JPH02219572A - Modified protease and production thereof - Google Patents

Abstract

PURPOSE:To produce a modified protease exhibiting suppressed cutaneous sensitization and antigenicity and having excellent stability and high activity by reacting a protease with a polysaccharide having bonded triazine ring and produced by reacting a polysaccharide with cyanuryl chloride. CONSTITUTION:A polysaccharide bonded with triazine ring is synthesized by reacting a polysaccharide (preferably dextrin, pullulan, etc.) with cyanuryl chloride preferably at pH8-11. The amount of triazine ring in the bonded polysaccharide is preferably >=3X10<-4>mol/l. The bonded polysaccharide is made to react with a protease originated preferably from microorganism (especially from genus Bacillus) at a weight ratio of >=3/1. The obtained modified protease can be purified by ultrafiltration. gel-filtration, liquid chromatography, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) 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 in detergents, pharmaceuticals such as digestive agents and anti-inflammatory agents, cosmetics, meat tenderizers, silk scouring, and beer manufacturing processes. It is widely and effectively used in various industrial fields.

However, proteases are used in detergents and cosmetics.

However, when applying to other types of medicines, proteases are proteins of foreign origin to the human body, so
It has been pointed out that it exhibits antigenicity and skin sensitization, and may cause strong irritation to some people.

Another problem is that the stability of protease is insufficient. In addition to denaturation, autolytic decomposition occurs especially in media with high moisture content or in dosage forms such as aqueous solutions.
Currently, it is difficult to supply a stable product because it quickly loses its activity when stored at room temperature.

To resolve safety issues such as antigenicity of enzymes, for example, to suppress antigenicity and improve and extend blood half-life for the purpose of injecting therapeutic enzymes into the body.

Method for modifying uricase and asparaginase with polyethylene glycol (Japanese Patent Publication No. 42658/1983),
A method of modifying streptokinase with polyethylene glycol (Japanese Unexamined Patent Publication No. 118789/1989) has been proposed. Furthermore, it has been shown for enzymes such as chymotrypsin that modification that contributes to intramolecular crosslinking is effective in solving stability problems. (Biochim
ica et geophysica277-283(
1117g). Same, 485.1-12 (11177))
Furthermore, manganese-type superoxide dismutase,
Those bound with water-soluble polymers such as polysaccharides, polyethylene glycol, and proteins have suppressed antigenicity and
It has been shown that thermal stability is improved (Tokusei Sho 58-
tsess issue).

However, there is no known product that suppresses antigenicity, skin sensitization, etc. and improves stability of proteases for practical use. Among these, skin sensitization is a sensitive reaction, so it is extremely difficult to suppress it. Furthermore, since the substrate of protease usually has a high molecular weight, depending on the method and degree of modification, the activity of protease may almost disappear or the thermostability may decrease.

(Problems to be Solved by the Invention) In order to use proteases widely in fields such as detergents, cosmetics, and pharmaceuticals, the present inventors have developed various types of proteases with the aim of maintaining high activity while ensuring safety and stabilization. The present invention was completed as a result of intensive search and research into modification methods.

That is, one object of the present invention is to provide a modified protease that has suppressed skin sensitization and antigenicity, excellent stability, and high activity.

Another object is to provide a method for producing the modified protease.

(Means for Solving the Problem) The above object is to synthesize a modified protease in which a protease and a polysaccharide are bonded via a triazine ring, and a triazine ring-linked polysaccharide by reacting the polysaccharide with cyanuric chloride. This is achieved by a method for producing a modified protease, which then reacts the triazine ring-linked polysaccharide with a protease.

Next, the present invention will be explained in detail.

Examples of proteases used in the present invention include animal-derived proteases such as trypsin and chymotrypsin, and microbial-derived proteases. All of the modified proteases of the present invention have suppressed antigenicity and skin sensitization, and also have greatly improved stability. but,
The relative stability varies depending on the protease. In terms of stability, many proteases derived from microorganisms are superior to proteases derived from animals. Therefore, good results can be obtained by preferably using a protease derived from a microorganism, particularly preferably a protease derived from the genus Bacillus.

Examples of polysaccharides used in the present invention include natural polysaccharides and their derivatives such as agarose, guar gum, inulin, starch, dextran, pullulan, xanthan gum, carrageenan, pectin, alginic acid, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, etc. Examples include. Among them, dextran and pullulan have low solution viscosity and are easy to perform in one reaction even if they have a fairly high molecular weight.
The performance of the obtained modified protease is also excellent in that it is uniform and stable.

Unless the molecular weight of the polysaccharide is extremely small,
Although the stability of the modified protease gives good results, it may not be able to completely suppress antigenicity and skin sensitization, so it is preferable to use one with an average molecular weight of 10.000 or more. , particularly preferably 40. Q
It is O0 or more.

The magnitude of the suppressive effect and the degree of stabilization of the antigenicity and skin sensitization of the primary protease in the modified protease vary depending on the type, molecular weight, binding amount, and state of the polysaccharide used. In general, increasing the binding amount improves safety and stability, but the protease tends to be inactivated, and the activity of modified proteases obtained by carrying out modifications to achieve the desired purpose is often extremely low. However, the modified protease of the present invention has very high activity even when the modification rate is increased considerably. This is a major feature of the present invention, which cannot be obtained with conventional techniques. In addition, a feature of the present invention is that it has sufficiently high stability and safety even when the modification rate is low; however, there is a correlation between the degree of modification and the modification rate.

From these points, it is preferable that the modification rate of the surface amino groups of protease is 30% 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-linked polysaccharide, and then reacting this with a protease. The synthesis reaction of the triazine ring-bonded polysaccharide is preferably carried out at a pH of 8 to 11.

The obtained triazine ring-bonded polysaccharide may be separated and purified by adding a poor solvent under acidic conditions, if necessary.

When the amount of triazine rings introduced into the triazine ring-bonded polysaccharide is small, the modification rate of amino groups decreases.The triazine mtt in the triazine-ring-bonded polysaccharide is preferably 3 X 10-'Sif 1 or more.

In the binding reaction between protease and triazine ring-linked polysaccharide, it is preferable to use the triazine ring-linked polysaccharide at a weight ratio of at least three times that of the protease.

Although it is possible to obtain a modified protease with considerably high stability even when the stability is less than 3 times, it may not be possible to obtain a modified protease with complete suppression of antigenicity and skin sensitization. Also,
Even if an excessive amount of polysaccharide is added, the performance of the resulting modified protease will be saturated, so the amount used is preferably 20 times or less.

After the binding reaction between protease and polysaccharide, the target product of stable quality can be obtained by adding lysine, glycine, aminoethanol, etc. to the excess active groups of the polysaccharide and performing post-treatment. .

Furthermore, the obtained modified protease can be purified by ultrafiltration, gel filtration, liquid chromatography, or the like.

(Effects of the Invention) The modified protease of the present invention has almost or completely suppressed antigenicity and skin sensitization, and has extremely high thermal stability. In addition, the decrease in activity due to modification is small, and the activity is extremely high. Furthermore, it maintains its stability even in a system containing a high concentration of surfactant, making it extremely useful.

In addition, since its self-decomposition and inactivation is suppressed, it has excellent storage stability in aqueous systems and can be effectively used in detergents, cosmetics, pharmaceuticals, and the like.

In the present invention, measurement of thermostability, antigenicity, skin sensitization, and surface amino groups of proteases.

Evaluation was performed using the method described below.

(υ Measurement of thermal stability Dissolve the modified protease in 50 mM phosphate buffer (pH 6, 8), Q, 5 ml protein/ml
This was used as the test solution. The test solution was heated to 60°C.
After incubation for a period of time, the enzyme activity in the test solution was measured.

(2) Evaluation of antigenicity Modified protease solution at a predetermined concentration (O ~ 1 ml pro
tein/me) Q, 4 ml + C1 0.4 ml of antiserum prepared separately in advance was added and incubated at 80°C for 2 hours. The generated precipitate is subjected to centrifugal force 7
75mM phosphoric acid [(1)H7,
8) After washing 3 times with tml, 0.1 N NaOH3
ml was added to dissolve this, and the absorbance at 285 nm was measured.

(3) Skin sensitization evaluation maximization method (
Bertil, Mand Albert, M.
J, Invest.

Derm, 52 (3), 268 (1989
) Based on ) skin A! Fli1 production test was conducted.

The induction and induction concentrations were set to be 0.02% by weight of protein for both protease and modified protease. The degree of skin sensitization was evaluated using the average score determined by the method shown below.

(, 4) Measurement of the surface amino group modification rate of protease using the method of Haynes et al.
,H,etal.

nsochemistry, 6. 841 (1a
trinitrobenzenesulfonic acid (
The amount of unreacted amino groups on the surface of the modified protease was measured as the reaction amount of TNB8), and the modification rate of the surface amino groups was calculated from the ratio to the amount of surface amino groups of the unmodified product.

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

(Example 1) Dextran (average molecular weight 6-9X10') IF was 50
Dissolved in rrl of water. To this, 1, which was dissolved in 35 ml of water-acetone mixed solvent (volume ratio 1:2.6) at room temperature,
3.5-Trichlorotriazine (cyanuric chloride) 11/
was added dropwise over 8 minutes while adjusting the pH to 8 to 11. 1)
Adjustment of H was performed using IN NaOH.

After dropping, add 0.1 N Hog to adjust the pH to 5.
Adjusted to. Add this to Ashiton sooml.

The precipitated crystals were filtered and washed with acetone to obtain activated dextran.

Next, 20 mf of protease derived from Bacillus licheniformis, manufactured by Kunovo, trade name Esperase (hereinafter referred to as Esperase), was added to a 0.1 M boric acid solution (pH 9.2).
Dissolve in 10 ml and add 0 of the above activated dextran to this.
．． 21 was added and the reaction was carried out at 4°C for 24 hours. After adding glycine + omy to this reaction solution and treating it for 2 hours, the solution was purified by ultrafiltration.

After concentration, it was freeze-dried.

The surface amino group modification rate of the obtained modified protease was 15
The % enzyme activity retention rate was 80%.

(Example 2) In Example 1, dextran (average molecular weight 6-9X
Instead of adding pullulan (average molecular fi50)
,000) A similar operation was performed using Biopra\-ase (manufactured by Nagase Biochemical Co., Ltd.) as a protease,
The product was obtained.

The surface amino group modification rate of the obtained modified protease was 18
%, and the activity retention rate was 15%.

(Example 3) The same operation as in Example 1 was performed using methylcellulose instead of adding dextran to obtain a product.

The surface amino group modification rate of the obtained modified protease was 68
%, and the activity retention rate was 72%.

(*Example 4) The same operation as in Example 1 was performed using inulin (average molecular weight ff 160,000) instead of adding dextran and chymotrypsin (manufactured by Sigma) as the protease to obtain a product.

The surface amino group modification rate of the obtained modified protease was 11
%, and the activity retention rate was 70%.

(Comparative Example 1) Monomethoxypolyethylene glycol (average molecular ms,
o o o > s, o y, p-nitrophenyl chloroformate 0.6f in anhydrous acetonitrile 30ml
dissolved in. Add triethylamine 0.3f to this,
After stirring this solution at room temperature and 26°C overnight, 200 ml of diethyl ether was added and kept at 4°C overnight to precipitate crystals. After the precipitated crystals were separated in a furnace, they were recrystallized with a diethyl ether-acetonitrile mixed solvent, thoroughly washed with diethyl ether, and dried under reduced pressure to obtain 4.5 g of white crystals of activated polyethylene glycol.

Next, esperase somp was adjusted to pH 7.8.
After dissolving in 20 ml of mM potassium phosphate buffer and adding 100 ml of the activated polyethylene glycol obtained above, the mixture was stirred at 4°C overnight.

After adding 100 rllF of glycine to the obtained reaction solution and treating it for 5 hours, the solution was purified by ultrafiltration, concentrated, and freeze-dried.

The activity retention rate of the obtained modified protease was 28%.

(Comparative Example 2) Esperase somp was dissolved in 0.1 M phosphate buffer (PH
6,5) After dissolving in 25 ml, 1.5 ml of a 1.4% aqueous glutaraldehyde solution was added dropwise over 25 minutes with stirring at room temperature. After stirring the solution for an additional 2 hours, 10 ml of glycine was added and treated for 5 hours.

After adding 10 ml of sodium borohydride to the resulting reaction solution for reduction treatment, it was purified by ultrafiltration.

Concentrate and lyophilize.

The activity retention rate of the obtained modified protease was 48%.

(Comparative Example B) Carboxymethyl cellulose was dissolved in 200 mft-20 ml of water. Add lN1101 to this and pH 4
, 75, then 1-ethyl/L/-8-(3-dimethylaminotukpyr)carbodiimide hydrochloride a8omI and 20 mf of esperase were added.

The mixture was stirred at 4°C for 2 hours. Add 120 μl of acetic acid to this reaction solution.
After adding 120 ml of monoethanolamine and stirring for 20 minutes, the solution was purified by ultrafiltration.

It was concentrated and lyophilized.

The activity retention rate of the obtained modified protease was 52%.

(Comparative Example 4) Dextran (average molecular weight 6-9X10) 0.51
Dissolved in 00ITV water and added sodium periodate O,! 21 was added and reacted at 60°C for 5 hours. Then, 50m/ml of ethylene glycol was added to stop the reaction.
Unreacted substances were removed by ultraviolet filtration. Furthermore, 76mM phosphorus [! After washing with buffer solution (pH 7, 8>), it was concentrated to 10 rrl. Esperase somp was added to this and incubated at 4°C for 2
After reacting for 4 hours, reduction treatment was performed with NaBH4,
The purified fR was concentrated by ultrafiltration and then lyophilized.

The enzyme activity retention rate of the obtained modified protease was 45%.

Examples 1 to 4 above. The antigenicity of the modified proteases obtained in Comparative Examples 1 to 4 is shown in FIG. 1, and the test results of thermal stability and skin sensitization are shown in Table 1.

FIG. 1 is a graph showing the relationship between the amount of modified proteases obtained in Examples and Comparative Examples added as an antigen and the immune complex ffi (absorbance at 280 nm) resulting from the antigen-antibody reaction generated as a result of the addition. It is clear that the modified protease of the present invention is very useful, as it has high activity with little decrease in activity due to oxidation.

(Example 5) In the modification operation of Example 1, activated dextra a) Remaining activity rate after heat treatment of modified protease solution b) Average evaluation score From the results in Table 1, polysaccharide and protease were found to have a triazine depth. It was observed that the modified protease bonded via the conjugate had a markedly improved thermal stability as compared to the modified protease obtained in the comparative example, and also had the effect of significantly reducing antigenicity and skin sensitization. Furthermore, from the results in Table 2, it was found that by appropriately selecting the reaction ratio between activated dextran and protease, the thermal stability of the modified product was significantly improved, and the skin sensitization was significantly reduced or eliminated. It was also found that these effects were particularly large when the active dextran/protease ratio was 3 or more.

(Example 6) Among the modification reaction operations of Example 1, the triazine ring introduction reaction of dextran was carried out. From the results shown in Table 3 of dextran, it was found that the amount of triazine ring introduced into dextran considerably influences the thermal stability and skin sensitization suppressing effect of the modified protease.

(Example 1) The modified proteases obtained in Examples 1 to 4 and Comparative Examples 1 to 4 were subjected to a thermal stability test in a 10% surfactant coexistence system. The results are shown in Table 4.

Table 4 From the results shown in Table 4, it was found that the modified protease in which a polysaccharide was bonded via a triazine ring had remarkable thermal stability even in a surfactant coexistence system.

[Brief explanation of the drawing]

Figure 1 shows the amounts of modified proteases obtained in Examples and Comparative Examples added as antigens and the immune complexes generated by antigen-antibody reactions as a result of addition. (280nm
It is a graph showing the relationship between the absorbance of

Claims (2)

[Claims] (1) A modified protease characterized in that a protease and a polysaccharide are bonded via a triazine ring. (2) A method for producing a modified protease, which comprises reacting a polysaccharide with cyanuric chloride to synthesize a triazine ring-linked polysaccharide, and then reacting the triazine ring-linked polysaccharide with a protease.