JPH0657151B2 - Enzyme stabilization method - Google Patents

Description

The present invention relates to a method for stabilizing enzymes, and more particularly to a method for stabilizing proteases in detergents.

[Prior Art] In recent years, an enzyme-containing detergent that takes into account the proteolytic action of an enzyme has been developed for the purpose of washing stains derived from biological proteins (stain, blood, etc.) that cannot be washed sufficiently with ordinary detergents. It is in the spotlight.

The enzyme-containing detergent is a general detergent base material, for example, an anionic surfactant as a detergent main ingredient, and sodium tripolyphosphate, sodium sulfate, carboxymethylcellulose, diatomaceous earth, etc. added as a builder, and an enzyme blended therein. As an enzyme, an alkaline protease that is active under the pH conditions of an aqueous detergent solution is generally used.

[Problems to be Solved by the Invention] Anionic surfactants, which are the main ingredients of detergents, are accompanied by unfavorable properties such as partial inactivation of the enzyme and a tendency to weaken the stability of the enzyme. Therefore, practically, it is desired to minimize the activity decrease during storage, and various stabilization methods have been proposed.

Since enzymes are usually stable in the dry state, they can be stabilized relatively easily when used as granules, but they tend to become unstable in the presence of water, and especially in the case of liquid detergents, there are various proposals. The reality is that it has not been fully resolved.

[Means for Solving Problems] The present inventors searched for a compound that suppresses the inactivation of the enzyme contained in various detergents, and when the reversible inhibitor was bound to the enzyme, the solution was used. Further, they found that the stability of the enzyme was improved in the powder state, and completed the present invention.

That is, the present invention provides a reversible inhibitor that binds to a protease and inhibits the action of the protease.
The present invention provides a method for stabilizing the protease, which comprises adding 5 equivalents to an amount that does not actually show an inhibitory effect under the conditions of use.

In the enzyme stabilization method of the present invention, those generally used as reversible inhibitors of enzymes can be used, but in particular, peptide low molecular weight inhibitors such as chymostatin, leupeptin, antipain and phosphoramidon, soybean trypsin. Inhibitors (hereinafter abbreviated as STI) Streptomyces subtilisin inhibitors, high molecular weight protein inhibitors such as ovo inhibitors, and carbobenzyloxyphenylalanine (hereinafter abbreviated as Z-Phe), phenylboric acid, N, N -Compounds such as dibenzyl urea that have a non-biological origin and have a reversible enzyme inhibitory effect have an excellent effect on stabilizing the enzyme in the detergent, and when these are added together with the enzyme to the detergent, the enzyme and the inhibitor are complex. Form a long-lasting activity, the enzyme activity does not decrease much, and it can be sufficiently put into practical use as an enzyme-containing detergent. Found.

Proteolytic enzyme (protease) used for detergent
There are various types of industrial resources, such as the food industry,
Any of the proteases used in various fields such as leather industry, pharmaceutical industry, textile industry, and seafood processing industry can be used, but particularly alkaline protease active under alkaline conditions of detergent aqueous solution, for example, subtilisin-Karlsberg ( Subtilisin Carlsberg), Subtilisin
BPN '(hereinafter abbreviated as BPN'), API-21, etc. are enemies.

In the present invention, in addition to a reversible inhibitor of the enzyme, the storage stability of the enzyme can be improved by further using a calcium salt such as acetic acid, sulfuric acid, carbonic acid, hydrochloric acid, nitric acid and phosphoric acid calcium salt, preferably calcium sulfate salt. Can be further improved.

The addition amount of the reversible inhibitor of the enzyme used in the enzyme stabilization method of the present invention is a concentration that effectively acts as an inhibitor in a storage state and does not practically show an inhibitory effect under diluted actual use conditions. When AIP-21 is used as the enzyme and chymostatin is used as the inhibitor, the amount is in the range of 0.5 equivalent or more with respect to the enzyme, and the enzyme inhibitor concentration under actual use conditions is 1 μM or less.

Further, it is preferable that the calcium salt which is a stabilizing aid is used by adding 0 to 40% by weight to the enzyme.

Stabilization of an enzyme according to the present invention specifically involves mixing an enzyme and a reversible inhibitor of the enzyme to form an enzyme-inhibitor complex,
Further, if desired, a calcium salt is added to obtain a stabilized enzyme composition. The thus-obtained stabilized composition can be used for detergents or other purposes such as silk scouring, leather processing, and desizing of clothes.

[Example] Hereinafter, the effect of the enzyme stabilization method of the present invention will be described with reference to Examples.
The case of powder detergent and liquid detergent will be specifically described.

(1) Powder detergent 0.05 g of enzyme (BPN ') and an inhibitor / enzyme equivalent ratio of the inhibitor (chymostatin and Z-Ph shown in Table 1).
e) and a mixture of sodium sulfate are placed in a container, mixed well, and 5 g of detergent (commercial detergent: New beads) is added to this.
After mixing for 40 hours, 110 hours and 160 hours after placing in a thermo-hygrostat at 40 ° C and 80% relative humidity, the residual enzyme activity of each sample was measured (see Japanese Examined Patent Publication No. 60-55118). ),
The results shown in Table 1 were obtained. Table 1 also shows the results with the addition of calcium sulphate and without the use of inhibitors for comparison.

(2) Liquid detergent LAS 10% by weight, alkoxyethoxylate 33% by weight, triethanolamine 8% by weight, ethanol 8% by weight and water 41% by weight In a liquid detergent 30 ml, enzymes (BPN 'and trypsin) 30 mg, Inhibitors / enzyme equivalent ratios of the inhibitors (chymostatin, leupeptin, STI) as shown in Table 2 were added, mixed well and placed in a constant temperature bath at a temperature of 45 ° C for 48 hours, 90 hours and 187 After a lapse of time, the residual enzyme activity of each sample was measured, and the results shown in Table 2 were obtained. Table 2 also shows the results when no inhibitor was used for comparison.

From Tables 1 and 2, it is clear that the enzyme stability is improved by using the enzyme inhibitor, and the result is more remarkable by the addition of the calcium salt.

Claims (6)

[Claims] 1. A reversible inhibitor that binds to a protease and inhibits its action is added in an amount of 0.5 equivalent to the protease in an amount that does not actually show an inhibitory effect under the conditions of use. A method for stabilizing the protease, comprising: 2. The stabilization method according to claim 1, wherein the reversible inhibitor is a peptidic low molecular weight inhibitor. 3. The stabilizing method according to claim 1, wherein the reversible inhibitor is a proteinaceous high molecular weight inhibitor. 4. The stabilization method according to claim 1, wherein the reversible inhibitor is a compound of non-biological origin. 5. The stabilization method according to any one of claims 1 to 4, further comprising adding a calcium salt in an amount of up to 40% by weight with respect to the protease. 6. The stabilization according to claim 5, wherein the calcium salt is at least one salt selected from calcium salts of acetic acid, sulfuric acid, carbonic acid, hydrochloric acid, nitric acid, phosphoric acid and polyphosphoric acid. Law.