae, Kluyveromyces lactis, or K. fragilis. Is produced by.

Since the physicochemical properties of these enzymes differ considerably depending on their origins, they are used by selecting the usage situations in which each enzyme exerts its maximum activity.

For example, pharmaceuticals need to exhibit stable activity in acidic gastric juice when administered orally, so the optimum pH is on the acidic side, and due to the long history of use as a koji mold, it is safe. A problem-free β-galactosidase produced by Aspergillus oryzae is used.

<Prior Art> β-galactosidase is generally poor in stability as an enzyme preparation, and is very unstable particularly in an aqueous solution.

For example, when β-galactosidase derived from Aspergillus oryzae is stored in an aqueous solution, the maximum activity is 4 ° C.
It is known that it can be maintained for several months at, but it can be maintained at 37 ℃ for only a few days. [Journal of Biochemistry, Vol. 80, 11
Page 95, 1976. Therefore, only solid preparations that need to be stored in a cold place (15 ° C or less) are marketed as pharmaceutical products.

Although there is a demand for the development of liquid medicines because of the expansion of usage and ease of use, they have not yet been marketed.

As a method for stabilizing the β-galactosidase aqueous solution,
Regarding β-galactosidase derived from Escherichia coli, when cryopreserving an aqueous solution, a method of preventing deactivation of the enzyme at the time of freezing by adding about 0.1 to 5% of saccharide, gelatin or glycerin to the solution. (JP-A-58-81782).

Β- from yeast (Kluyveromyces fragilis) origin
Add 10-80% sorbitol to the galactosidase solution.
A method of adding a stable aqueous composition. (USP 446446
9) Step of separating and purifying β-galactosidase from the fermentation broth
In the liquid to be treated, as a stabilizer , Mn , Mg Over
And at least one sulfur-containing amino acid and phosphate ion are allowed to coexist.
How to do.

(Japanese Patent Publication No. 49-20515) and the like are known.

It is also known that β-galactosidase has a remarkable difference in physicochemical properties as an enzyme molecule depending on the type (origin) of the microorganism that produces them, when the individual enzymes are compared in detail.

Table 1 compares the literature values of various properties of 9 kinds of β-galactosidases with different origins, and the molecular weights are
10.5 to 54 (10,000), optimum pH is 3.2 to 8.3, optimum temperature is 46 to 80
(° C) and Km values show large variations with 0.72 to 9.8 (mM to ONPG).

As described above, β-galactosidase has marked differences in properties depending on the origin, so in actual use, a physicochemical property suitable for the purpose of use is selected, and a stabilization method suitable for the enzyme is selected. Is currently being adopted.

* Producing bacteria E. collie: Escherichia coli. K. fragilis: Kluyveromyces fragilis. S. punomonia: Streptococcus pneumoniae. A. oryzae: Aspergillus oryzae. A. niger: Aspergillus niger. Penicillum multicolor. T. Thermophilus: Thermus thermophilus. B. Stearothermophilus: Bacillus stearothermophil
us. ** References Methods in Enzology
ymology) 5, p. 212, 1962. Agricultural and Biological chemistr
y) 36, 570-577, 1975. Biochemistry Volume 3, 1535
Page, 1964. Journal Abu Biochemistry (Journal
of Biochemistry) 77, 241-247, 1975. Method in Entimology Volume 28, 728-734
Page, 1972. Agricultural and Biological Chemistry Vol. 43, pp. 943-950, 1979. Ibid. 47, 2533-2540, 1983. JP-A-56-154991. Canadian Journal of Microbiology Volume 22, 8
Pages 17-825, 1976. <Problems to be Solved by the Invention> An object of the present invention is to provide a stable β-galactosidase aqueous composition derived from Aspergillus oryzae.

Currently, the only commercially available β-galactosidase as a drug is a solid preparation derived from Aspergillus oryzae.

As described above, solid preparations of β-galactosidase are obliged to be stored in a cold place. Therefore, the current situation is that the distribution process and handling of pharmaceutical products containing this enzyme as a main drug are greatly restricted.

Further, when a solid preparation is given to, for example, an infant or an infant, it is difficult to drink as it is, and therefore it is often troublesome to dissolve the solid preparation in water or milk before giving it.

In view of these points, it is desired to develop a liquid formulation which is stable from room temperature to high temperature, has little microbial contamination, and is easy to drink.

Under the circumstances described above, the present inventors have examined the use of various stabilizers, and found that stable Aspergillus
The present invention has been completed by succeeding in creating an aqueous composition of β-galactosidase derived from oryzae.

(2) Structure of the Invention The present invention is one in which one or more sugar alcohols selected from maltitol, xylitol and sorbitol are 50 to 50%.
The present invention relates to a stable β-galactosidase aqueous composition derived from Aspergillus oryzae, which is characterized in that it is contained at a concentration of 80% (W / W).

The sugar alcohols used in the present invention can be obtained as powders or aqueous solutions at low industrial cost.

Β-derived from Aspergillus oryzae used in the present invention
Galactosidase has been confirmed to be safe due to its long history of use, and there is no concern about side effects.

The concentration of sugar alcohol used is preferably 50 to 80% (W / W).

If it is less than 50%, the stabilizing effect is poor, and if it exceeds 80%,
In terms of stability, there is no great difference, but the sugar alcohol solubility exceeds the upper limit, making handling difficult.

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

Example Aspergillus β-gal. (Production No. 914153) 685,000 U was dissolved in 500 ml of 1% sorbitol, and this solution was used as a Diaflow hollow fiber system (Amicon, DC2, USA).
Type, HIP30-2 as hollow fiber cartridge
Concentration and dialysis (using distilled water as external dialysis solution) with 0 type), and further ultrafiltration (using PM30 overmembrane made by Amicon Inc. in the US) to obtain 68 ml of enzyme solution (10,000 U / ml, 96.5 mg protein / m)
l) got. 1.0 ml of this enzyme solution was added to 3.45 g of sorbitol and 0.5 ml of distilled water and dissolved to obtain an aqueous β-galactosidase composition having an enzyme concentration of 2020 U / ml and a sorbitol concentration of 70% (W / W).

This water-based composition is about 1 ml for each milk given to infants.
Addition to the extent that diarrhea has a therapeutic and prophylactic effect.

In this case, compared to conventional solid agents (powder, granules, etc.), it is easier to add to and mix with milk, and even when added to milk heated to 50 to 60 ° C, it hardly deactivates. It shows the characteristics.

In addition, almost all microorganisms such as mold, bacteria, and yeast do not grow in this aqueous composition, except for very limited microorganisms such as osmotic yeast Kluyveromyces luxii (K. rouxii). , It can be used safely in terms of hygiene.

In addition, if desired, fragrances that are acceptable as food additives
Of course, dyes, etc. can be added as appropriate.

(3) Effect of the Invention The β derived from Aspergillus oryzae is stable according to the present invention.
-A galactosidase aqueous composition is provided.

Below, test examples are shown to explain the effects of the present invention.

Test Example 1. (Stability comparison test of enzymes of different origins) Test method 50% (W / W) of commercially available β-galactosidase with different origins of enzymes (enzyme-producing microorganisms) to a concentration of 20 units / ml
It was dissolved in sorbitol aqueous solution and 70% (W / W) sorbitol aqueous solution, sealed in a test tube at 5 ml and kept warm in a 70 ° C water bath, and the enzyme activity was measured after 2 hours. The residual activity ratio of each test enzyme was calculated in comparison with the activity.

One unit (U) of enzyme was the amount of enzyme that hydrolyzes 1 μmol of O-nitrophenyl β-D-galactopyranoside (hereinafter referred to as ONPG) per minute. However, since the physicochemical properties of the enzyme differ depending on the origin, the enzyme activity was measured under conditions such that the pH, temperature, and substrate concentration were optimized for each enzyme, as described below.

Test Enzymes The following four types of commercially available β-galactosidases having different origins were used as test enzymes.

1) β-galactosidase produced by Escherichia coli.

(Commercial product manufactured by US Sigma Company, Catalog No. T6008 Grade 6, hereinafter referred to as "E. coli β-gal") 2) β-galactosidase produced by Kluyveromyces lactis.

(Lactase GODO-YNL, a commercial product manufactured by Godo Sake Co., Ltd., hereinafter referred to as "yeast β-gal.L") 3) β-galactosidase produced by Kluyveromyces fragilis.

(Commercial product manufactured by US Sigma Company, Catalog No. G-7013 Grade 12, hereinafter referred to as "yeast β-gal.F") 4) β-galactosidase produced by Aspergillus oryzae.

(Commercial item manufactured by Shin Nippon Chemical Industry Co., Ltd., hereinafter referred to as "Sumilacto L""Aspergillusβ-gal.") Test Results The results of this test are shown in Table 2.

That is, Aspergillus β-gal dissolved in an aqueous solution of 50% sorbitol and 70% sorbitol was hardly inactivated when kept at 70 ° C for 2 hours. In contrast, Escherichia coli β-gal, yeast β
-Gal.L and F are both unstable, and sorbitol 5
In the case of 0% (W / W) solution, the enzyme activity disappeared completely, 70%
Even in the case of (W / W) solution, the residual activity rate is 25.3%,
It was a very low value of 66.1% and 3.6%.

Moreover, as is clear from the results in Table 2, even if β-galactosidase originates from a microorganism belonging to the same genus (Kluyveromyces), the species of the microorganisms that produced them (lactis and Fragilis) differ. Then, the stabilization effect of each enzyme (yeast β-gal.L and yeast β-gal.F) by the addition of sorbitol showed a large difference.

In the present invention, the activity of each enzyme was measured by the following method so that the pH, temperature and substrate concentration were optimized for each enzyme.

Activity measurement of Escherichia coli β-gal. That is, 1.25 ml of 0.1M sodium phosphate buffer (pH 7.3), 3.36M2-
A solution consisting of 50 μm of mercaptoethanol, 50 μm of 30 mM magnesium chloride aqueous solution, and 50 μm of the enzyme solution under test at 37 ° C. for 3
After incubation for 30 minutes to activate the enzyme, 0.1 mM containing 34 mM ONPG was added.
Add 100 μM sodium phosphate buffer (pH 7.3) and incubate at 37 ℃ for 10 minutes, then add 1 mM ethylenediaminetetraacetate (hereinafter referred to as EDTA) disodium
The reaction was stopped by adding 0.5 ml of 1M sodium carbonate to 420 nm.
The absorbance was measured to determine the amount of O-nitrophenol produced in this solution.

Method for measuring the activity of yeast β-gal.L and F Methods such as Wendorf [Journal of Milk and Hood Technology (Journal of
Milk and Food Technology) 34, 451, 1971] was slightly modified. That is, 3 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 27 mM ONPG, 5 mM manganese chloride aqueous solution
After mixing 1 ml and 1 ml of the enzyme solution to be tested and incubating at 37 ℃ for 10 minutes
1 ml of 1 M sodium carbonate solution containing 1 mM EDTA disodium
Was added and centrifuged at 3000 rpm for 5 minutes to obtain a supernatant.
Absorbance at m was measured.

Aspergillus β-gal. Activity measurement method 3.5 ml of 5.7 mM ONPG solution adjusted to pH 4.5 and 0.5 ml of test enzyme
The mixture was kept at 30 ° C. for 10 minutes, the reaction was stopped by adding 1 ml of 1M sodium carbonate solution, and the absorbance of this solution at 420 nm was measured to determine the amount of O-nitrophenol produced.

In the above methods 1) to 3), 1 unit (U) of enzyme is 1
The amount of enzyme that hydrolyzes 1 μmol of ONPG per minute was used.

Test Example 2 (Stability Comparison Test by Type of Sugar Alcohol) Aspergillus β-gal. Was sufficiently dialyzed against distilled water to be desalted, and then the concentration was 430 U in various sugar alcohol aqueous solutions having a concentration of 50% (W / W). Dissolve each to 5 g, put each solution in a test tube for 5 g, and measure the enzyme activity after keeping it warm in a 70 ° C water bath for 30 minutes.
The residual enzyme activity was calculated by comparing with the activity before the incubation.

The results of this test are shown in Table 3.

Test Example 3 (Stability Comparison Test of Sugar Alcohol Concentrations by Concentration) Aspergillus β-gal.
10% (W / W), 30% (W / W), 50% (W / W), 70% (W / W) and 80% (W / W) Sorbitol aqueous solution with a concentration of 430 U / g Each solution was sealed in a test tube, and the enzyme activity after 30 minutes of incubation in a 70 ° C water bath was measured, and the residual enzyme activity rate was calculated by comparing with the activity before incubation.

For comparison, a similar test was conducted using water instead of the sorbitol aqueous solution.

The results of this test are shown in Table 4.

Test Example 4 (Heat resistance test) Two kinds of β-galactosidases produced by microorganisms belonging to Aspergillus oryzae species but having different strains were each brought to a predetermined concentration in a 70% (W / W) concentration sorbitol aqueous solution. Dissolve and seal in a test tube with a volume of 5 ml.
The enzyme activity after incubation for 1 minute and 2 hours was measured, and the residual enzyme activity was calculated by comparing with the activity before incubation.

The test enzymes and enzyme concentrations are as follows.

(A) Test enzyme Sample 1: (Aspergillus β-gal.) Sample 2: [β-galactosidase produced by Aspergillus oryzae U-8 (Microtechnology Research Institute No. 7378)] (b) Enzyme concentration Sample 1 : 766U / ml Sample 2: 272U / ml The results of this test are shown in Table 5.

As is clear from Table 5, the β-galactosidase aqueous composition of the present invention showed heat resistance with little activity decrease even at a high temperature of 80 ° C. for a relatively short time.

Claims (1)

[Claims] 1. One or more sugar alcohols selected from maltitol, xylitol and sorbitol are used.
A stable β-galactosidase aqueous composition originating from Aspergillus oryzae, which is characterized by containing at a concentration of 80% (W / W).