JP2902747B2 - Method for producing modified protease - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a modified protease chemically modified with a polysaccharide.

(Prior art) In general, proteases originating from animals, plants, or microorganisms are used in various pharmaceuticals such as detergents, digestives and anti-inflammatory agents, cosmetics, meat softeners, silk refining, and beer manufacturing processes. It is widely and effectively used in industrial fields.

However, when proteases are applied to detergents, cosmetics, or certain pharmaceuticals, they are antigenic and sensitizing to the skin because they are heterogeneous proteins to the human body, and may cause strong irritation to some people. It has been pointed out that. Another problem is that the stability of the protease is not sufficient. In particular, in mediums with a high water content or in dosage forms such as aqueous solutions, in addition to denaturation, autolysis digestion occurs, and when stored at room temperature, it is quickly deactivated, so it is difficult to supply stable products. is there.

To solve safety problems such as the antigenicity of enzymes, for example, in order to administer therapeutic enzymes in vivo, uricase and asparaginase are treated with polyethylene glycol to suppress antigenicity and improve blood half-life. Modification method (Japanese Patent Publication No. Sho 61-42558) and a method of modifying streptokinase with polyethylene glycol (JP-A-57-1187).
No. 89) has been proposed. In addition, it has been shown for enzymes such as chymotrypsin that modification that contributes to intramolecular crosslinking is effective for solving the stability problem. (Biochimicaet Biophysica Acata, 522, 277-283
(1978). Id., 485, 1-12 (1977)). Furthermore, it has been shown that those obtained by binding water-soluble polymers such as polysaccharides, polyethylene glycol, and proteins to manganese-type superoxide dismutase have reduced antigenicity and improved thermostability. JP-A-58-16685).

However, there is no known protease capable of improving its stability as well as suppressing its antigenicity and skin sensitization with respect to protease, and has been put to practical use. Above all, since skin sensitization is a sharp reaction, it is extremely difficult to suppress it. Further, since the substrate of the protease is usually of a high molecular weight, the activity of the protease is almost completely lost or the thermal stability is reduced depending on the modification method and the degree thereof.

The present inventors have widely used proteases in the fields of detergents, cosmetics, pharmaceuticals, and the like. Therefore, proteases and polysaccharides have already been linked via a triazine ring as one that acquires safety and stability while maintaining high activity. And a method for producing a modified protease by reacting a polysaccharide with cyanuric chloride to synthesize a triazine ring-bonded polysaccharide, and then reacting the triazine ring-bonded polysaccharide with the protease. . (Japanese Patent Application No. 1-
41065) The stability of the modified protease obtained in this way in aqueous solution is significantly better than that of the original enzyme,
Depending on the formulation of detergents, cosmetics, pharmaceuticals, etc.
May not show sufficient stability. As a result of investigating the cause, there is some variation in the stability of the resulting modified protease depending on the preparation conditions, and this difference may be amplified and appear in a dosage form of a system that easily causes inactivation of the enzyme over time. found. In addition, the degree of suppression of sensitization of the obtained modified enzyme was sometimes incomplete.

Further, it has been desired to improve the efficiency of introducing cyanuric chloride into dextran as an effective active group slightly lower.

(Problems to be Solved by the Invention) In view of the above problems of the existing modified protease, the present inventors have made intensive studies and as a result, completed the present invention. It is an object of the present invention to provide a method which is industrially, reproducibly and economically excellent in producing a water-soluble modified protease having the stability and safety of the above and which can be used in a wide variety of dosage forms.

(Means for Solving the Problems) The above-mentioned object is to produce a modified protease by reacting a triazine ring-bonded polysaccharide obtained by reacting a polysaccharide with cyanuric chloride with a protease to produce a modified protease. Reactive chlorine content of 0.4 as linked polysaccharide
修飾 1.2 mmol / g, and reacted in the presence of a reactive base of a triazine ring polysaccharide at a molar ratio of 2 or more with respect to the reactive amino group of the protease. In the production method and the reaction between the triazine ring-bonded polysaccharide and the protease, the triazine ring-bonded polysaccharide is added to the protease in a weight ratio of 3%.
This is achieved by the manufacturing method according to claim 1, which is used twice or more times.

 Hereinafter, the present invention will be described in detail.

As the protease used in the present invention, for example,
Examples include animal-derived proteases such as trypsin and chymotrypsin, and microorganism-derived proteases. All of the modified proteases of the present invention have reduced antigenicity and skin sensitization, and have greatly improved stability. However, the stability is relatively different depending on the protease. In terms of stability, many proteases derived from microorganisms are superior to proteases derived from animals. Therefore, good results are obtained by using preferably a protease derived from a microorganism, particularly preferably a protease derived from the genus Bacillus.

Examples of the polysaccharide used in the present invention include agarose,
Examples include natural polysaccharides such as guar gum, inulin, starch, dextran, pullulan, xanthan gum, carrageenan, pectin, and alginic acid, and derivatives thereof, and hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. Among them, dextran and pullulan are excellent in that the solution viscosity is low and the reaction operation is easy even when those having a considerably high molecular weight are used, and the performance of the obtained modified protease is uniform and stable.

If the molecular weight of the polysaccharide is not particularly small, the stability of the modified protease will give good results, but the antigenicity and skin sensitization may not be completely suppressed. It is preferable to use those having 10,000 or more, and particularly preferably 40,000 or more.

In the modified protease, the magnitude of the effect of suppressing the antigenicity and skin sensitization of the original protease and the degree of stabilization are as follows:
It varies depending on the type, molecular weight, binding amount and state of the polysaccharide used. In general, when the amount of binding is increased, the safety and stability are improved, but the protease tends to be inactivated. In many cases, the activity of the modified protease obtained by performing modification until the objective is achieved is extremely low. However, the modified protease of the present invention has a very high activity even when the modification rate is considerably increased. Conversely, even if the modification rate is low, the modified protease of the present invention has a sufficiently high stability and safety, which is also a feature of the modified protease. From these points, the modification rate of the surface amino group of the protease is preferably 30% or more, more preferably 50% or more, as measured by the TNBS method.

The modified protease of the present invention can be obtained by reacting a polysaccharide with cyanuric chloride to synthesize a triazine ring-bonded polysaccharide, and then reacting this with a protease.

In particular, in order to reproducibly produce a modified protease having a high degree of stability and safety, it is important that the triazine ring-bonded polysaccharide has an active group density of a certain level or more.

However, when the active group density is excessively high, the polysaccharide is insolubilized by self-crosslinking or gelation occurs partially. In an extreme case, there is a phenomenon that the modification rate of the enzyme is rather lowered, and a sufficient sensitizing effect cannot be obtained.

The active group density in this case is the amount of reactive chlorine bound to the triazine ring in the activated polysaccharide, and this amount can be determined by a silver nitrate titration method described later.

For the reasons described above, the reactive chlorine content of the triazine ring-bonded polysaccharide needs to be in the range of 0.4 to 1.2 mmol / g, and preferably 0.5 to 1.0 mmol / g.

In order to control the preparation process of such an activated polysaccharide and synthesize it with good reproducibility, the solvent of the cyanuric chloride solution is a non-aqueous solvent that is water-miscible and has no reactivity with cyanuric chloride, The reaction is carried out by adding a cyanuric chloride solution to the aqueous polysaccharide solution and controlling the pH at the time of the reaction to preferably 7.5 to 9.5, more preferably 8.0 to 9.0. Suitable solvents for the cyanuric chloride solution include acetone, tetrohydrofuran, dioxane, dimethylsulfoxide and the like, and acetone is particularly preferred because of its ease of use and easy removal of the solvent. By setting the conditions in this manner, an active reactive chlorine group can be introduced into the polysaccharide with excellent reproducibility, the reaction rate between the polysaccharide and cyanuric chloride is high, and the amount of cyanuric chloride used can be reduced. The reaction is preferably performed at around room temperature. In addition, since the active reactive chlorine group introduced into the polysaccharide is decomposed as the reaction proceeds, the reaction is preferably completed within 30 minutes. The reaction is stopped under acidic conditions of about pH 3.
If necessary, a poor solvent such as acetone may be added under acidic conditions for separation and purification. In the binding reaction between the triazine ring-bonded polysaccharide and the protease, the triazine ring-bonded polysaccharide is used at least three times in weight ratio to the protease, and the amount of reactive chlorine in the triazine ring-bonded polysaccharide is determined by the reactivity of the protease. It is preferable that the molar ratio is twice or more with respect to the amino group, and it is particularly preferable that the amount of reactive chlorine with respect to the reactive amino group is five or more. That is, the larger this value is, the higher the modification ratio of the enzyme is, and particularly when the ratio is 5 times or more, a very stable modified protease can be obtained. In addition, even if the above-mentioned weight ratio and molar ratio conditions of the activated polysaccharide and the protease are not satisfied, a quite stable modified protease may be obtained, but the suppression of antigenicity and skin sensitization is still unsatisfactory. Is insufficient. As the modification rate increases, the stability tends to increase, but the activity yield slightly decreases. Therefore, when formulated into a dosage form that contributes to the stabilization of the enzyme and used, a slightly lower modification rate can be selected.

The protease used may contain other protein components or a low molecular weight amino group-containing substance depending on the degree of purification. Amino groups derived from these also react with the activated polysaccharide and affect the modification rate of the protease. Therefore, as the reactive amino group of the protease in the present invention,
It means the sum of the surface amino groups of the protease itself and the amino groups derived from the mixed substance.

Then, in an aqueous solution of a low molecular weight compound having an amino group to treat the remaining active groups, preferably 50 to 50
Work up at 75 ° C. The low molecular weight compound having an amino group used in the present invention is not particularly limited, but it is hardly a source of sensitization from the viewpoint of safety, such as amino acids such as glycine, alanine, lysine, serine, and glutamic acid, and monoethanolamine. Substances that do not adversely affect the structure of the protease are preferred.

The number of triazine ring-bonded halogen atoms remaining in the modifying enzyme decreases with treatment time, but this rate tends to increase with increasing temperature and pH. The pH value of the aqueous solution is usually in the range of 5 to 10, but it is preferably 6.5 to 9.5 as a condition for avoiding denaturation and deactivation of the enzyme and obtaining high treatment efficiency.

The required processing time varies depending on the processing temperature and pH conditions.
When the content of the bound halogen atom is more than 500 ppm, the powder is partially gelled when stored for a long time even at room temperature, the solubility in water is reduced, and the activity is also reduced, but the content of the bound halogen atom is reduced. By controlling the content to 500 ppm or less, such a problem can be suppressed. In addition, by selecting the temperature conditions of the present invention, the inactivation of the enzyme during the treatment can be almost avoided. By performing this post-treatment, an object of stable quality can be obtained even in a powder state.

The resulting modified protease can be purified by ultrafiltration or gel permeation chromatography, and the like. Powdering methods further include removing the solvent under reduced pressure, freeze-drying,
Precipitation by addition of a poor solvent with ethanol or the like can be used.

(Effect of the Invention) By the method of the present invention, it is possible to produce a modified protease that has little or no antigenicity and skin sensitization and has extremely high heat stability with good reproducibility. . Further, the decrease in activity due to modification is small, and the activity is very high. Since it is stable even in a system containing a surfactant at a high concentration and suppresses self-decomposition inactivation, it is suitable for blending into various aqueous formulations.

Furthermore, even in the form of a solid such as a powder, it does not cause insolubilization or decrease in activity during storage, and can withstand heat sterilization at 105 ° C, so that it can be applied to a wide range of dosage forms, and can be used as a detergent
It can be used effectively for cosmetics, pharmaceuticals, etc.

In the present invention, the measurement and evaluation of heat stability, skin sensitization, and the content of amino groups and halogen atoms on the surface of protease were carried out by the following methods.

(1) Measurement of thermal stability (aqueous) The modified protease was dissolved in a 50 mM phosphate buffer (pH 6.8), and a solution prepared at 0.5 mg protein / ml was used as a test solution. After incubating the test solution at 60 ° C. for 6 hours or at 40 ° C. for 3 months, the enzyme activity in the test solution was measured and compared with the activity before the treatment to determine the residual ratio. The stability in the model dosage form was evaluated in the same manner, and the activity remaining rate after treatment at 60 ° C. for 6 hours was evaluated.

(2) Evaluation of skin sensitization Maximization method [Bertil, Ma
nd Albert, MK, J. Invest. Derm., 52 (3), 268 (196
9)], a skin sensitization test was conducted.

The induction and induction concentrations were set so that both the protease and the modified protease had a protein amount of 0.05% by weight.
The degree of skin sensitization was evaluated by the average evaluation point obtained by the method shown below.

(3) Measurement of Protease Surface Amino Group Modification Rate The measurement of the reactive amino group of the protease was carried out by the method of Haynes et al. [Haynes, R. et al., Biochemistry,
6 , 541 (1967)] as the reaction amount of trinitrobenzenesulfonic acid (TNBS), and the modification rate of the modified protease was determined by measuring the amount of unreacted amino groups on the enzyme surface and comparing it with the surface amino group amount of the unmodified form. The modification ratio of the surface amino group was calculated from the ratio.

 The amount of protease was determined from the absorbance at 280 nm.

(4) Measurement of Reactive Chlorine Content in Activated Polysaccharide 100 mg of a sample is dissolved in 4 ml of water, 1 ml of 0.5M-NaHCO ₃ is added, and heat treatment is performed at 100 ° C. for 30 minutes. 7% chromic acid aqueous solution
Add 0.5 ml, dilute further with water, and titrate with 0.1 N silver nitrate aqueous solution. (V ₁ ml) As a control, titration was also performed without alkali decomposition treatment. (V ₀ ml) The corresponding chlorine amount was calculated from the value of (v ₁ −v ₀ ), and was calculated as the reactive chlorine content. .

 Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 125 g of dextran (average molecular weight 4 × 10 ⁴ ) was dissolved in 2.5 l of water. To this, various amounts of 1,3,5-trichlorotriazine (cyanuric chloride) dissolved in 600 ml of acetone were dropped and mixed, and reacted under the conditions shown in Table 1, respectively. However, the pH was adjusted using 1N NaOH, and the temperature was 18 to 22.
° C.

After the addition was completed, 0.1N HCl was added to adjust the pH to 3. This was added to 20 l of acetone, and the precipitated crystals were collected and washed with acetone to obtain activated dextran.

Next, 10 g of the activated dextran thus obtained was added to water 1
In 100 ml of water, a protease derived from Bacillus licheniformis (an enzyme produced by Novo, and purified from Esperase (trade name)) dissolved in 10 ml of water and added thereto, followed by 100 ml of 0.2 M borate buffer (pH 9.2). Was added and reacted at 25 ° C. for 20 hours. However, the protease has a reactive amino group content of 0.6 mm.
ole / g, and the amount of reactive chlorine / the amount of reactive amino group was set to be 10 in a molar ratio.

After the modification reaction, 1.3 g of glycine was added to and dissolved in the reaction system, followed by heat treatment at 60 ° C. for 30 hours, followed by removing each low-molecular substance four times by ultra-pass operation, washing, concentrating and freeze-drying. Yielded a light brown powder. The activity, protein surface amino group modification ratio, skin sensitization and aqueous stability of these modified protease powders were evaluated. The results are shown in Table 1.

From Table 1, the amount of reactive chlorine in the activated polysaccharide was 0.4 to 1.2.
It can be seen that when the amount is mmol / g, a modified protease excellent in both stability under aqueous conditions and skin sensitization can be prepared. Also, the amount of reactive chlorine in the activated polysaccharide is 1.2 mmol / g.
In the case where the ratio exceeds 2, gelation often occurs as in Comparative Example 2, which is not preferable.

Example 2 10 g of activated dextran prepared under the same conditions as in Experimental Example 2 of Example 1 was dissolved in 100 ml of water, and purified esperase was added and reacted in the same manner as in Example 1.

However, purified Esperase has a reactive amino group content of 0.72 mM
le / g, and the amount of reactive chlorine / reactive amino group was set as shown in Table 2.

Table 2 shows the evaluation results of the obtained modified protease.

From Table 2, it is necessary that the molar ratio of the amount of reactive chlorine / the amount of reactive amino group be 2 or more in order to prepare a modified protease excellent in both stability and safety. It is clear that a more preferable result is obtained when the ratio is 5 or more.

Example 3 A modified protease was prepared according to the method of Example 1. Table 3 shows the amounts of cyanuric chloride, reaction pH, and reaction time used for preparing activated dextran. As the protease, purified Esperase having a reactive amino group content of 0.55 mmol / g was used, and in the modification reaction, activated dextran in an amount 8 times the weight of the enzyme was subjected to the reaction.

Table 3 shows the evaluation results of the obtained modified protease.

As is evident from Table 3, the modified protease prepared under the conditions of the present invention shows excellent stability and safety.

Example 4 0.2% of the modified protease obtained in Experimental Example 8 of Example 3 was added to various basic dosage forms shown in Table 4 at 60 ° C.
The activity after heat treatment for 6 hours was measured, and the stability was compared with the original enzyme.

As evident from the results in Table 4, the modified protease shows excellent stability even in these model dosage forms.

 ────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-156468 (JP, A) JP-A-62-175175 (JP, A) JP-A-58-16685 (JP, A) 500309 (JP, A) Prikl. Biokhim. Mik robiol. , 15 (1) 1979, p. 82 −87

Claims (2)

(57) [Claims] 1. A process for reacting a triazine ring-bonded polysaccharide obtained by reacting a polysaccharide with cyanuric chloride and a protease to produce a modified protease, wherein the reactive chlorine content as the triazine ring-bonded polysaccharide is used. Is 0.4 to 1.2
A water-soluble modified protease, characterized in that the protease is used in the presence of a reactive base of a triazine ring-bonded polysaccharide at a molar ratio of at least 2 times the amount of the reactive amino group of the protease, using a mol / g thereof. Manufacturing method. 2. The water-soluble compound according to claim 1, wherein the triazine ring-bonded polysaccharide is used at least three times in weight ratio with respect to the protease in the reaction between the triazine ring-bonded polysaccharide and the protease. Method for producing modified protease.