JPS6031478B2 - Method for producing pure chondroitinase B - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing chondroitinase B, which is a chondroitin sulfate type B degrading enzyme. The present invention relates to a method for producing pure chondroitinase B, which is characterized by subjecting it to column talomatography using heparin-immobilized agarose as a separation agent.

Chondroitin sulfate type B is a type of acidic mucopolysaccharide found together with heparitin sulfate in the skin and blood vessels of animals, and its water-retaining properties smooth cell metabolism.
On the other hand, since it is found in blood vessel walls, it is assumed that it plays an important role in the body, such as being involved in the blood clotting prevention mechanism, but much remains unknown regarding its structure, physical properties, biological activity, etc. For example, although its structure has been speculated, it has not been established.

It is extremely desirable to clarify the structure of chondroitin sulfate type B in order to explore the role of chondroitin sulfate type B in the body and clarify its physiological mechanism, and it is hoped that these will be clarified. It is believed that this will greatly contribute to the promotion of the development of medicinal and cosmetic uses for chondroitin sulfate type B.
In this way, in research on chondroitin sulfate type B, there are high expectations for chondroitinase B, which breaks down chondroitin sulfate type B without undue damage due to the mild conditions, and chondroitinase B plays a major role. .

Regarding chondroitinase B, bacteria of the genus Flavobacterium were cultured in a medium supplemented with chondroitin sulfate (
Hoffman, P. , etal. :J. Bjol. C
235, 3066, 1960), chondroitinase B is produced within the bacterial cells, and regarding the separation and purification of chondroitinase B produced within the bacterial cells, the agarose of Michelasie et al. A method using open air migration as a support (Y.M.Mjche
lacci & C. P. Dietrieh:B. B.
R. C. , 56, 973 (1974)).

However, since this method uses lightning electrophoresis, it cannot be said to be a reliable method for separating and purifying chondroitinase B in large quantities. The present inventors resolved the difficulties of the conventional method and conducted intensive research on a method for separating and purifying a large amount of highly pure chondroitinase B that does not contain other enzymes. The present inventors have discovered that a large amount of highly purified chondroitinase B can be easily obtained by performing column chromatography using heparin-immobilized agarose treated with chondroitin sulfate type B, and have arrived at the present invention. .

That is, the purpose of the present invention is to easily obtain a large amount of highly purified chondroitinase B that does not contain other enzymes, and by carrying out separation and purification according to the method of the present invention, You can easily reach that goal.

Next, the present invention will be explained in more detail.

In the present invention, a partially purified chondroitinase B solution is further purified by column chromatography using heparin-immobilized agarose treated with chondroitin sulfate type B as a separation agent, and highly purified chondroitinase B is obtained. Regarding the production method, the partially purified solution of chondroitinase B in the present invention refers to the extract obtained by destroying chondroitinase B-producing microbial cells, for example, by treating it with protamine sulfuric acid and then adding hydroxyapatite. Chondroitinase B-containing fraction obtained by partial purification by column chromatography, gel method, fractional precipitation method, etc.
Among these, fractionation by hydroxyapatite column chromatography is preferred.

Examples of chondroitinase B-producing microorganisms include Flavobacterium genus bacteria, especially Flavobacterium heparinum (FlaVo heparinum). When parin is added, chondroitinase B is accumulated in the bacterial cells at a high concentration.

Therefore, as the partially purified solution of chondroitinase B of the present invention, the extract obtained by destroying Flavobacterium cells cultured in a medium supplemented with chondroitin sulfate or hebalin is treated with protamine sulfate, Partially purified by column chromatography using apatite is most suitable. When Flavobacterium heparinum is cultured in a medium supplemented with chondroitin sulfate, chondroitinase B, chondroitinase C, and chondroitinase AC are produced in the bacterial cells.
Various enzymes such as are produced. Furthermore, even if a medium to which heparin is added instead of chondroitin sulfate is used, various enzymes such as chondroitinase B, chondroitinase C, heparinase, heparitinase, and chondroitinase AC are produced within the bacterial cells. The bacterial cells obtained in this way are destroyed by normal methods such as ultrasonication,
The pH of the resulting extract was adjusted, protamine sulfate was added, and the supernatant obtained by centrifugation was loaded onto a hydroxyapatite column, and then the concentration of sodium chloride added to the buffer solution was adjusted to 0.3M or When elution is performed while linearly increasing the buffer concentration to 0.18 without adding sodium chloride, a fraction containing chondroitinase B is eluted.

At this time, when bacterial cells cultured using a medium supplemented with chondroitin sulfate are used, chondroitinase C and chondroitinase AC are extracted from the bacterial cell extract cultured using a medium supplemented with heparin. When using heparinase, heparinase and heparinase coexist in the fraction.
The hydroxyapatite used in this fractionation may be prepared from the eye or a commercially available product.

The buffer used in this fractionation may be any buffer as long as it has a pH around neutrality.

Chondroitinase B may be deactivated in a pH range other than neutral, which is not preferable. Also, the concentration of the buffer solution is 0.
Must be less than 09M. Concentrations exceeding 0.09M are not preferred because chondroitinase B may be eluted.

Also, the concentration of sodium chloride added to the buffer solution is 0.
Concentrations exceeding 3M or buffer concentrations exceeding 0.13 are not preferred because the amount of heparitinase or chondroitinase AC contaminating increases. Next, the fraction was loaded onto a column packed with heparin-immobilized beaded agarose to which chondroitin sulfate type B was bound, and the concentration of sodium chloride added to the buffer solution was adjusted to 0.1%. By carrying out elution while linearly increasing the buffer concentration to 0.08M or 0.08M, highly pure chondroitinase B free from other coexisting enzymes can be easily obtained.

As the agarose for immobilizing heparin used in the main purification, any agarose having a carboxyl group, epoxy group, or amino group as a functional group may be used, but agarose formed into beads may be used. Particularly preferred.

Agarose with such functionality can be prepared from the eye or commercially available, such as A
H-Sepharose abrasive, Epoxy-activated S
epharose) string, activated cyanogen furomide pharose (CNBr-ActivatedSepharo)
There is no problem in using ``se'' or string (all of the above are trademarks of Pharmacia Fine Chemicals, Sweden).

The method of immobilizing heparin on such agarose is the already known method of Inman (J.K. Inman:
Me ratios in Enzymology, 34, 3
0, 1974).

That is, heparin-immobilized agarose can be easily obtained by adding a heparin solution to a suspension of agarose having a functional group, adding a carbodimide thereto and causing a reaction, and then fractionating the agarose. The heparin-immobilized agarose thus obtained is treated by adding chondroitin sulfate type B.

That is, a column is filled with heparin-immobilized agarose and treated with chondroitin sulfate type B by flowing a chondroitin sulfate type B solution. In addition, this treatment may be performed by immersing heparin-immobilized agarose in a chondroitin sulfate type B solution. The buffer used at this time has a concentration of 0.
Any one may be used as long as it is less than 3M and has a pH in the range of 6 to 8.

If the concentration of the buffer solution is 0.3M or higher, the amount of bound chondroitin sulfate type B will decrease, which is not preferable. Furthermore, a pH of less than 6 or more than 8 is not preferred because the amount of chondroitin sulfate type B bound decreases. Only by using heparin-immobilized agarose and agarose further treated with chondroitin sulfate type B,
Chondroitinase B can be purified with good yield and high purity.

Use of heparin-immobilized agarose treated with chondroitin sulfate type B provides a high yield. In addition, purification using heparin-immobilized agarose that has been further treated with chondroitin sulfate B type not only allows highly pure chondroitinase B to be obtained efficiently, but also allows chondroitin sulfate to be added as needed during continuous use. It can be easily regenerated by B-type treatment, and there is no need to start from immobilizing heparin on agarose, so introducing a simple regeneration method in the middle of the process allows continuous purification over a long period of time. It has advantages. The fractions were then loaded onto a column packed with heparin-immobilized agarose, preferably in the form of beads, further treated with chondroitin sulfate type B, and the concentration of sodium chloride added to the buffer was adjusted to 0.18. By performing elution while linearly increasing the concentration of the buffer solution up to 0.0 molar concentration, high purity chondroitinase B containing no other coexisting enzymes can be obtained.
can be easily obtained.

In this case, the concentration of sodium chloride exceeds 0.19M, or the concentration of the buffer solution exceeds 0.19M. Concentrations exceeding M are not preferred because other coexisting enzymes may be eluted and mixed. Any buffer used for elution may be used as long as the concentration is 0.005M or less and the pH is around neutral.

If the concentration exceeds 0.009, chondroitinase B may come out, and if the pH is other than neutral, chondroitinase B will be deactivated, which is not preferable.

In this way, chondroitinase B purified using heparin-immobilized agarose to which chondroitin sulfate type B was bound showed a purification degree about 11 times higher than that before purification, and it was also recovered. The rate was as high as approximately 61%.

Chondroitinase B thus obtained decomposes only chondroitin sulfate type B, and does not decompose other chondroitin sulfate types A and C, heparitin sulfate, etc. In addition, the optimum temperature for its action is 20:00, and the optimum temperature for its action is p.
H is 8.0, and chondroitinase 8 (
Y. M. Mich-elacci & C. P. Ditr
ich:J. Biol. To Chem 251, 1154
, 1976). Next, the present invention will be explained in more detail with reference to Preparation Examples, Examples, and Comparative Examples, but the present invention is not limited thereto unless it exceeds the gist thereof.

Preparation example 1 Culture of bacterial cells Bepton 1.5%, meat extract 0.45%, yeast extract 1
．． 0%, malt extract 1.0%, chondroitin sulfate C type (S content 7.1%)
．． 0) Flavobacterium heparinum ATCCI3
125 was inoculated, the temperature was raised to 30 C, shake culture was carried out for 2 feeding hours, and the culture solution was added to the same liquid tower to a concentration of 3%.
3000, 2-plate time shaking culture was carried out.

Preparation Example 2 Culture of bacterial cells Beptone 1.5%, meat extract 0.45%, yeast extract 1
．． Liquid beach soil (pH 7.0%) containing 0% and malt extract 1.0%.
0) Flavobacterium heparinum ATCCI31
25 was inoculated and shake cultured for 2 feeding hours at 3000, and the culture solution was added to the same liquid medium to a concentration of 3%.
Culture was carried out at 000.

Heparin (manufactured by Hani Chemical Co., Ltd., 150
International Unit/9) was added to give a concentration of 0.02%, and culture was continued for an additional 6 hours. Preparation Example 3 Preparation of bacterial cell extract Flavobacterium heparinum ATCCI3125 obtained in Preparation Example 1 was centrifuged according to a conventional method to collect cocoons, washed with 0.7M acetate buffer (pH 7.0)
Suspend 5 microorganisms on 30 bars and cycle at 10 kilocycles/sec.
Ultrasonic treatment was performed for 5 minutes, and the cell walls were removed by centrifugation to obtain a bacterial cell extract.

Preparation Example 4 Preparation of Cocoon Body Extract Flavobacterium heparinum ATCCI3125 obtained in Preparation Example 2 was treated in the same manner as in Preparation Example 3 to obtain a bacterial cell extract.

Preparation Example 5 Preparation of heparin-immobilized agarose Heparin (manufactured by Hanui Chemical Co., Ltd., 15 international units/female) 5
Add 1 part of the solution containing 9 of 0 to 1 part of the suspension containing 1 of AH-Sepharose, the pH of which was previously adjusted to 4.75, and add 1 part of the solution containing 9 of 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide to Add 0.5' of a solution containing 9, pH
The mixture was kept at room temperature overnight while keeping the temperature at 4.75, and washed after passing through the oven to obtain heparin-immobilized agarose at 1.05.

Example 1 Preparation of Crude Chondroitinase B Fraction The bacterial cell extract obtained in Preparation Example 3 was dialyzed against running water overnight at 4°C using a Visking tube. Add sodium acetate to make it 0.1M, adjust the pH to 6.5, add protamine sulfate 75, leave for 1 hour, centrifuge to remove the precipitate, and add water to double the volume. Dilute and use Bernardi's method (G.B or rMrdi:Me ratio.

d in E rudder Mm. logic, 22, 325, 197
A column (2.2 x 8.0 skin) packed with hydroxyapatite prepared according to 1) was pre-equilibrated with 0.09M phosphate buffer (pH 6.8) and then loaded. Add sodium chloride to the solution to reduce its concentration to 0. The elution was carried out at a flow rate of 35 per hour, increasing linearly to arc 4, and the eluate was divided into 250 to 600 'boxes of 35' per hour.
Chondroitinase B was obtained at 0'. Chondroitinase B in this fraction had a total activity of 901 units, with a protein content of 14 units per female.
It was 8 units. The activity of chondroitinase B was determined by adding 0.1 volume of 10mM calcium acetate solution to 0.2 volume of enzyme solution, and adding 0.1 volume of substrate solution containing 10w9 of chondroitin sulfate type B per 1 volume. Added, kept at 20oo for 1 hour, then immediately boiled for 2 minutes, added 0.0 carp hydrochloric acid 22
．． 5 and centrifuged, and the absorption of the supernatant obtained in the 23-year-old skin was measured.

As a blank test, the same treatment was performed using water instead of the enzyme solution, and the absorbance was measured. At this time, when the difference in absorbance was 1, it was defined as 1 unit. In addition to chondroitinase B, chondroitinase C and chondroitinase AC were detected in this fraction.

Example 2 Chondroitin sulfate type B treatment of heparin-immobilized agarose 1.05 μl of the heparin-immobilized agarose obtained in Preparation Example 3 was added to a 0.005 M phosphate buffer (pH 6.
8) Suspend in 10 cells, pour this into a column (1 x 5 cells) and fill it with heparin-immobilized agarose, then load 3 volumes of the same buffer containing chondroitin sulfate type B 50 female, Excess chondroitin sulfate type B was removed by flowing 100 tubes at a flow rate of 35 mm per hour.

Chondroitin sulfate type B recovered in the washing solution was 10 compared to 9 of 50 loaded chondroitin sulfate type B.
It was 9.

From this, subtract 40 c of chondroitin sulfate B
This means that the type is bound to heparin-immobilized agarose. Chondroitin sulfate type B is obtained by Bittar and Muir's carbazole method (J.Bit r, J.MMui).
r: Anal. Biochem. , 4, 330, 196
2) Chondroitin sulfate type B aqueous solution 0.5
Add concentrated sulfuric acid containing 0.95% borax to
Maintain the temperature at 0.00 for 1 minute, add 0.125% carbazolethanol solution, maintain at 10000 for 15 minutes, and after cooling, measure the absorbance of the 53-marked skin, and determine chondroitin from the amount of uronic acid obtained. The amount of sulfuric acid B type was calculated.

Example 3 Purification of Chondroitinase B 350' of the crude chondroitinase B solution obtained in Example 1 (total activity ~901 units, 148 units/female protein) was added to water using a Visking tube. After dialysis at 4°C, it was loaded onto a heparin-immobilized agarose column (1 x 5 skin) treated with chondroitin sulfate type B obtained in Example 2, and 0.008M phosphate buffer (pH 6.8) was added. Add sodium chloride to the solution and increase the concentration linearly up to 0.18M at 30% per hour.
Elution was carried out at a flow rate of 100 ml, and chondroitinase B was obtained in the 200 ml fraction of the eluate No. 350 to 550 ml.

Chondroitinase B in this fraction had a total activity of 55 units, a recovery rate of about 60%, and a high degree of purification of about 11 times, with 1.623 units per protein 19. Moreover, chondroitinase C and chondroitinase AC were not found in this fraction. Example 4 Preparation of Crude Chondroitinase B Fraction The bacterial cell extract obtained in Preparation Example 4 was subjected to dialysis and protamine sulfuric acid treatment in the same manner as in Example 1, and then subjected to hydroxyapatite column chromatography. Chondroitinase B was obtained in the 150th fraction above eluate Nos. 400-550.

The total activity of chondroitinase B in this fraction was 54 peak units, and 15 peak units per protein 19. Furthermore, heparinase and heparitinase were detected in this fraction. Example
5 Purification of Chondroitinase B 150' of crude chondroitinase B solution obtained according to Example 4 (total activity 540 units, 15 units/female protein)
Column chromatography was performed in the same manner as in Example 3 using heparin-immobilized agarose treated with chondroitin sulfate type B, and eluates Nos. 350 to 550'
Chondroitinase B was obtained in a fraction of 200 birds.

Chondroitinase B in this fraction had a total activity of 33 units, a recovery rate of about 60%, and a high degree of purification of about 11 times, with 1.650 units per protein Imp. Moreover, heparinase and heparitinase were not found in this fraction.
Comparative Examples 1-2 Chondroitinase B fraction obtained by fractionation using hydroxyapatite secreted 175 units (total activity 45 units,
After dialyzing 148 units/double 9 protein), it was loaded onto a column (lx5 skin) packed with heparin-immobilized agarose and heparin-immobilized agarose further bound with chondroitin sulfate type B, and 0.00% Add sodium chloride to phosphate buffer (pH 6.8) and increase the concentration linearly to 30M per hour to 0.19M.
Elution was carried out at a flow rate of 100 mL to obtain chondroitinase B that does not contain other coexisting enzymes.

The results are as follows. Kono, The values in the chondroitinase column are expressed in units of chondroitinase B. Comparative Example 1 used heparin-immobilized agarose.

Comparative Example 2 is a heparin-immobilized agarose and an o. It was loaded with 3000M phosphate buffer (PH6.8), treated with chondroitin sulfate type B, washed to remove excess chondroitin sulfate type B, and then used.

As is clear from the above results, comparatively only 49 units of chondroitinase B were obtained with 2, and the recovery rate was 10.
%, whereas in Comparative Example 2, a total amount of 270 units of chondroitinase B was obtained, showing a high recovery rate of 60%.

Claims (1)

[Scope of Claims] 1. Pure chondroitinase B, characterized in that a partially purified chondroitinase B solution is subjected to column chromatography using heparin-immobilized agarose treated with chondroitin sulfate type B as a separating agent. manufacturing method. 2. A patent in which the partially purified solution of chondroitinase B is a chondroitinase B-containing solution obtained by treating the extract obtained after destroying chondroitinase B-producing microbial cells with protamine sulfuric acid, and then fractionating it using hydroxyapatite. The manufacturing method according to claim 1. 3. The method according to claim 2, wherein the chondroitinase B-producing microbial cells are Flavobacterium microbial cells cultured in a medium supplemented with chondroitin sulfate or heparin.