JPS61139384A - Production of stable enzyme solution - Google Patents

Abstract

PURPOSE:To produce a stable aqueous solution of an enzyme capable of preventing contamination of microorganism, inexpensively circulatable on the market, by adding a sugaralcohol to an enzyme to prevent the enzyme from precipitating, when the enzyme is dissolved in an aqueous solution of ethyl alcohol. CONSTITUTION:Amylase, protease, lipase, lipoxydase, lysozyme, catalase, glucose oxidase, or cell wall dissolving enzyme is dissolved in an aqueous solution of ethyl alcohol. In the operation, at least >= about 5%(W/W) sugaralcohol is added to the enzyme, so precipitation of the enzyme is prevented, and enczymatic activity is stabilized. Sorbitol, maltitol, or mannitol is used as the sugaralcohol.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for producing an enzyme solution, and more specifically, when dissolving an enzyme in an aqueous ethyl alcohol solution, sugar alcohol is added to prevent precipitation of the enzyme. This invention relates to a method for producing a novel and useful aqueous solution of #alcohol, thereby stabilizing enzyme activity.

(Prior art) In recent years, various enzymes such as amylase, protease, lipase, and cellulase, which originate from animals, plants, and microorganisms, as well as lysozyme, catalase, glucose oxidase, and lipoxidase, have been developed.
It has come to be used in a wide variety of industrial fields such as pharmaceuticals, food, feed, and leather.

These enzymes are usually unstable in aqueous solution, so
Most enzyme preparations are commercialized and distributed in the market in a solid state such as powder or granules. Therefore, when using these enzyme preparations, only the necessary amount is weighed out and dissolved in water to form a uniform solution or suspension before use. This method is often inconvenient, such as requiring techniques for accurate weighing or long mixing times.

If the supplied enzymes are in a long-term stable aqueous solution state,
Adjustment of the amount used, dilution, dissolution, and mixing operations become easy, and in some cases, it can be used as is, so it is thought that the ease of use of enzyme preparations will be dramatically increased.

(Problem to be solved by the invention) However, some enzymes in an aqueous solution maintain their activity stability if stored at low temperatures; Denaturation of the enzyme is generally more likely than in some cases.

Furthermore, if it is stored for a long period of time even at low temperatures, it may cause hygienic problems such as bacterial growth over time, which often makes it impossible to put it to practical use.

As a method for preventing bacterial growth in this aqueous enzyme preparation, sterile filtration using a filter or the like is usually performed, but this method requires a large amount of expense and is often not economically viable.

Furthermore, if the enzyme aqueous solution is frozen and stored at a temperature below freezing, the stability of the enzyme is generally maintained, but thawing treatment and frozen storage equipment are required before use, which increases costs in practice. The structure is complicated to use, and at present it has not been put into practical use at an industrial level.

(Means for Solving the Problems) Therefore, in order to obtain a stable enzyme aqueous solution that can prevent microbial contamination and can be marketed at low cost, the inventors of the present invention have conducted intensive studies and found that ethyl alcohol is added to the enzyme aqueous solution. It has been found that by adding a sugar alcohol and a sugar alcohol in combination, it is possible to prepare an enzyme preparation in a stable solution state, which was difficult to prepare in a simple aqueous solution state.

The present invention will be explained in detail below.

From the perspective of preventing microbial contamination of enzyme aqueous solutions, the present inventors have
We have found that an ethyl alcohol aqueous solution form that can prevent the growth of microorganisms such as bacteria in an enzyme solution is ideal. However, if the ethyl alcohol concentration is 30% or more, it will not freeze even when stored at freezing temperatures, so there is no need for thawing. However, we were faced with the problem that either the solution did not dissolve completely, or it formed a precipitate over time, making it impossible to achieve the intended purpose.

The precipitation of the precipitate in this way is due to the precipitation recovery of the enzyme,
This can be easily understood from the fact that organic solvents such as ethyl alcohol are used as a means of purifying crystals, and it was thought that it would be impossible to achieve the original purpose.

However, as a result of various studies on the conditions for preventing the precipitation of the enzyme in an ethyl alcohol aqueous solution and stabilizing the enzyme activity, the inventors of the present invention unexpectedly found that the enzyme was in an ethyl alcohol aqueous solution with a high concentration of 4%. The present inventors have discovered that even if sugar alcohol is dissolved, the precipitation can be prevented and enzyme activity can be maintained stably by adding sugar alcohol thereto, leading to the completion of the present invention.

The common concentration of sugar alcohol and ethyl alcohol in the present invention is determined by the mutual relationship between the respective concentrations of sugar alcohol and ethyl alcohol. The concentration also needs to be 10% (VXN) or more. Furthermore, the enzymes used in the present invention are not limited by their origin, degree of purification, or form of preparation.

(Example) Experimental Example 1 Experimental method Neutral protease of microbial origin was dissolved in phosphoric acid solution (pH 7,
After dissolving in 0), D-sorbitol (Japanese Pharmacopoeia Standard) and ethyl alcohol were added, and the mixture was stored under refrigeration at 2 to 4°C. Each concentration of the prepared enzyme ethyl alcohol aqueous solution was neutral protease 0.3%, ethyl alcohol 40%, D
- Sorbitol 0, 15, and 30% (Izuremo v
X,) It was torn. The precipitate formation rate is determined by the absorbance (280 nm) of the supernatant after centrifugation in an enzyme ethyl alcohol aqueous solution.
) was measured, calculated according to the following formula and the following table, and represented in the table.

The results are shown in Table 1. When neutral protease was used in a 40% ethyl alcohol aqueous solution without the addition of D-sorbitol, a precipitate was precipitated within one day, but by using D-sorbitol in combination, the formation of this precipitate could be suppressed. Moreover, this inhibitory effect was more effective when the D-sorbitol concentration was 30% than 15%. It was also found that the residual activity rate of neutral protease decreases with the formation of precipitates, and that the residual activity rate can be increased by keeping the precipitate formation rate as low as possible.

Based on this fact, it was found that there is a high correlation between the precipitate formation rate of the enzyme in an aqueous ethyl alcohol solution and the residual activity rate of the enzyme, so the precipitate formation rate should be measured in subsequent experiments. Experimental Example 2 Experimental Method A neutral protease of microbial origin was used as a guideline for determining the stability of enzyme activity. An ethyl alcohol aqueous solution was prepared according to the method, and the precipitate formation rate was measured after storage for 90 days. Ethyl alcohol concentration is 30.40 and 50%, D ~ sorbitol fi degree is 0.5.1 O12
0 and 30% (both 'X'). All other experimental conditions, measurement methods, and notations were in accordance with Experimental Example 1.

Experimental Results Table 2 Table 2 shows the relationship between the rate of precipitation of neutral protease in an aqueous ethyl alcohol solution and the concentration of D-sorbitol added. In 30%, 40%, and 50% aqueous ethyl alcohol solutions, the neutral protease that was dissolved in the absence of D-sorbitol precipitated in the form of a large amount of precipitate. On the other hand, by adding a small amount of D-sorbitol (both at a concentration of 5%), the formation of this precipitate could be considerably suppressed, and when D-sorbitol was added at a concentration of 20% or more, the formation of the precipitate could be almost suppressed.

Experimental Example 3 Experimental Method A neutral protease of microbial origin was prepared into an ethyl alcohol aqueous solution according to Experimental Example 1, and the precipitate formation rate was measured after storage for 90 days. All experimental conditions, measurement methods, and descriptions other than the ethyl alcohol concentration and D-sorbitol concentration were in accordance with Experimental Example 1.

Experimental Results Table 3 Relationship between the rate of precipitation of neutral protease in an ethyl alcohol aqueous solution and the ethyl alcohol concentration *Due to microbial growth? 'f? , '/@ generation was recognized.

The results are shown in Table 3. By adding D-sorbitol, the formation of precipitates could be suppressed, as can be seen from the above-mentioned experimental examples.

The precipitate formation rate increased in proportion to the ethyl alcohol concentration of the ethyl alcohol and coal aqueous solutions.

Experimental Example 4 Experimental Method Neutral protease of microbial origin was cleaved in ethyl alcohol aqueous solutions of various concentrations to which 15% D-sorbitol was added according to Experimental Example 1, and then stored at 7 to 9°C, and changes in the total number of viable bacteria. was measured. The total number of viable bacteria was measured by the plate dilution method according to a conventional method, and the culture temperature was 3.
The seeds (cultured for 7 days, 5 days, and 2 days, respectively) were used, and the results obtained by obtaining the maximum total number of viable bacteria among them were displayed.

(See next page) Experimental Results Table 4: Change in Total Viable Bacteria Number in Ethyl Alcohol Aqueous Solution of Neutral Protease” The results are shown in Table 4. Even if the enzyme solution is stored at a low temperature, after one month, a slight increase in the total number of viable bacteria is observed in the solution that does not contain ethyl alcohol, and after six months, the enzyme solution is completely consumed by the proliferated bacteria. It turned cloudy.

However, when the ethyl alcohol concentration exceeds 10%,
No growth of microorganisms was observed, and it was proved that using an ethyl alcohol aqueous solution is extremely useful from a sanitary standpoint.

Example 1 Ethyl alcohol aqueous solution (30% and 10% (:lZ) containing 0.3% () of lysozyme chloride (egg white lysozyme)
) Phosphoric acid ff1ff [p87.0) 100 g 5-30% (vX,) of various sugar alcohols such as D-sorbitol (Japanese Pharmacopoeia standard product), maltitol, and D-mannitol (food additive standard product) It was mixed and added so that The lysozyme chloride preparation thus obtained was
After being stored for 6 months in a refrigerator kept at a temperature of 0.degree. C., the precipitate formation rate was measured by applying the measurement method and notation method used in Experimental Example 1. The results are shown in Table 5, and even in the ethyl alcohol aqueous solution formulation of lysozyme chloride, the addition of D-sorbitol, maltitol, and D-mannitol was effective in preventing the precipitation of lysozyme chloride from the ethyl alcohol aqueous solution. admitted.

Table 5 Precipitate formation prevention effect of lysozyme chloride in ethyl alcohol aqueous solution by blending various sugar alcohols Example 2 Lysozyme chloride (egg white lysozyme) 0.05% (2)
Highly concentrated aqueous solutions of ethyl alcohol containing (50% and 6%)
0% (X) phosphate buffer pH 7,0) 10011,
Either D-sorbitol or maltitol from 10 to
It was added in a proportion of 25% (X). The lysozyme chloride preparation thus obtained was stored under the same conditions as in Example 1,
Precipitate production rate was measured according to Experimental Example 1. The results are shown in Table 6, and even in the preparation of a highly concentrated ethyl alcohol aqueous solution of lysozyme chloride, the addition of D-sorbitol or maltitol was effective in preventing the precipitation of lysozyme chloride from the ethyl alcohol aqueous solution. .

(See next page) Table 6 Effect of preventing precipitate formation in a highly concentrated ethyl alcohol aqueous solution of lysozyme chloride by blending various sugar alcohols Sakakita Example 3 Diastase (Japanese Pharmacopoeia standard product) 0.3% (X) and an ethyl alcohol aqueous solution containing 0.1% (x) of glucose oxidase (commercially available crude product)
-40% (X) To 1501, D-sorbitol (Pharmacopoeia standard product) was added to 0 to 30% (z). The precipitate production rate of the thus obtained enzyme preparation was measured in the same manner as in Example 1.

The results are shown in Table 7, and the effect of preventing precipitation of both enzymes from the ethyl alcohol aqueous solution was observed by incorporating D-sorbitol in the ethyl alcohol aqueous solution preparation of both enzymes, diastase and glucose oxidase.

Table 7 Effect of preventing precipitate formation in ethyl alcohol aqueous solution of diastase and glucose oxidase by blending D-sorbitol ethyl ID-
Precipitate formation rate [elementary alcohol sorbitol (6
After a month la degree% (X) Concentration% (X) elapsed);
As 5 ±1ze
2〇 -0 0
(Microbial growth) O ×
10
+ Kishi 40 2
0 -'-ze 30
- Example 4 α-amylase (commercially available powder product) 40% aqueous ethyl alcohol solution (X) containing 0.3% (VX), phosphate buffer p
H7,0) 100 It was blended with D-sorbitol (Japanese Pharmacopoeia standard product) at a concentration of 30% (x) to obtain #discarded solution.

After storing the main product in a refrigerator at 7 to 9° C. for 90 days, its precipitate formation and enzyme activity were measured using conventional methods, but no precipitate formation was observed, and sufficient enzyme activity remained.

Example 5 To 150 Il of 50+wM phosphate buffer (pH 1,0), lipase (commercially available powder product) 0.2% (vXN), ethyl alcohol 40% ($), D-sorbitol (food additive standard product) 30% (X ) to obtain an enzyme solution. The sample was stored at 7 to 9°C for 6 months, but no precipitate was observed.

Example 6 After dissolving cellulase (commercial product) in 30mM phosphate buffer (pf 17.0) 1006 to a concentration of 0.3% (vXI), ethyl alcohol (30%C%) D-sorbitol (food additive standard product) ) 30% (X) to obtain an enzyme solution. Although the sample was stored at 7 to 9°C for 3 months, no formation of precipitate was observed.

Example 7 Lipoxidase (commercial product> 300g
Ethyl alcohol and D-sorbitol were dissolved in 1-phosphoric acid 5 & buffer solution (pH 7.0) to a final concentration of 40% (
%) and 30% (X) to obtain an enzyme solution. After storing the sample at 7 to 9° C. for 3 months, the formation of precipitate was measured using a conventional method, but hardly any formation of precipitate was observed.

Example 8 Catalase (commercial powder product) 40% containing 0.2% (x)
(X) Ethyl alcohol aqueous solution (contains phosphate buffer pH 7)
,0) 1001 was mixed with D-sorbitol (Japanese Pharmacopoeia Crystal) at a concentration of 30% (vX, ) to obtain an enzyme solution.The drug was stored at 7 to 9°C for 3 months, but no precipitate appeared. No formation was observed.

Example 9 4 containing 0.3% ("X) of cell wall lytic enzyme of microbial origin
0% (z) ethyl alcohol aqueous solution (including phosphate buffer)
D-sorbitol (food additive standard product) is 30% <X
> to obtain an enzyme solution. Add this solution to 5 to 1
The product was stored at 0''C, and the presence or absence of precipitate formation was measured after 3 months, but almost no precipitate formation was observed.

Claims (3)

[Claims] (1) A method for producing a stable enzyme ethyl alcohol aqueous solution, which is characterized by adding a sugar alcohol when dissolving the enzyme in the ethyl alcohol aqueous solution. (2) Enzymes include amylase, protease, lipase, cellulase, lipoxidase, lysozyme, catalase,
The manufacturing method according to claim 1, which is glucose oxidase or a cell wall lytic enzyme. (3) The production method according to claim 1 or 2, wherein the sugar alcohol is sorbitol, maltitol or mannitol.