JPS6154398B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing heparitinase, which is a heparitin sulfate degrading enzyme, and more specifically, a partially purified solution of heparitinase is subjected to column chromatography using heparin-treated heparin-immobilized agarose as a separating agent. The present invention relates to a method for producing pure heparitinase. Heparitin sulfate is a type of acidic mucopolysaccharide that is found wherever chondroitin sulfate B is present, such as in the skin and blood vessels of animals, and the significance of its existence is largely unknown. Much remains unknown about chondroitin sulfate B, which coexists with chondroitin sulfate, but its water-retaining properties facilitate cell metabolism, and since it is found in blood vessel walls, it is thought to be involved in the blood clotting prevention mechanism. Therefore,
Heparitin sulfate is also presumed to have the same function as chondroitin sulfate B in living organisms, but there are many unknowns about its structure, physical properties, biological activity, etc., and for example, its structure is not necessarily clarified. It is extremely desirable to clarify the structure of heparitin sulfate in order to explore the role of heparitin sulfate in the body and clarify its physiological mechanisms. It is expected that certain uses of heparitin sulfate, such as pharmaceutical and cosmetic uses, may be developed. As described above, in research on heparitin sulfate, there are great expectations for heparitinase, which can degrade heparitin sulfate without causing unnecessary damage under mild conditions, and heparitinase plays a major role. Regarding heparitinase, it is known that heparitinase is produced within Flavobacterium bacteria by culturing them in a medium supplemented with heparin (Linker, A.,
et al; J.Biol.Chem., 240 , 3724, 1965), hydroxyapatite was used for the isolation and purification of heparitinase produced within bacterial cells.
Hovingh et al.'s method using column chromatography (P. Hovingh & A. Linker: J. Biol. Chem.,
245 , 6170, 1970) are known. However, in this separation method, chondroitinase B and chondroitinase AC produced by the bacterial cells are contaminated, so it is necessary to remove these enzymes. The present inventors solved the drawbacks of the conventional method and conducted intensive research on a method for isolating and purifying a large amount of highly pure heparitinase that does not contain other acidic mucopolysaccharide-degrading enzymes. We have discovered that a large amount of highly pure heparitinase can be easily obtained by subjecting the purified solution to column chromatography using heparin-fixed agarose, which has been further treated with heparin, as a separation agent, resulting in the present invention. did. That is, the purpose of the present invention is to easily obtain a large amount of highly purified heparitinase that does not contain other acidic mucopolysaccharide-degrading enzymes, and the purpose of the present invention is to perform separation and purification according to the production method of the present invention. By doing so, you can easily reach that goal. Next, the present invention will be explained in more detail. The present invention is a method for producing highly pure heparitinase by further purifying a partially purified heparitinase solution by column chromatography using heparin-treated heparin-immobilized agarose as a separating agent. The partially purified solution of heparitinase refers to an extract obtained by destroying heparitinase-producing bacterial cells, for example, after treating it with protamine sulfuric acid and then partially purifying it by hydroxyapatite column chromatography, gel filtration method, fractional precipitation method, etc. It refers to a heparitinase-containing fraction obtained by purification, and among these, fractionation by hydroxyapatite column chromatography is preferred. Heparitinase-producing bacteria include bacteria of the genus Flavobacterium, especially Flavobacterium heparinum.
However, when heparin is added to the culture medium during culture, heparitinase is accumulated in the bacterial cells at a high concentration. Therefore, as the partially purified solution of heparitinase of the present invention, hydroxyapatite was used, which was obtained by treating an extract obtained by destroying Flavobacterium cells cultured in a medium supplemented with heparin with protamine sulfuric acid. Partially purified by column chromatography is most suitable. When Flavobacterium heparinum is cultured in a medium supplemented with heparin, various enzymes such as heparitinase, chondroitinase B, chondroitinase AC, and heparinase are produced as inducible enzymes within 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, protamine sulfate is added, and the supernatant obtained by centrifugation. When the solution is loaded onto a hydroxyapatite column and elution is performed while linearly increasing the concentration of sodium chloride in the buffer solution, heparitinase is eluted. At this time, even if elution is performed while linearly increasing the buffer concentration without adding sodium chloride, the heparitinase fraction will similarly elute. However, the heparitinase-containing fraction obtained in this way also contains chondroitinase B, chondroitinase
AC, etc. coexist. The fractions were then loaded onto a column packed with heparin-immobilized agarose that had been further treated with heparin, and the concentration of sodium chloride added to the buffer solution was increased to 0.15M or the concentration of the buffer solution was increased to 0.08M.
By performing elution while linearly increasing the amount of heparitinase to 100%, it is possible to easily obtain highly pure heparitinase that does not contain other coexisting enzymes. 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 shaped into beads is particularly preferred. Agarose having such a functional group may be prepared and used in-house, but commercially available ones such as AH-Sepharose 4B, Epoxy-Activated Sepharose 6B,
Active cyanogen bromide cephalose (CNBr
There is no problem in using Activated Sepharose) 4B or 6B (both of which are trade names of Pharmacia Huain Chemicals, Sweden). The method of immobilizing heparin on such agarose is the already known method of Inman (JK
Inman: Methods in Enzymology, 34 , 30,
(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 carbodiimide to the suspension, causing a reaction, and then fractionating the agarose. The heparin-immobilized agarose thus obtained is further treated by adding heparin again. That is, a column is filled with heparin-immobilized agarose, a heparin solution is passed through the column, and the column is further treated with heparin. In addition, this treatment may be performed by immersing heparin-immobilized agarose in a heparin 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 one may be used. A buffer concentration of 0.3M or higher is not preferable because the amount of heparin bound decreases. Moreover, heparin becomes unstable at a pH range of less than 6, and the amount of binding decreases when it exceeds 8, which is not preferable. Only by using heparin-immobilized agarose and further heparin-treated agarose,
Heparitinase can be purified with good yield and high purity. If heparin-immobilized agarose on which heparin is simply immobilized is used, the yield will drop significantly, but if this heparin-immobilized agarose is further treated with heparin, a high yield can be obtained. In addition, purification using heparin-immobilized agarose that has been further heparin-treated not only allows highly purified heparitinase to be obtained efficiently, but also facilitates continuous use by appropriately performing heparin treatment. Since it is not necessary to start from immobilizing heparin on agarose, it has the great advantage that continuous purification over a long period of time is possible by introducing a simple regeneration method midway through. . The fractions were then loaded onto a column packed with heparin-immobilized beaded agarose that had been further treated with heparin, and the concentration of sodium chloride added to the buffer was adjusted to 0.15M or without the addition of sodium chloride. By performing elution while linearly increasing the buffer concentration to 0.08M, highly pure heparitinase that does not contain other coexisting enzymes can be easily obtained. In this case, if the concentration of sodium chloride exceeds 0.15M or the concentration of the buffer exceeds 0.08M, coexisting chondroitinase AC may be eluted and mixed, which is not preferable. 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 0.005M, heparitinase may be eluted or mixed in, and It is not preferable in the PH range because heparitinase is inactivated. In this way, heparitinase purified using heparin-immobilized agarose further treated with heparin showed a high degree of purification, about 8 times that of the heparinase before purification, and also showed a high recovery rate. . The heparitinase obtained in this way decomposes only heparitin sulfate and other acidic mucopolysaccharides,
For example, heparin, chondroitin sulfate A, B, C
Do not disassemble each type. In addition, its optimal temperature for action is 42°C, and its optimal pH for action is 7.0, and the heparitinase of Hovingh et al. (P. Hovingh & A. Linker: J. Biol.
Chem., 245 , 6170, 1970). 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 Peptone 1.5%, meat extract 0.45%, yeast extract
Liquid medium containing 1.0% and malt extract (PH7.0)
Flavobacterium heparinum ATCC13125
was inoculated and cultured with shaking at 30°C for 20 hours. The culture solution was added to the same liquid medium at a concentration of 3% and cultured at 30°C. After 18 hours of culture, heparin (manufactured by Hanui Chemical Co., Ltd., 150 international units/mg)
It was added to a concentration of 0.02% and cultured for an additional 6 hours. Preparation Example 2 Preparation of Bacterial Cell Extract Flavobacterium heparinum ATCC13125 obtained in Preparation Example 1 was centrifuged according to a conventional method to collect bacterial cells, and washed with 0.7M acetate buffer (PH7. 0) 5 g of bacterial cells were suspended in 30 ml, sonicated at 10 kilocycles/second for 15 minutes, and centrifuged to remove cell walls to obtain a bacterial cell extract. Preparation Example 3 Preparation of Crude Heparitinase Fraction The bacterial cell extract obtained in Preparation Example 2 was dialyzed against running water overnight at 4°C using a Wiesking tube, and 0.1 M was added to 80 ml of the dialyzed solution. Add sodium acetate to adjust the pH to 6.5, add 75 mg of protamine sulfate, leave for 1 hour, centrifuge to remove the precipitate, add water to dilute to 2 times, and use Bernardi's Method (G.
Bernardi: Method in Enzymology, 22 , 325,
A column (2.2 x 8.0 cm) packed with hydroxyapatite prepared according to the method (1971) was equilibrated and loaded with 0.05 M phosphate buffer (PH6.8), and then sodium chloride was added to the same buffer. Then, elution was carried out at a flow rate of 35 ml per hour while linearly increasing the concentration to 0.3 M.
Heparitinase was obtained in 350 ml fractions. Its total activity is 1194 units and specific activity is 170.5 units/mg
It was hot with protein. In addition to heparitinase, this fraction contains chondroitinase B and chondroitinase AC.
etc. were detected. Preparation example 4 Preparation of heparin-immobilized agarose Heparin (manufactured by Hanui Chemical Co., Ltd., 150 international units/mg)
1 ml of a solution containing 50 mg was prepared in advance to have a pH of 4.75.
Add 0.5 ml of a solution containing 15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 6 ml of a suspension containing 1 g of AH-Sepharose 4B, keep at room temperature overnight while keeping the pH at 4.75, and wash after washing. 1.05 g of heparin-immobilized agarose was obtained. Example 1 Heparin treatment of heparin-immobilized agarose 1.05 g of heparin-immobilized agarose obtained in Preparation Example 4 was dissolved in 0.005 M phosphate buffer (PH6.8) 10
ml, poured into a column (1 x 5 cm) and filled with heparin-immobilized agarose, and then loaded with the same buffer containing 7500 international units of heparin.
Excess heparin was removed by flowing approximately 100 ml of the same buffer solution at a flow rate of 35 ml per hour. The heparin recovered in the wash was 1500 International Units compared to the 7500 International Units of heparin loaded.
From this, an additional 6000 international units were subtracted and bound to the heparin-immobilized agarose. Heparin activity was measured according to the section on ``Nikkyomaru'' heparin sodium activity measurement method. Example 2 Purification of heparitinase 350 ml of crude heparitinase solution obtained in Preparation Example 3 (total activity 1194 units, 170.5 units/mg protein)
4°C against water using a Wiesking tube.
After dialysis, the column was loaded onto the heparin-treated heparin-immobilized agarose column (1 x 5 cm) obtained in Example 1, and sodium chloride was added to 0.005M phosphate buffer (PH6.8). , increasing its concentration linearly up to 0.15M per hour.
Perform elution at a flow rate of 30 ml, and elute 350 to 600 ml.
Heparitinase was obtained in a 250 ml fraction. The total activity of heparitinase in this fraction was 717 units, and the recovery rate was approximately 60 units.
% and activity per mg of protein was 1360.3 units, indicating a high degree of purification of about 8 times. The fraction also contains chondroitinase B and chondroitinase.
No AC was found. The activity of heparitinase is 10mM in 0.2ml of enzyme solution.
Add 0.1 ml of calcium acetate solution, add 0.1 ml of substrate solution containing 10 mg of heparitin sulfate per ml, keep at 42°C for 1 hour, immediately boil for 2 minutes, add 2.5 ml of 0.06N hydrochloric acid, and centrifuge. The absorption at 232 nm of the supernatant obtained 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. Comparative Examples 1 to 2 175 ml of heparitinase fraction obtained by fractionation using hydroxyapatite (heparitinase activity 598 units, 170 units/mg protein; 30 units of heparitinase mixed) was dialyzed and then treated with heparin-immobilized agarose and further Examples The heparin-treated heparin-immobilized agarose obtained in step 1 was loaded onto a column (1 x 5 cm) packed with the heparin-immobilized agarose.
Add sodium chloride to 0.005M phosphate buffer (PH6.8) and elute at a flow rate of 30ml per hour while increasing the concentration linearly to 0.15M to obtain heparitinase free of other coexisting enzymes. Ta. The results were as shown in Table 1. Comparative Example 1 used heparin-immobilized agarose. Comparative Example 2 contains heparin-immobilized agarose and further contains 50 mg of heparin (150 international units/mg)
The tube was loaded with 3 ml of 0.005M phosphate buffer (PH6.8), treated with heparin, washed to remove excess heparin, and then used.

[Table] As is clear from the above results, in Comparative Example 1, the heparitinase obtained by this operation was 60 units (recovery rate 10%, heparitinase mixing rate was 5%, the same as before the operation), whereas Comparative Example 2: 359 units (recovery rate 60%, heparitinase mixed rate 0.06%)
This shows that according to the present invention, extremely pure heparitinase can be obtained at a high rate.

Claims (1)

[Scope of Claims] 1. A method for producing pure heparitinase, which comprises subjecting a partially purified heparitinase solution to column chromatography using heparin-immobilized agarose further treated with heparin as a separating agent. 2. The production according to claim 1, wherein the partially purified heparitinase solution is a heparitinase-containing solution obtained by treating an extract obtained after destroying heparitinase-producing microbial cells with protamine sulfuric acid, and further fractionating using hydroxyapatite. Law. 3. The production method according to claim 2, wherein the heparitinase-producing microbial cells are Flavobacterium microbial cells cultured using a medium supplemented with heparin.