JPH07265075A - Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition - Google Patents

Abstract

PURPOSE:To obtain a stable modified protease composition, excellent in thermal and preservation stabilities and useful as a cosmetic, a detergent, a medicine, etc., by adding calcium to a modified protease prepared by binding a protease to polysaccharides through a triazine ring. CONSTITUTION:This composition is obtained by reacting polysaccharides with cyanuric chloride, further reacting the resultant active polysaccharides with a protease and adding a trace amount of calcium to the prepared modified protease. The obtained composition is stable even in a wet state and capable of manifesting the stability even when dissolved in an aqueous medium without causing the turbidity or precipitates even in aspects of properties as a product. The calcium is added in an amount of preferably about 0.01-1wt.%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a highly stable modified protease composition suitable for use in cosmetics, detergents and pharmaceuticals.

[0002]

BACKGROUND OF THE INVENTION Enzymes are widely used in various industrial fields such as detergents, scouring of textile products, decomposition of oils and fats, starch, food processing, pharmaceuticals, clinical tests, biosensors, cosmetics, and conversion / production of useful substances. Has been. One problem in measuring such utilization is that the stability of the enzyme is generally low, and the requirement is often not satisfied.
That is, most of the enzymes are easily denatured and inactivated by being heated, under extremely high pH conditions or, conversely, under low pH conditions, in the presence of a mixture of a surfactant, an organic solvent and the like, and further stored for a long time. . In particular, in the case of protease, it is difficult to supply stable products because in addition to denaturation, it undergoes autolytic digestion in dosage forms such as high-moisture content media and aqueous solutions, and is rapidly inactivated during storage at room temperature. There's a problem. Also, the enzymes used are of different origin to the human body,
When it is applied to medicines, cosmetics, detergents, etc., its antigenicity, skin sensitization and irritation become problems.

Chemical modification of enzymes has been attempted as a method for coping with these problems. For example, a method for improving clearance and antigenicity in blood by modifying uricase, asparaginase, streptokinase, etc., which are used as therapeutic enzymes, with polyethylene glycol (Japanese Patent Publication No. Sho 61).
No. 42558, JP-A-57-118789), a method of modifying superoxide dismutase with a polysaccharide or polyethylene glycol to suppress antigenicity and improve heat stability (JP-A-58-16685), Alternatively, chymotrypsin is modified to give intramolecular cross-linking, and stabilization is performed (Biochimica et Biophysica Act).
a 522,277 to 283 (1978), ibd 485,1 to 12 (1977)) and the like are disclosed.

[0004] In particular, regarding prostheses, the substrate is a polymer called a protein, and the modification generally impairs the activity to a large extent. In addition, it suppresses the skin sensitization and imparts a high degree of stabilization for practical use. Since no example was known, the present inventors investigated a method for simultaneously achieving both the purpose of suppressing skin sensitization and stabilizing, and as a result, a protease modified with a polysaccharide through a triazine ring and Proposal of its production method (Japanese Patent Laid-Open No. 2-219572, Japanese Patent Laid-Open No. 4-27388, Japanese Patent Laid-Open No. 4-88982, etc.) and chemical modification of a protease under appropriate conditions result in activity, stability, and safety. It has been reported that a modified enzyme excellent in physical properties such as solubility and water solubility can be obtained.

It is widely known that metal ions, particularly calcium in the case of protease, are effective for stabilizing the enzyme. Although such effects differ depending on the type of enzyme, methods for converting amino acid residues on the surface of the enzyme to increase its affinity for metal ions have also been studied for the purpose of stabilization (Biochemistry 27,8311-8317 (1988), etc.). .

[0006] Although it is effective to increase the affinity for metal ions by such a method, it is difficult to secure a high degree of stability by using protease alone because it exhibits an autodigestion phenomenon. Also, calcium (Ca)
Addition of a metal ion such as, etc. to a high concentration may impair its value as a product in some cases. That is, it may cause precipitation or turbidity under alkaline conditions or due to the formation of an insoluble substance with the mixture, or may reduce the product function in the case of detergents containing surfactants, etc. Is desirable.

[0007]

DISCLOSURE OF THE INVENTION The inventors of the present invention contain various components of the above-mentioned modified protease (Japanese Patent Application Laid-Open Nos. 2-219572, 4-27388, 4-88982, etc.). As a result of diligent studies to prepare a more highly stable system for use in actual products, the present invention has been achieved. It was found that an enzyme composition excellent in heat stability and storage stability in a wide range of conditions can be obtained without affecting.

[0008]

The present invention provides a modified protease composition in which a polysaccharide and a protease are bound to each other through a triazine ring, and a stable modified protease composition characterized by containing calcium. It solves the problem.

One aspect of the present invention is a stable enzyme-containing aqueous solution containing the modified protease composition.

The modified protease of the present invention can be produced basically according to the method described in the above-mentioned patent proposed by the present inventors. For example, the activated polysaccharide is synthesized by adding and mixing an acetone solution of cyanuric chloride to an aqueous polysaccharide solution. At this time, the pH is maintained at 8 to 9 with an aqueous NaOH solution or the like for the purpose of increasing the modification rate and ensuring quality such as stability. Immediately after completion of the activation reaction, adjust the pH to about 3 with hydrochloric acid or the like to make the activator relatively stable, add it to a large amount of acetone solution, precipitate it, separate it as a solid, wash and dry it to remove the activated polysaccharide. obtain. Alternatively, if the activator is not separated once and proceeded directly to the reaction with the enzyme, the acidified activator solution is purified by ultrafiltration or dialysis to remove unbound cyanuric chloride decomposition products, acetone solvent, etc. . When the activated solution is purified by dialysis, it is preferable to use a hollow fiber type dialysis tube. The polysaccharide is not particularly limited, but dextran and pullulan are preferable in that the solution viscosity is low, the reaction operation is easy, and the modification reaction can proceed uniformly. The molecular weight is 10,000.
The above is preferable.

The modification of the protease with the activated polysaccharide can be carried out by mixing and reacting the two in a buffer solution having a pH of about 7-9. As a protease,
Although not particularly limited, a microbial protease derived from Bacillus is preferably used. As the buffer solution, a borate buffer solution, a carbonate buffer solution or the like can be preferably used. At this time, the reactive chlorine content of the activated polysaccharide is 0.4 to
In addition to 1.2 mmol / g, this reactive chlorine amount is more than twice the molar ratio of the reactive amino groups of the protease, and the activated polysaccharide is more than three times the weight ratio of the protease. By doing so, a safe and stable water-soluble modified protease can be obtained with good reproducibility. The modified protease thus obtained still has some active residues on the triazine ring, so it may cause insolubilization when it is made into a powder state.
This deactivation treatment is carried out at about 0 ° C. to adjust the amount of chlorine derived from the triazine ring to 500 ppm or less based on the modifying enzyme. On this occasion,
It is possible to efficiently proceed by adding amino acids such as glycine, alanine, β-aminopropionic acid, lysine and serine, taurine, monoethanolamine and the like.
Further, usually, these amino compounds are added in an amount of 5 to 25% by weight based on the polysaccharide-enzyme conjugate.

[0012] The above-mentioned modified protease proposed by the present inventors has a completely suppressed autodigestibility, is water-soluble, and is itself very stable in an aqueous medium. Since various components are usually contained therein, the stability may be slightly impaired. However, Ca,
Stability can be improved by adding a polyvalent metal such as Mg, Ba, or Sr. In particular, Ca shows a remarkable stabilizing effect, and the modified protease itself also shows high stability in a water-containing powder state. One of the reasons for this is considered to be the increase in chelating ability due to the active group treating agent bonded to the hydroxyl group of the triazine ring residue or the triazine ring residue, and there are considerable differences depending on the type of active group treating agent. It is preferable to use any one of glycine, β-aminopropionic acid, taurine, or a mixture thereof as an active group treating agent in the preparation of protease. The content of the polyvalent metal is preferably 0.01% by weight or more in the modified protease from the viewpoint of the stabilizing effect, and it must be bound to the modified protease by a coordinate bond or an ionic bond. However, if the above conditions are met,
In addition, solid metal salt powder and the like may be contained. Generally, when a protease is used by adding it to the dosage form, the stability increases as the Ca ion concentration increases, but under alkaline conditions or when Ca ions bind to the components in the dosage form, turbidity / precipitation occurs. There is a drawback that causes. In contrast,
In the case of this modified protease, the content of metal ions is 0.0
If it is 1 to 1% by weight, such trouble does not occur in normal use, and a highly stable and clear product can be obtained. When there is no restriction on the appearance of the product and there is no particular problem in terms of performance such as turbidity or precipitation, the content of metal ions may be 1% by weight or more.

Stabilization of modified proteases with Ca
Although it is structurally considered to be due to the coordination of Ca ions to the protease, in order to add calcium to bring about such a state, the modified protease should be added to an aqueous solution containing 0.01% by weight or more of Ca ion relative to the amount of modified protease. After dissolving, the solvent may be removed. At this time, the solution pH is 6.0 to 1 from the viewpoint of its effect and enzyme deactivation.
It is preferably 0.0, and more preferably 7.0.
It is 9.5. A buffer salt may be added for pH adjustment, but it is necessary to select one that does not cause turbidity or insoluble precipitation by binding with Ca ions. Prior to removal of the solvent, if necessary, purification can be performed once by dialysis, ultrafiltration, etc. to remove the buffer salts and the like, but since the modified protease of the present invention has a high chelating ability, the Ca ion concentration should be slightly increased. Even if such an operation is added by setting it, the content of calcium in the modified protease is usually 0.01% by weight.
The above can be done.

The calcium-containing modified protease composition of the present invention can be stably used by adding it to an aqueous dosage form product, but a solvent other than water may be mixed in the dosage form depending on the purpose. Good. The Ca ion concentration in the dosage form system varies depending on the amount (concentration) of the modified protease unless otherwise added. For example, when a modified protease containing 0.2% of Ca ions was dissolved at a concentration of 10 mg / ml, the Ca ion concentration in the system was 0.5 mmol, but it was much more stable than the Ca ion-free system. In addition to exhibiting excellent effect, it has the same Ca content as in phosphate buffer.
Since it does not give turbidity even in a system which produces an insoluble salt, it can be used for a wide range of purposes.

[0015]

INDUSTRIAL APPLICABILITY The modified protease composition of the present invention in which a polysaccharide and a protease are bound to each other through a triazine ring is not only highly stable even in a wet state, but also in an aqueous medium. Even when it is dissolved in water and used, it shows extremely high stability even when the Ca ion concentration in the system is low, and it does not cause turbidity or precipitation in terms of product properties. It is extremely useful in other industrial applications.

[0016]

EXAMPLES The effects of the present invention will be described in detail below with reference to examples.

Example 1 5 l of 4% dextran aqueous solution (average molecular weight 4 × 10 ⁴ ).
At room temperature, 1.5 l of an acetone solution containing 5% of cyanuric chloride was added while keeping the pH at 7.5 to 9.5, and then the pH was adjusted to 3. This solution was added to an acetone bath, and the precipitated activated dextran was collected by filtration. 2 l of 0.1 M borate buffer (pH 9.0) in which 90 g of the activated dextran was dissolved was added to a protease powder derived from Bacillus lentus (previously belonging to Bacillus licheniformis) (Novo Nordisk, 10g of Esperase ^™ ) was added and reacted at 25 ° C for 20 hours, then 20g of glycine was added, and 35 ° C at 60 ° C.
Heat treatment was performed for a period of time, and the protease was modified with dextran. After the reaction, the aqueous solution of polysulfone was used as a dialyzer (PS-1.6UW, manufactured by Kuraray Co., Ltd.).
It was purified by dialysis with a membrane area of 1.6 m ² ). As processing conditions, dialysate (using ion exchange water) flow rate 1 l / min,
Flow rate of reaction solution: 85 ml / min, discharge flow rate: 63 ml
/ Min, the outlet side pressure was 100 mmHg. When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), the decomposition product of cyanuric chloride and its glycine derivative which were impurities in the reaction solution were not detected at all. The solvent was distilled off under reduced pressure to obtain a dextran-modified protease as a powder from the aqueous solution. The Ca content in the modified protease determined by fluorescent X-ray analysis was 450 ppm, but this Ca was derived from the starting enzyme. The Ca content in the raw material enzyme was 12300 ppm. 50m
100 ml of M sodium borate buffer (pH 8.5)
5 g of the modified protease obtained above is dissolved in 25 ml of calcium chloride (dihydrate), and then dissolved in a cuprophane dialysis tube.
It was dialyzed 3 times against 0 l. The modified protease was recovered in powder form by distilling off the water solvent under reduced pressure. Fluorescent X
The Ca content in the modified protease determined by line analysis was 1350 ppm (abbreviated as DMP + Ca).
In addition, 50 mM sodium borate buffer (pH 8.5)
5 g of the above modified protease was dissolved in 100 ml and placed in a cuprophane dialysis tube, and 5 mM-E of pH 8.5 was added.
DTA / 2Na salt (ethylenediamine tetraacetic acid / 2Na
(Salt) solution was dialyzed 3 times against 10 l of ion-exchanged water
A modified protease sample with Ca removed was obtained in powder form by dialysis in 1 and subsequent evaporation of the aqueous solvent under reduced pressure. C of this modified protease (abbreviated as DMP-Ca)
The a content was 80 ppm. DMP + C obtained above
a, DPM-Ca and unmodified Esperase were evaluated for stability under moist heat and solution conditions. The conditions and the results are shown in Table 1. The experiment was carried out in a sterile condition. Under all of the conditions, the modified form of the unmodified enzyme shows remarkably high stability, but it is clear that there is a significant difference depending on the Ca content.

[0018]

[Table 1] Experimental condition: (A) Powder, 40 ° C., 75% RH, 3 months. (B) 0.1 M phosphate buffer (pH 7.0), 70
C, 1 hour, modified enzyme concentration 2 mg / ml, (concentration of unmodified enzyme is 0.2 mg / ml).

Example 2 Ca removal modified protease sample prepared in Example 1 (D
MP-Ca, Ca content 80 ppm) was dissolved in 0.1 M borate buffer (pH 9.2) at a concentration of 1 mg / ml. After adding calcium chloride to this and adjusting to various Ca concentrations, it heat-processed at 75 degreeC and 1 hour, and evaluated residual activity. The results are shown in Table 2. Compared with a system in which Ca ions are removed (blocked) by adding ethylenediaminetetraacetic acid (EDTA) salt, a remarkable stabilizing effect is recognized particularly in a system containing 0.01% by weight or more of Ca.

[0020]

[Table 2] * 1: Ca content ratio to modified protease. * 2: EDTA ・ 2Na salt was added (5 mM concentration).

Example 3 After dextran and protease were bound in the same scheme as in Example 1, various amino compounds shown in Table 3 were added in place of glycine to perform post-treatment, and further, in accordance with Example 1. Then, dialysis purification, pulverization, and Ca addition treatment were performed to obtain each Ca-containing modified protease sample. However, the amount of amino compound used was 0.25 mol in all cases. Each Ca
Table 3 also shows the result of quantifying the Ca content of the modified protease sample containing by the fluorescent X-ray analysis. Each sample 10
0 mg was dissolved in 25 ml of water (concentration 0.4% by weight), calcium chloride solution was added to adjust the Ca content to 1800 ppm based on the modified protease, and water was added to make 50 ml. Furthermore, 0.2M phosphate buffer (p
H7.2) 50 ml was added to prepare a 0.1 wt% solution of each modifying enzyme. Neither turbidity nor the like was observed. Table 3 shows the results of evaluating the residual activity by subjecting this buffer system to heat treatment at 70 ° C for 1 hour. However, the stability of the unmodified enzyme Esperase shown in the comparison was evaluated in a 0.01 wt% solution. It is clear that the modification significantly improves the stability, but it is found that particularly good results are obtained when glycine, taurine, or β-aminopropionic acid is used as the amino compound. .

[0022]

[Table 3]

Example 4 Ca removal modified protease sample prepared in Example 1 (D
MP-Ca, Ca content 80ppm) 100mg,
It is dissolved in 0.1 M borate buffer (pH 8.5) in 10 ml, and calcium chloride (dihydrate) is added at various concentrations to dissolve it, and then placed in a cuprophane dialysis tube to prepare 3 parts per 1 l of ion-exchanged water. He dialyzed twice. The modified protease was recovered in powder form by distilling off the water solvent under reduced pressure. Ca content in the modified protease determined by X-ray fluorescence analysis and 0.1 M phosphate buffer (pH 7.
The dissolution state when 2% by weight was dissolved in 2) was evaluated. The results are shown in Table 4. The modified enzyme composition of the present invention gave a clear aqueous solution even in a phosphate buffer, but those containing 1 wt% or more of Ca produced turbidity.

[0024]

[Table 4]

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Claims (4)

[Claims] 1. A modified protease composition in which a polysaccharide and a protease are bound to each other via a triazine ring and which contains calcium and is stable. 2. A modified protease in which a polysaccharide and a protease are bound to each other through a triazine ring is reacted with cyanuric chloride to obtain an activated polysaccharide, and then the activated polysaccharide is reacted with the protease. The modified protease composition according to claim 1, which is the obtained modified protease. 3. The modified protease is treated with an aqueous solution containing at least one selected from glycine, β-aminopropionic acid, and taurine after the reaction between the activated polysaccharide and the protease, to give a triazine ring residue. The modified protease composition according to claim 2, wherein the amount of chlorine derived from the modified protease is 500 ppm or less based on the modified protease. 4. A stable enzyme-containing aqueous solution containing the modified protease composition according to claim 1.