JPS6031479B2 - Method for producing pure chondroitinase C - Google Patents

Description

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

Chondroitin sulfate type C is a type of acidic mucopolysaccharide found in the connective tissue of animals, and its water-retaining properties are thought to smooth cell metabolism and maintain tissue flexibility. There are many unknowns about its structure, physical properties, biological activity, etc., and although its structure has been speculated, it has not been established.

It is extremely desirable to clarify the structure of chondroitin sulfate C type in order to pursue the role of chondroitin sulfate C type in the body and clarify its physiological mechanism. It is believed that this will greatly contribute to the expansion of the types of uses for pharmaceuticals, food additives, and cosmetics. In this way, in research on chondroitin sulfate type C, Chondroitin was able to break down chondroitin sulfate type C without causing unnecessary damage due to the mild conditions.

There are great expectations for chondroitinase C, and chondroitinase C plays a major role. Concerning chondroitinase C, Michelasie et al.

It has been discovered that the enzyme is produced in cells of the genus Flavobacterium cultured in diluted soil supplemented with ditin sulfate or heparin, and has been isolated and purified by an open air electrophoresis method using agarose as a support. (Y.A. Michelac
ci & c. P. Dietrich: J. Bio1.
Chem. , 251, 1154 (1976)). However, since this method uses open air electrophoresis, it cannot be said to be a suitable method for separating and purifying chondroitinase C in large quantities. The present inventors solved the problems of the conventional method and conducted intensive research on a method for separating and purifying a large amount of highly pure chondroitinase C that does not contain other enzymes. A large amount of highly purified chondroitinase C can be easily obtained by subjecting the partially purified liquid of C to column chromatography using chondroitin sulfate type B-immobilized agarose treated with chondroitin sulfate type B as a separating agent. This discovery led to the present invention.

That is, the purpose of the present invention is to easily obtain a large amount of highly purified chondroitinase C that does not contain other enzymes, and the purpose of the present invention is to perform 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 solution of chondroitinase C is further purified by column chromatography using chondroitin sulfate type B-immobilized agarose treated with chondroitin sulfate type B treatment as a separating agent, and highly pure chondroitinase C is obtained. Regarding the production method, the partially purified solution of chondroitinase C in the present invention refers to the extract obtained by destroying chondroitinase C-producing microbial cells, for example, by treating it with protamine sulfuric acid and then applying hydroxyapatite column chromatography. Chondroitinase C-containing fractions are obtained by partially purifying the chondroitinase C fraction by chromatography, gel filtration, fractional precipitation, etc., and fractionation by hydroxyl/gtite column chromatography is particularly preferred.

Examples of chondroitinase C-producing microorganisms include Flavobacterium genus bacteria, especially Flavobacterium heparinum (FlaVo heparinum). When acid is added, chondroitinase C accumulates at a high concentration within the bacterial cells.

Therefore, the partially purified solution of chondroitinase C of the present invention is obtained by destroying Flavobacterium cells cultured in a medium supplemented with chondroitin sulfate or heparin. Partially purified by column chromatography using apatite is most suitable. When Flavobacterium heparinum is cultured in a medium supplemented with chondroitin sulfate, various enzymes such as chondroitinase C, chondroitinase B, and chondroitinase AC are produced within the bacterial cells. Furthermore, even if a medium to which heparin is added instead of chondroitin sulfate is used, various enzymes such as chondroitinase C, chondroitinase B, heparinase, heparitinase, and chondroitinase AC are produced in the bacterial cells. The bacterial cells thus obtained are destroyed by a conventional method such as ultrasonication, the pH of the resulting extract is adjusted, and protamine sulfate is added.
The upper 7 blue solution obtained by centrifugation was loaded onto a hydroxyapatite column, and then the concentration of sodium chloride added to a 0.09 M buffer was increased to 0.2 M, or the buffer was adjusted without adding sodium chloride. The concentration of the liquid is 0.19
If you perform melting while directly raising it to M, it will be a condo.

A fraction containing nitinase C is eluted. At this time, when an extract of bacterial cells cultured using a medium supplemented with chondroitin sulfate was used, chondroitinase B was detected, whereas an extract of bacterial cells cultured using a medium supplemented with heparin was used. In some cases, heparinase coexists in the fraction. The hydroxyapatite used in this fractionation may be prepared visually or may be a commercially available product.

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

Chondroitinase C may be deactivated at a pH other than neutral, which is not preferable. Also, the concentration of the buffer solution is 0.
Must be less than 0.05M. Concentrations exceeding 0.08M are not preferred because chondroitinase C may be eluted.

When performing this fractionation, if the concentration of sodium chloride added to the buffer solution exceeds 0.2 M, or if the concentration of the buffer solution exceeds 0.19 M, the amount of contamination of chondroitinase B or heparinase may increase. This is not desirable because it increases

Next, the fraction was loaded into a ram filled with bead-shaped agarose immobilized with chondroitin sulfate type B and chondroitin sulfate type B bound to it, and the concentration of sodium chloride added to the buffer solution was reduced to 0. .19
or reduce the concentration of the buffer to 0.M or without adding sodium chloride. By carrying out elution while linearly increasing the elution to the point of elution, it is possible to easily obtain highly pure chondroitinase C that does not contain other coexisting enzymes. The chondroitin sulfate B type used in the main purification has a carboxyl group, epoxy group, or amino group as a functional group for chondroitin sulfate B type immobilization.
As the agarose for immobilizing the type, any agarose having an amino group, a carboxyl group, or an epoxy group as a functional group may be used, but agarose shaped into beads is particularly preferred.

Agarose having such functional groups can be prepared from the eye or commercially available, such as A
H-Sepharose Princess, Epoxy-activated
Sepharose) clothing, active cyanogen bromide Sepharose (CNBr-Activa)
There is no problem in using lights (rose) or clothes (all of which are trademarks of Pharmasha Fine Chemicals, Sweden). The method of immobilizing chondroitin sulfate type B on such agarose is the already known method of Inman (J.K. Inman: Memo
(Ship mEmymolo Hyō, 34, 30, 1974).

That is, by adding chondroitin sulfate type B solution to a suspension of agarose having a functional group, adding carbodiimides to this, reacting, and fractionating the agarose, chondroitin sulfate type B immobilized agarose can be easily obtained. Obtainable. Chondroitin sulfate type B is further added to the thus obtained chondroitin sulfate type B-immobilized agarose for treatment.

That is, a column is filled with chondroitin sulfate type B-immobilized agarose, and a chondroitin sulfate type B solution is passed therethrough to treat the column with chondroitin sulfate type B. This treatment may also be carried out by immersing chondroitin sulfate type B-immobilized agarose in a chondroitin sulfate type B solution. The buffer used at this time has a concentration of less than 0.3M and has a pH of
As long as it is in the range of 6 to 8, any may be used.

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. Chondroitinase C can be purified with high yield and high purity only by using cosdroitin sulfate type B-immobilized agarose and agarose further treated with chondroitin sulfate type B.

Condo treated with this chondroitin sulfate type B. The use of agarose immobilized with nitin sulfate B type gives a high yield. In addition, when purified using chondroitin sulfate type B-immobilized agarose treated with chondroitin sulfate type B in this way, not only can highly pure chondroitinase C be efficiently obtained, but also chondroitin sulfate can be used as needed during continuous use. It can be easily regenerated by processing with type B, and there is no need to start by immobilizing chondroitin sulfate type B on agarose, so continuous purification over a long period of time is possible by introducing a simple regeneration method midway through. It has the great advantage of being Next, the fraction was loaded onto a column packed with chondroitin sulfate type B-immobilized agarose, preferably in the form of beads, which had been further treated with chondroitin sulfate type B, and added to a buffer solution with a concentration of 0.008 M or less. By performing elution while linearly increasing the concentration of sodium chloride to 0.19 and the concentration of the buffer solution to 0.08 M without adding sodium chloride, a high-purity sample containing no other coexisting enzymes is obtained. Chondroitinase C can be easily obtained.

In this case, if the concentration of sodium chloride exceeds 0.18M or the concentration of the buffer exceeds 0.08K, other coexisting enzymes may be eluted and mixed, which is not preferable.

Any buffer may be used for elution 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. In this way, chondroitinase C purified using column chromatography that separates chondroitin sulfate type B-immobilized agarose treated with chondroitin sulfate type B is about 35 times that of the chondroitinase C before purification. It shows a high degree of purification and gives a high recovery rate.

Chondroitinase C thus obtained decomposes only chondroitin sulfate type C, and does not decompose other chondroitin sulfate types A and B, heparitin sulfate, etc.

In addition, its maximum operating temperature is 20 oo, and its operating maximum pH is 20 oo.
8.0, chondroitinase C (Y
．． M. Michelacci & C. P. Diemch
:J. Biol. To Chem 251, 1154, 19
76). Next, the present invention will be explained in more detail with reference to Adjustment Examples, Examples, and Comparative Examples, but the present invention is not limited thereto unless it exceeds the gist thereof.

Adjustment example 1 Culture of bacterial cells Bepton 1.5%, meat extract 0.45%, yeast extract 1
．． 0%, malt extract 1.0%, chondroitin sulfate type C (S amount 7.1%)
7.0) Flavobacterium heparinum ATCCI
3125 was inoculated and cultured on a shaker for 20 hours at 30oo.The culture solution was added to the same liquid medium at a concentration of 3%, and shaker culture was carried out for 2 feedings at 30oooo.

Adjustment example 2 Culture of bacterial cells Beptone 1.5%, meat extract 0.45%, yeast extract 1
．． 0%, malt extract 1.0% (pH 7.
0) Flavobacterium, Heparinum ATCCI31
25 was inoculated, and shaken culture was carried out 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 0.000, and 0.0 heparin (manufactured by Hani Chemical Co., Ltd., 150 international units/female) was added at the 1st hour of culture.
The mixture was added to a concentration of 2% and cultured 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 bacterial cells, washed with 0.7M acetate buffer (pH 7.0). )
Five microbial cells were suspended in a 30-liter tube, subjected to ultrasonication at 10 kilocycles/second for 15 minutes, and centrifuged to remove cell walls to obtain a bacterial cell infusion.

Preparation Example 4 Preparation of Bacterial Cell 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 chondroitin sulfate type B immobilized agarose Chondroitin sulfate type B (S content 7.2%) 9 of 50
A, whose pH had been adjusted to 47.5 in advance, was placed on top of the solution 1 containing A.
To a suspension containing 60% of H-Sepharose, 0.5% of a solution containing 15% of 1-ethyl-3-(3-dimethylaminopropynopropylene)carbodiimide was added, and the pH was adjusted to 4.
．． Keep it at room temperature overnight while keeping it at 75°C, then wash it in the oven and put it in a condo.

1.05 g of agarose immobilized with nitin sulfate B type was obtained. Example 1 Preparation of Crude Chondroitinase C Fraction The bacterial cell extract obtained in Preparation Example 3 was dialyzed against running water overnight at 4°C using a Visking tube, and the dialysis fluid was 80% Sodium acetate was added to give a concentration of 0.1 to 4, and after adjusting the concentration to 6.5, 75% of protamine sulfate was added and left to stand for 1 hour. was diluted 2 times, and the method of Bernardi (G.
EmMmology, 22, 325, 1971)
．． 2
6.8) for equilibration and post-loading, then add sodium chloride to the same buffer and elute at a flow rate of 35 secretions per hour while increasing the concentration linearly to 0.2M. Chondroitinase C was obtained in fraction 250' of the 150th to 400th secreted fluid.

The total activity of chondroitinase C in this fraction was 600 units, and 58.9 units per protein dose. The activity of chondroitinase C was measured using enzyme solution o. 2. Add 0.1% of 10mM calcium acetate solution and add to this.

Substrate solution containing 10 mp/M of nitin sulfate type C 0
．． The mixture was heated at 370°C for 1 hour, then immediately boiled for 2 minutes, added with 0.0% hydrochloric acid, and centrifuged.The resulting supernatant was measured for absorption at 23°C. As a blank test, perform the same treatment using water instead of the enzyme solution and measure the absorbance. At this time, when the difference in absorbance was 1, it was defined as 1 unit. In addition to chondroitinase C, chondroitinase B was detected in this fraction.

Example 2 Treatment of chondroitin sulfate type B-immobilized agarose with chondroitin sulfate type B-immobilized chondroitin sulfate type B-immobilized agarose obtained in Preparation Example 3.
0.05M phosphate buffer (pH 6.8) 10
This was poured into a column (1 x 5 arcs) and filled with chondroitin sulfate type B immobilized agarose, and then the same buffer solution 3' containing chondroitin sulfate type B type 50 9 was loaded. Excess chondroitin sulfate type B was removed by flowing 100% buffer solution at a flow rate of 35% per hour.

The chondroitin sulfate type B recovered in the washing solution was 15 compared to 9 of the loaded chondroitin sulfate type B of 50.
It was 9.

From this, subtract 35/9 chondroitin sulfate B
This means that the type is bound to the chondroitin sulfate B type-immobilized agarose. Chondroitin sulfate type B is obtained by Bitter and Muir's carbazole method (J.Bit).
ni r&day. M. Muir: Anal. Biochem.
, 4, 330, 1962), add 3' of concentrated sulfuric acid containing 0.95% boric acid to 0.5 of a chondroitin sulfate type B aqueous solution, keep it at 100 oo for 10 minutes, and add 0.12
Add 0.1 ml of 5% carbazolethanol solution,
Maintain at 0qo for 18 minutes, measure absorbance of 53 blood after cooling,
The amount of chondroitin sulfate type B was calculated from the amount of uronic acid obtained.

Example 3 Purification of Chondroitinase C 250 g of the crude chondroitinase C solution obtained in Example 1 (total activity: 60 mm, protein: 58.9 mm) was purified using a Visking tube. and 4℃ against water
After dialysis, the column was loaded onto a chondroitin sulfate B type-immobilized agarose column (1 x 5 cm) treated with chondroitin sulfate B type obtained in Example 2, and added to 0.009M phosphate buffer (pH 6.8). Chondroitinase C was added to fraction 125 of the eluate 125-250' by elution at a flow rate of 30 m/hr while adding sodium chloride and increasing its concentration linearly to 0.18 M. Obtained.

Chondroitinase C in this fraction had a total activity of 439 units;
The recovery rate was approximately 72%, and the purity was high, with 2.050 molecules per protein 1 and 38 years old. Moreover, chondroitinase B was not found in this fraction. Example 4
Preparation of Crude Chondroitinase C 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/power column chromatography. Chondroitinase C was obtained in fractions 200 to 400 of the eluate.

The total activity of chondroitinase C in this fraction was 36 units, 6 units per 9 units of protein. Heparinase and chondroitinase B were also detected in this fraction. Example 5 Purification of Chondroitinase C 200 grams of the crude chondroitinase C solution obtained in Example 4 (total activity: 36 units, 6 units/protein) was added to chondroitin sulfate B in the same manner as in Example 3. Column chromatography was performed using type-treated chondroitin sulfate type B-immobilized agarose, and the eluate No. 12
Chondroitinase C was obtained in the first 125 minutes of 5 to 250 minutes.

The total activity of chondroitinase C in this fraction was 252 units, the recovery rate was about 70%, and the purity was about 36 times higher, with 2.160 units per protein 19. Furthermore, heparinase and chondroitinase B were not found in this fraction. Comparative Examples 1-2 Chondroitinase C fraction obtained by fractionation using hydroxyapatite 125% (total activity 30 units,
After dialyzing 29 units/female protein), columns filled with chondroitin sulfate type B-immobilized agarose and chondroitin sulfate type B-immobilized agarose further bound with chondroitin sulfate type B (1 to 5 skins).
0.009K phosphate buffer (pH 6.8) and elution was carried out at a flow rate of 30% per hour while increasing the concentration linearly to 0.18M. Chondroitinase C that does not contain other enzymes
I got it.

The results were as shown in Table 1.・Shigon-oh, the value in the chondroitinase C column is chondroitinase C.
Expressed in units of.

Comparative Example 1 used chondroitin sulfate B type immobilized agarose.

In Comparative Example 2, chondroitin sulfate B type immobilized agarose was further loaded with 0.009 phosphate buffer (pH 6.8) 3' containing chondroitin sulfate B type 50, treated with chondroitin sulfate B type, and washed. All in all, an excessive number of condos.

It was used after removing the ditin sulfate type B. As is clear from the results, in Comparative Example 1, a total amount of only 24 units of chondroitinase C was obtained, and the recovery rate was 8%, whereas in Comparative Example 2, a total amount of 213 units of chondroitinase C was obtained. was obtained, showing a high recovery rate of 72%.

Claims (1)

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