JP2919912B2 - Modified protease and method for producing the same - Google Patents

Description

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

(Prior art) In general, proteases originating from animals, plants, or microorganisms are used for various pharmaceuticals such as detergents, digestives and anti-inflammatory agents, cosmetics, meat softeners, silk scouring or 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 Acta, 522, 277-283 (1
978). 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-
However, it has been found that the modified protease thus obtained has high stability in an aqueous solution, but when it is powdered and stored, it becomes insoluble over time, resulting in a decrease in activity.

(Problems to be Solved by the Invention) The present inventors have made intensive studies in view of the above-mentioned problems of the existing modified protease, and as a result, completed the present invention. Another object of the present invention is to provide a modified protease which does not cause insolubilization and decrease in enzyme activity even when stored in a powder state for a long period of time. Other objects and advantages of the present invention will become apparent from the following description.

(Means for Solving the Problems) That is, in the present invention, a protease and a polysaccharide are bonded via a triazine ring, and the content of a halogen atom bonded to the triazine ring is 500 ppm or less. After synthesizing a modified protease and a triazine ring-bonded polysaccharide by reacting the polysaccharide with cyanuric chloride, the triazine ring-bonded polysaccharide is reacted with the protease, and then the reaction product is converted into an amino group. Is achieved in an aqueous solution of a low molecular weight compound having the formula (1), whereby the content of halogen atoms bonded to the triazine ring is reduced to 500 ppm or less.

 Next, 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.
The synthesis reaction of the triazine ring-bonded polysaccharide is preferably carried out at pH 8 to 11, and more preferably at pH 8 to 9.

Further, as this synthesis method, a method of adding cyanuric chloride to a polysaccharide aqueous solution as a solution of a non-aqueous water mixed solvent such as acetone is preferable, and a highly active activated compound can be obtained with good reproducibility. The resulting triazine ring-bonded polysaccharide is
If necessary, separation and purification may be carried out by adding a poor solvent under acidic conditions.

If the amount of the triazine ring introduced into the triazine ring-bonded polysaccharide is small, the modification rate of the amino group decreases, and the degree of stabilization of the modified protease and suppression of antigenicity tend to be reduced accordingly. The amount of the triazine ring in the saccharide is preferably 3 × 10 ⁻⁴ mol / g or more.

In the binding reaction between the protease and the triazine ring-bonded polysaccharide, it is preferable to use the triazine ring-bonded polysaccharide at a weight ratio of at least three times that of the protease. Even if it is less than 3 times, a modified protease having a considerably high stability can be obtained, but there are cases where the modified protease cannot be obtained completely in suppressing antigenicity and skin sensitization. Further, since the performance of the modified protease obtained even when the polysaccharide is excessively added is saturated, it is preferable to use the modified protease in an amount of 20 times or less.

Next, in an aqueous solution of a low molecular weight compound having an amino group, post-treatment is preferably performed at 50 to 75 ° C. The low molecular weight compound having an amino group used in the present invention is not particularly limited, but includes amino acids such as glycine, alanine, lysine, serine and glutamic acid, and monoethanolamine and the like.
From the viewpoint of safety, a substance that hardly becomes a sensitizing source and does not adversely affect the structure of the modified protease is preferable.

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 processing temperature is preferably from 50 to 75 ° C. If the temperature is too low, it takes a long time to reduce the content of the bound halogen atom to 500 ppm or less. Also, if the temperature is too high,
During the necessary processing, the enzyme deactivation proceeds in parallel,
Activity decreases. Also, 55-
Most preferably, it is performed in the range of 70 ° C.

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) The modified protease of the present invention has almost or completely suppressed antigenicity and skin sensitization, and has remarkably high heat stability. 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) to obtain 0.5 mg protein / ml, which was used as a test solution. After the test solution was incubated at 60 ° C. for 6 hours, the enzyme activity in the test solution was measured, and the residual ratio was determined by comparing the activity with the activity before the treatment.

(2) Measurement of thermal stability (powder) The modified protease powder was dried and heat-treated at 60 ° C. for 7 days, and then dissolved in 50 mM phosphate buffer (pH 6.8). The activity was measured and compared with the activity before the dry heat treatment to determine the activity retention. When a complete solution was not obtained, a uniform dispersion was prepared and used for measurement.

(3) Evaluation of skin sensitization Maximization method [Bertil, Ma
nd Albert, M, K., 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 0.02% by weight. The degree of skin sensitization was evaluated by the average evaluation point obtained by the method shown below.

(4) Measurement of Shrinkage Ratio of Protease Surface Amino Groups [Haynes, et al., Bioche
mistry, 6 , 541 (1967)], the amount of unreacted amino groups on the surface of the modified protease was measured as the reaction amount of trinitrobenzenesulfonic acid (TNBS). Was calculated.

(5) Measurement of halogen atom content Fluorescence X
The content of halogen atoms was determined by a line analysis method.

 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 of water. This was dissolved in 625 ml of acetone at room temperature
25 g of 1,3,5-trichlorotriazine (cyanuric chloride) at pH
The solution was added dropwise over 8 minutes while adjusting to 7 to 9. pH adjustment is 1NN
Performed using aOH.

After the addition was completed, 0.1N HCl was added to adjust the pH to 3.
This was added to acetone 20 and the precipitated crystals were passed.
After washing with acetone, 144 g of activated dextran was obtained.

Next, 9 g of the activated dextran thus obtained was added to 90 parts of water.
and 1 g of Bacillus licheniformis-derived protease <produced by Novo, trade name Esperase> (hereinafter referred to as Esperase) dissolved in 10 ml of water.
100 ml of a 0.2 M borate buffer (pH 9.2) was added, and the mixture was reacted at 25 ° C for 18 hours.

Next, after adding and dissolving 1.2 g of glycine to the aqueous solution of the modified protease, the solution was divided into 40 ml portions, and heat-treated at 60 ° C for 0 to 36 hours. After removing and washing, the mixture was concentrated and freeze-dried to obtain a brown powder.

These modified protease powders were subjected to dry heat treatment at 60 ° C. for 7 days, and the water solubility, activity, and aqueous stability of each of the treated powders were evaluated. The activity, chlorine content, surface amino group modification rate, and skin sensitization of the sample before the dry heat treatment were measured and evaluated. Table 1 shows the above results.

Table 1 shows that the modified enzyme with a high chlorine content is gelled (water-insolubilized) by dry heat treatment, whereas the sample with an active chlorine content of 500 ppm or less by heat treatment does not cause gelation by dry heat treatment. It is clear that the decrease in activity is small.

No decrease in activity yield or induction of sensitization due to this heat treatment is observed, and no decrease in thermal stability in an aqueous system is observed.

In addition, the chlorine content is 500 ppm or less for 24 hours and
From the value of the activity yield of the heat-treated product for 36 hours (the modified enzyme of the present invention), it can be seen that the decrease in activity due to the treatment is very small.

It has been confirmed that the activity of the modified enzyme of the present invention is 10% or less even when subjected to dry heat sterilization at 105 ° C. for 1 hour, and no change in physical properties is observed. The utility is clear.

Example 2 In Example 1, Esperase (1 g) was modified using pullulan (average molecular weight: 5 × 10 ⁴ ) instead of dextran as the polysaccharide. However, the inactivation treatment of the active chlorine group of the triazine ring was performed at 63 ° C. for 24 hours after the addition of glycine, purified by the ultrafiltration method, and lyophilized to obtain 9.5 g of the modified protease. Activity yield 62%, chlorine content 17
It was 5 ppm. No skin sensitization was observed, and the average score was 0. When this product was subjected to a storage test for 6 months at 40 ° C., the solubility in a phosphate buffer (pH 6.8) was good, and no decrease in activity was observed.

Evaluation of aqueous thermal stability of powder after test (60 ° C, 6 hours)
Had an activity retention of 99%, and no change was observed.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-60-156468 (JP, A) JP-A-55-500309 (JP, A) Prikl. Biokhim. Microbiol. , 15 (1) (1979), p. 82-87 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. a) a protease derived from the genus Bacillus is linked to a polysaccharide via a triazine ring; b) the modification rate of the surface amino group of the protease is 30% or more; The residual amount is 500 ppm or less by weight. D) Retains water solubility even after being stored in a solid state at 60 ° C. for 7 days.
Further, a water-soluble modified protease having no decrease in titer. 2. A polyazine is reacted with cyanuric chloride to synthesize a triazine ring-bonded polysaccharide, then the triazine ring-bonded polysaccharide is reacted with a protease, and the reaction product is subsequently converted to a low molecular weight compound having an amino group. A method for producing a water-soluble modified protease, comprising heating in an aqueous solution of (1) to adjust the content of halogen atoms bonded to the triazine ring to 500 ppm or less.