JPH034791A - Stabilization of enzyme - Google Patents

Abstract

PURPOSE:To improve high-temperature stability of enzyme by blending an aqueous solution of a protease with >= specific amount of a polysaccharide. CONSTITUTION:An aqueous solution of a protease such as pepsin, chymotrypsin, trypsin or pancreatin is blended with >=0.05wt.%, preferably 0.5-10wt.% polysaccharide (preferably hyaluronic acid, alginic acid or salt thereof) and the enzyme is stabilized.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for stabilizing proteolytic enzymes.

(Conventional Technical Field) Enzymes are being increasingly used in many fields as catalysts that carry out specific and selective reactions at room temperature. Proteolytic enzymes are also effectively used in medicines, detergents, cosmetics, and silk scouring. However, these biocatalysts have low stability against heat and pH changes, which limits their applications.

In particular, if the form containing the enzyme is water-containing or an aqueous solution, there is a problem that the activity quickly decreases during storage. Measures are being taken to:

Enzyme inactivation is generally considered to be denaturation inactivation based on structural changes due to thermal movement, but in the case of proteolytic enzymes, inactivation also occurs due to mutual decomposition of the enzymes (autolysis).

Many proposals have already been made regarding the stabilization of proteolytic enzymes in consideration of such enzyme inactivation factors. For example, a method of adding casein, gelatin, etc. to an aqueous solution of a proteolytic enzyme (Japanese Patent Publication No. 41-182);
A method in which an enzyme is covalently bonded to a soluble polymer and further proteins such as albumin and gelatin are added to the aqueous solution (Japanese Patent Application Laid-Open No. 122796/1983), and a method in which calcium ions, sodium formate, and alcohol are added to a liquid detergent containing an enzyme. Method of adding etc. (Special Publication No. 58-11198
(Japanese Patent Publication No. 59-217799) and the like are known.

However, depending on the purpose, the above-mentioned existing methods have limitations on their formulations, and depending on the protein oil, there is a problem in that the decomposition of the protein oil may generate off-flavors.

Also, in terms of its effects, it has been recognized that it is effective to a certain degree, but a satisfactory effect has not yet been obtained. In particular, the stabilizing effect under high temperature conditions is low, and the activity is said to be significantly impaired when exposed to high temperature atmosphere during distribution and storage processes.

(Problems to be Solved by the Invention) The present inventors have completed the present invention as a result of intensive research to solve the above-mentioned problems. In particular, it is an object of the present invention to provide a method for stabilizing enzymes that significantly improves high-temperature stability and has a wide range of applications.

(Means for Solving the Problem) The above object is achieved by a method for stabilizing an enzyme, which is characterized by adding 0.06% or more of polysaccharide to an aqueous solution of a proteolytic enzyme.

The proteolytic enzymes used in the present invention are not particularly limited, but include animal-derived pepsin, chymotrypsin, trypsin, bancreatin, etc., plant-derived papain, microbial-derived proteolytic enzymes produced by Aspergis, Rhizopus, and Bacillus, etc. can be mentioned.

The polysaccharides of the present invention include gum arabic, guar gum,
Xanthan gum, locust bean gum.

Starch, dextran, pullulan, alginic acid.

Natural polysaccharides such as hyaluronic acid, carrageenan, pectin, and chitosan, salts and derivatives thereof, cellulose derivatives such as carboxymethyl cellulose, methyl cellulose, ethyl cellulose, and hydroxypropyl cellulose, and salts thereof, etc., are water-soluble. Although any substance can be used, hyaluronic acid, alginic acid, and their salts are preferred from the viewpoint of their effectiveness and ease of use. The concentration of the polysaccharide used varies depending on the type and molecular weight of the polysaccharide, but the higher the concentration, the greater the effect tends to be, and it is necessary to be at least O,OS weight %. Although the upper limit is not particularly limited, it is preferably 0.05 to 205 to 20% by weight or 0.5 to 10% by weight, considering that the effect will be saturated even if added excessively, physical properties such as viscosity, solubility, etc. Used within the range of

(Effects of the Invention) According to the method of the present invention, enzymes in an aqueous solution are stabilized, but the degree of stabilization depends on the type and concentration of the enzyme as well as the type and concentration of the polysaccharide. Furthermore, in the method of the present invention, 30°C
It is characterized by its remarkably high stabilizing ability in the above-mentioned high temperature range, and under certain conditions, its activity is hardly lost even when stored at 50°C for 24 hours.

The method of the invention can also be applied to chemically modified molecules or enzymes bound to insoluble carriers. Furthermore, a major feature is that not only can it be added together with other stabilizers, but it also exhibits its effect even when added to each fence surfactant aqueous solution system.

As mentioned above, the method of the present invention greatly improves the stability of enzymes in aqueous media, especially high temperature stability, has a wide range of applications, and is a safe method, so it can be used in cosmetics, medicines, detergents, etc. Not only can it be suitably applied to chemical products, but also
Trouble caused by exposure of these products to high temperatures during distribution and storage can be avoided.

In addition, when applying these proteolytic enzymes to food manufacturing and processing processes, and when using them for synthetic reactions,
This is a useful method.

Hereinafter, the present invention will be specifically explained with reference to Examples.

In addition, the activity measurement and preparation of test mulberry and solution in Examples were performed as follows.

[Activity measurement]

Protease activity was measured by the following method based on Anson's modified method.

Casein solution sml in test tube (18X180mox)
After leaving it for 10 minutes at sO ± 0.5°C, accurately measure and add 1 ml of the sample solution, and immediately shake and stir. This solution was left at 50 ± 0.5°C for exactly 1°, then 15ml of trichloroacetic acid test solution was added, shaken, and heated again at 50°C.
After leaving it for 50 minutes at 0±o, s 'c,
31, φsem). Accurately measure 2 rne of filtrate, add 5 ml of O, 56M sodium carbonate reagent and 1.0 ml of Folin reagent. After adding Qmll and leaving it for 30 minutes at 40°C, e
Measure the absorbance At at e o nm. Separately, as a blank test, accurately measure 1 ml of the test solution, add 5.0 ml of trichloroacetic acid, and shake. b0 Furthermore, add 6 ml of casein solution and shake to 50 ± 0.
．． Leave it at 5°C for 30 minutes, then repeat the same procedure to measure the absorbance.

The protease titer is calculated using the following formula. Under these conditions, the amount of enzyme that causes an increase in the non-protein folin reagent colored substance equivalent to the amount of tyrosine of 1 μf per minute is defined as 1 protease activity unit. 0 protease titer (units/
f) = (At-mu b) Measure the quality. After accurately cooling the milk casein corresponding to 1.2Of the dried product, adjusting the pH to 8.0 with 1N sodium hydroxide test solution, and adding water to 20
0m1 and °15&.

2) Trichloroacetic acid test solution 1.8 g of trichloroacetic acid and 1.8 g of anhydrous sodium acetate,
Add 6N acetic acid rt, smI and water to 8f and rlQOm
g and b0 3) 0.68M sodium carbonate test solution Add water to anhydrous sodium carbonate 68.5 F and dissolve.
4) Folin reagent phenol reagent (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) may be diluted 2 times and used.

Example 1 The proteolytic enzyme esperase (manufactured by Novo) produced by Bacillus licheniformis was purified and the effect of additives on stability at 50°C was investigated. The results are shown in Table 1.

As is clear from Table 1, monosaccharides and disaccharides commonly used as stabilizers also have a slight stabilizing tendency.
It can be seen that the stabilizing effects of polysaccharides, particularly sodium alginate and hyaluronic acid, are extremely high.

Example 2 The proteolytic enzyme biobrase conc. (Nagase Seikagaku Kogyo ■!!) derived from Bacillus was purified, and the same study as in Experimental Example 1 was conducted. The results are shown in Table 2.

Table 2 however Additive concentration enzyme Heat treatment pH 1% 40 units 1me 50'C 8.0 hr, however Additive concentration 1% Arsenine 40 units 1ml Heat treatment 60℃ 15hr.

pH 8.0 As is clear from the same table, polysaccharides, especially sodium alginate and hyaluronic acid, had a significant stabilizing effect on this enzyme.

Example 3 Enzymes prepared in the same manner as in Example 1 were dissolved in various solutions, and the effect on stability when 0.5% by weight of hyaluronic acid was added thereto was investigated at 50°C. This result is the third
Shown in the table.

From Table 3, it can be seen that the effect of addition is expressed even in other stabilizers and surfactants.

Example 4 The 37°C stability of an enzyme prepared in the same manner as in Example 1 was investigated in systems with and without the addition of 1% hyaluronic acid.

The activity residual rate after 10 days was 85% without the addition, but it became 82% with the addition of 1.0% hyaluronic acid.

Example 5 Table 4 shows the results of examining the relationship between the enzyme concentration, the concentration of hyaluronic acid added, and the 50°C stability.

Table 4 The results of the study are shown in Table 5.

From Table 6, it can be seen that a particularly large stabilizing effect is observed at a hyaluronic acid concentration of 0.06% or higher, and that the stabilizing effect increases as the concentration increases, and that the lower the enzyme concentration is, the greater the stabilizing effect is.

Example 6 In the same manner as in Example 5, stability at 31° C. Although the effect was not very great at 50° C., the stabilizing effect by adding hyaluronic acid was clearly observed.

Claims (2)

[Claims] (1) Add 0.0% polysaccharide to the aqueous solution of proteolytic enzyme
A method for stabilizing an enzyme, characterized by adding 5% by weight or more. (2) A method for stabilizing an enzyme, which comprises adding alginic acid and/or hyaluronic acid or a salt thereof to an aqueous solution of a proteolytic enzyme.