JPH1169973A - Stabilization of enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving a long term preservation stability of an enzyme aqueous solution containing a cellulase and an amylase. SOLUTION: A polyol of 20-50 wt.%, a preservative of 0.01-1 wt.%, and salts of 2-10 wt.% are added to an enzyme aqueous solution and the pH of the aqueous solution is regulated to 7-8 to stabilize the enzyme in the enzyme aqueous solution containing the enzyme of 0.1-30 wt.% in the method for stabilizing the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for stabilizing an enzyme. More specifically, the present invention relates to a method for improving the long-term storage stability of an aqueous enzyme solution containing cellulase or amylase.

[0002]

2. Description of the Related Art Aqueous enzyme products are widely used in the industry where a large amount of enzymes are used because of their excellent handleability as compared with powder products.
However, generally such enzyme products in aqueous solution form
There is a problem that the product is extremely unstable, has poor storage stability, and lowers activity as compared with powder products. Conventionally, blending of various additives has been attempted for the purpose of improving the stability of an aqueous enzyme solution product. For example, attempts have been made to add a preservative such as salt or a polyhydric alcohol such as glycerol, but the effect is insufficient. Furthermore, a method of adding a stabilizer obtained by combining a polyhydric alcohol and gelatin and / or casein and ethyl alcohol (Japanese Patent Publication No.
No. 1-152), a method in which an enzyme is covalently bonded to a soluble polymer, and albumin or the like is further added to the aqueous solution (Japanese Patent Application Laid-Open No. 57-122795), a method in which dicarboxylic acid and reducing inorganic salts are added ( Japanese Patent Publication No. 59-21779
No. 9) has been proposed. According to these methods, although a certain degree of effect is recognized in the short term, the effect is not sufficient, and there is a problem that the enzyme activity is remarkably inferior, especially during long-term storage such as two months or more.

[0003]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems and to provide a method for remarkably improving the long-term storage stability of an aqueous enzyme solution containing cellulase or amylase. The present inventors have found that the combined use of a stabilizer and the adjustment of pH are extremely effective, and have completed the present invention. That is, the present invention relates to an aqueous enzyme solution containing 0.1 to 30% by weight of an enzyme, in which 20 to 50% by weight of a polyol, 0.001 to 1% by weight of a preservative, 2 to 10% by weight of a salt are added, and the pH of the aqueous solution is adjusted. It is a method for stabilizing an enzyme, which is adjusted to 7 to 8.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The enzymes that are the object of the present invention include cellulase,
Examples include carbohydrases such as amylase and pectinase, esterases such as lipase, protease, and lyase. In particular, it is suitable for use in carbohydrases such as cellulase and amylase. In the present invention, the enzyme content in the enzyme aqueous solution is 0.1 to 30% by weight, preferably 0.1 to 30% by weight.
5 to 10% by weight. The higher the concentration, the higher the activity of the obtained stabilized aqueous solution, and the more desirable. However, an enzyme aqueous solution having a high concentration of more than 30% by weight is liable to form a precipitate, which is not preferable in handling the product.

[0005] Examples of the polyol to be added to the enzyme aqueous solution of the present invention include ethylene glycol, propylene glycol, glycerol, sorbitol, mannitol and maltitol, and it is particularly preferable to use glycerol. The addition amount of the polyol is 20 to 50% by weight, preferably 30 to 40% by weight in consideration of the viscosity and the solubility of other components.
It is. If the amount is less than 20% by weight, the effect is small, and if the amount exceeds 50% by weight, the effect is saturated. In order to increase the enzyme activity of the product, it is desirable that the amount added be as small as possible within the above range.

The preservatives to be added to the enzyme aqueous solution of the present invention include benzoic acid, sodium benzoate, ethyl benzoate, phenoxyethanol, 5-chloro-2-methyl-
4-isothiazolic acid ("Caisson", manufactured by Rohm and Haas Co., Ltd.), 1,2-benzothiazolic acid ("Proxel", manufactured by Zeneca Corporation) and the like. The added amount of the preservative is 0.0001 to 1% by weight, and preferably 0.001 to 0.1% by weight in consideration of the solubility. If the amount is less than 0.0001% by weight, the effect is small. Even if added excessively, the effect is saturated, and the preservative is expensive and a high concentration of the preservative leads to inactivation of the enzyme. It is.

The salts added to the enzyme aqueous solution of the present invention include inorganic salts such as borate, sulfate, carbonate, phosphate, hydrochloride, citrate, tartrate, erysorbate, and the like.
Organic salts such as succinate, fumarate and the like can be mentioned, and metals constituting the salt include sodium, potassium, magnesium, calcium and the like. Particularly preferred are borates, sulfates and citrates. The salt is added in an amount of 2 to 10% by weight, and preferably 3 to 10% in consideration of solubility.
9% by weight. If the amount is less than 2% by weight, the effect is small, and even if added excessively, the effect is saturated.

[0008] The enzyme aqueous solution of the present invention is essentially required to have the above three kinds of stabilizers added thereto and to have a pH adjusted to 7 to 8. If the pH is less than 7 or more than 8, the storage stability decreases. The pH is adjusted to 7 to 8 by, for example, adding an aqueous solution of sodium hydroxide or acetic acid.

[0009]

According to the present invention, the long-term storage stability of the aqueous enzyme solution containing cellulase or amylase can be remarkably improved without reducing the enzyme activity even during long-term storage such as two months or more. .

[0010]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
In the following examples,% indicates% by weight. Example 1 Stabilizers shown in Table 1 were added to a cellulase concentrate or an amylase concentrate in the amounts shown in Table 1, and the pH values shown in Table 1 were respectively added.
An enzyme aqueous solution having the following formula was prepared. This enzyme aqueous solution
After storage for a month, the residual enzyme activity was examined. Table 1 shows the results. The cellulase concentrate or amylase concentrate used is as follows. Cellulase concentrated solution; aqueous solution containing a crude enzyme having cellulase activity (derived from Bacillus sp. KSM635-KNV (FERM P-13549)) (enzyme protein concentration: 2.5% by weight) Amylase concentrated solution; crude enzyme having amylase activity ( Aqueous solution containing Bacillus sp. KSM-AP1378 (derived from FERM BP-3048) (enzyme protein concentration; 0.6% by weight) The enzyme activity was measured by the following method. Measurement of cellulase activity: CMC (2.5%) 0.4 ml, 0.
0.1 ml of 5M glycine buffer (pH 9.0) and 0.
0.1 ml of enzyme solution is added to a 3 ml reaction solution, and
The reaction was performed for 20 minutes. After the reaction, the reducing sugar was quantified by the 3,5-dinitrosalicylic acid (DNS) method. That is, in order to measure the amount of reducing sugar produced, 1 ml of the DNS reagent was added to 1 ml of the reaction solution, and the mixture was heated at 100 ° C. for 5 minutes, cooled, and cooled.
It was diluted with 0 ml of deionized water and colorimetrically determined at a wavelength of 535 nm. The enzyme titer was defined as the amount of an enzyme that produces a reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions as one unit, and the fermentation productivity was expressed in units of enzyme per liter of culture solution. Measurement of amylase activity: 0.9 ml of a substrate solution obtained by dissolving Sigma soluble starch (final concentration 0.5% w / v) in glycine-NaOH buffer (pH 10, final concentration 40 mM).
0.1 ml of the enzyme solution was added thereto, and the mixture was reacted at 50 ° C. for 15 minutes. After the reaction, the reducing sugar was quantified by the 3,5-dinitrosalicylic acid (DNS) method. That is, the DNS reagent 1 was added to 1 ml of the reaction solution.
Add color at 100 ° C for 5 minutes and cool down.
The mixture was diluted with 1 l of deionized water and colorimetrically determined at a wavelength of 535 nm. The enzyme titer was defined as the amount of an enzyme that produces a reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions as one unit, and the fermentation productivity was expressed in units of enzyme per liter of culture solution.

[0011]

[Table 1]

Claims (3)

[Claims] 1. An enzyme aqueous solution containing 0.1 to 30% by weight of an enzyme, 20 to 50% by weight of a polyol and 0.005% of a preservative.
A method for stabilizing an enzyme, comprising adding 01 to 1% by weight, 2 to 10% by weight of a salt, and adjusting the pH of an aqueous solution to 7 to 8. 2. The method according to claim 1, wherein the enzyme is cellulase or amylase. 3. The method according to claim 1, wherein the salt is a borate, a sulfate or a citrate.