JPH06153947A - Chondroitinase abc, its production and medicine composition - Google Patents

Abstract

PURPOSE:To obtain a new enzyme useful as a therapeutic agent for herniated disk free from side effect because of absence of impurities, high purity, high specific activity and excellent stability, also useful as a reagent for research having high reproducibility. CONSTITUTION:Purified chondroitinase ABC not containing endotoxin, having contents of nucleic acid and protease of a detection limit. Molecular weight: 100,000 Dalton. Isoelectric point: pH 8.2-pH 8.5. Optimum pH and optimum temperature: optimum pH 8-8.2 and optimum temperature 37 deg.C in the case of chondroitin sulfate as a substrate in a tris-hydrochloric buffer solution. Influence by a metal; inhibition of activity with Zn<2+>, Ni<2+>, etc. End amino acids: Ala of N end and Pro of C end. Specific activity, >=300V/mg, etc. The enzyme is obtained by removing nucleic acid from an extracted solution of the enzyme yielded from a cell producing the enzyme and successively chromatographing the extracted solution with a weak cation exchange resin and a strong cation exchange resin.

Description

Detailed Description of the Invention

[0001]

The present invention has an extremely high degree of purification,
The present invention relates to a highly stable purified chondroitinase ABC, chondroitinase ABC crystals, and a method for producing the same. Also,
The present invention relates to a pharmaceutical composition containing such chondroitinase ABC as an active ingredient.

[0002]

2. Description of the Related Art Chondroitinase ABC (Chondroitin)
ase ABC) [EC 4.2.2.4] is an enzyme that decomposes hyaluronic acid, chondroitin sulfate, chondroitin, dermatan sulfate and the like into disaccharides containing unsaturated sugars. This enzyme is known to be produced from Proteus vulgaris (Proteusvulgaris) such as bacteria. Then, in order to obtain chondroitinase ABC, a method in which the cell disruption solution is sequentially subjected to streptomycin treatment, ammonium sulfate treatment, DEAE cellulose treatment and phosphocellulose treatment (J. Biol. Chem., 243 (7), 1523).
-1535 (1968)) or DEAE cellulose treatment, hydroxyapatite treatment, zinc-immobilized agarose treatment, and gel filtration treatment of the cell disruption solution (Agric.Biol.Che.
m., 50 (4), 1057-1059 (1986); JP-A-62-122588)
Etc. are known. On the other hand, recently chondroitinase ABC
Alternatively, attempts have been made to treat disc herniation by directly administering AC to the disc space, and its use as a therapeutic drug for disc herniation is expected [US Pat. No. 4,696,816, Clinical Orthopaedics, 253, 301-308]. (1990)]

In particular, chondroitinase ABC produced by Proteus vulgaris selectively removes chondroitin sulfate or dermatan sulfate side chains from proteoglycan and does not act on keratan sulfate, heparin or heparan sulfate, and It is considered to be suitable for industrial use due to its large production volume. Then, an enzyme preparation having chondroitinase ABC activity has been obtained from a culture of Proteus vulgaris by the above-mentioned method, but these all have a mixture of protease activity, endotoxin activity and nucleic acid. , Or unstable as an enzyme protein, it was unsuitable for administration as a therapeutic agent for disc herniation as described above or for use as a high-purity reagent [J. Biol.
m., 243 ( 7), 1523-1535 (1968), British Patent No. 1067253, Agric.Biol.Chem., 50 (4), 1057-1059 (1986), JP-A-62-122588. , JP-A-2-57180]. In particular, such mixture of impurities or poor stability may be a fatal drawback when using chondroitinase ABC as a drug.

[0004]

SUMMARY OF THE INVENTION The present invention has been made with the object of remedying such drawbacks.
That is, an object of the present invention is to provide a novel purified chondroitinase ABC, a crystallized product thereof, and a purified or crystalline chondrode thereof which can be used as a drug having high purity and high specific activity without impurities being mixed and having high stability. Itinase AB
It is intended to provide a method for producing C in a high yield. Further, the present invention is to provide a chondroitinase ABC-containing pharmaceutical composition containing such chondroitinase ABC as an active ingredient.

[0005]

[Means for Solving the Problems] The present inventors have aimed to solve the above-mentioned problems by using chondroitinase ABC.
As a result of repeated studies on the purification of the enzyme, the enzyme was extracted from the bacterial cells, the nucleic acid was removed from the enzyme extract, and the enzyme extract from which this nucleic acid had been removed was chromatographed by combining a weak cation exchange resin and a strong cation exchange resin. As a result, impurities such as endotoxin, nucleic acid, and protease are completely removed, and a single band is shown by electrophoresis (SDS-PAGE), and a single peak is obtained by high performance liquid chromatography (gel filtration, cation exchange). The present inventors have completed the present invention by finding that a purified purified chondroitinase ABC can be obtained. This chondroitinase ABC can be crystallized, its specific activity is about 3 times higher than that of conventional chondroitinase ABC preparations, it is stable even after long-term storage, and it is sufficiently used as a drug. It will be possible.

That is, the present invention relates to a purified chondroitinase ABC having a high degree of purification and high stability as will be shown later by its characteristics.

Further, the present invention relates to a method for producing purified chondroitinase ABC having a high degree of purification and high stability, which comprises the following steps. (i) a step of obtaining an enzyme extract from chondroitinase ABC-producing cells (step 1), (ii) a step of removing nucleic acids from the obtained enzyme extract (step 2), (iii) a nucleic acid was removed The enzyme extract is chromatographed using (a) a weak cation exchange resin to adsorb chondroitinase ABC, the adsorbed enzyme is eluted, and the eluate is chromatographed using a strong cation exchange resin. Treated with chondroitinase ABC
Of adsorbing and eluting (step 3-1), or (b)
Chromatographic treatment using strong cation exchange resin to adsorb and elute chondroitinase ABC, and eluate is subjected to chromatographic treatment using weak cation exchange resin to adsorb and elute chondroitinase ABC ( Step 3-2).

In the present invention, nucleic acid is removed from a bacterial cell extract containing chondroitinase ABC, and a chromatographic treatment is carried out using a combination of a weak cation exchange resin and a strong cation exchange resin. It is possible to obtain purified chondroitinase ABC which is pure and highly stable and has a specific activity about 3 times or more higher than that of chondroitinase ABC. Further, in the present invention, when the enzyme extract is obtained from the chondroitinase ABC-producing bacterial cells, the extraction efficiency is very good when the extraction is performed using a surfactant, and at the same time, the impurities are small and the specific activity of the target enzyme is high. An extract can be obtained. Further, in the present invention, chondroitinase AB
Chondroitinase ABC was extracted from C-producing cells using a surfactant, and the extract was chromatographed using either a weak cation exchange resin or a strong cation exchange resin to obtain chondroitinase ABC from the resin. The purified chondroitinase ABC having a high degree of purification can also be obtained by adsorbing the adsorbed enzyme on the above-mentioned enzyme and elution by continuously changing the ionic strength of the eluent by the gradient method. Furthermore, the present invention provides a highly purified chondroitinase AB as described above.
It can be produced by crystallizing C as a raw material and using a polyether having a structure having hydroxyl groups at both ends (for example, polyethylene glycol), has the characteristics of purified chondroitinase ABC, and is needle-shaped. Alternatively, it relates to a chondroitinase ABC crystal which is a columnar crystal. Such chondroitinase ABC and its crystals have high homogeneity, stable quality, high specific activity, and excellent storage stability (for example, at about 25-40 ° C, 1
Almost no decrease in activity even if left for months).

The method for producing the purified chondroitinase ABC and the chondroitinase ABC crystal of the present invention will be described in detail. Chondroitinase AB in the present invention
As the C-producing microbial cell, any microbial cell conventionally known to produce chondroitinase ABC can be used. For example, a chondroitinase ABC-producing bacterium belonging to Proteus bulgaris or the like is used. Specific examples of such bacteria include Proteus vulgaris.
NCTC 4636 (= ATCC 6896, = IFO 3988) can be mentioned. These cells are treated by the usual method (J. Biol. Chem. , 243 (7),
1523-1535 (1968), JP-A-62-122588, JP-A-2-5718
(See No. 0 publication), and collect wet bacterial cells from the culture solution.
This is suspended in a buffer solution having a pH around neutrality, and the enzyme is extracted from the suspension. Buffers around neutral are usually at pH
The cells are crushed by ultrasonic treatment using a buffer solution such as 6.0 to 8.0 1 to 100 mM phosphate buffer solution, Tris-hydrochloric acid buffer solution, acetate buffer solution, or treatment with a cell disruption device (Dino Mill etc.). , An enzyme solution containing chondroitinase ABC, protease, other enzyme, nucleic acid, protein and the like is extracted.

Further, in the extraction of chondroitinase ABC from bacterial cells, the extraction efficiency of the enzyme can be further enhanced by using a surfactant solution, for example, a buffer solution containing a surfactant. The surfactant used in the present invention may be any as long as it can efficiently extract the enzyme.

Nonionic surfactants are excellent as the surfactant used for extraction, and for example, polyoxyethylene alkyl ether, polyoxyethylene pt-octylphenyl ether, polysorbate and the like are preferable. Polyoxyethylene alkyl ethers include Emulgen-based surfactants, Liponox-based surfactants, Brij-based surfactants, and the like. These commercially available surfactants include Emulge
n 120, Emulgen 109 P, Liponox DCH, Brij 35, 78, 76,
96, 56, 58 or 98, Nikkol BL-9EX, BL-21 or BL-25. In addition, polyoxyethylene pt-octyl phenyl ether contains Triton surfactant, No
nidet type surfactant, Neutronyx type surfactant or
Examples include Conco surfactants. These commercially available surfactants include Triton X-100, X-45, X-114, X-10
2, X-165, X-305, X-405, NonidetP-40, Igepal CA-63
0, Neutronyx 605, Conco NIX-100 and the like can be exemplified. The polysorbate is preferably a sorbitan mono-9-octadecenoate poly (oxy-1,2-ethanediyl) derivative, such as Tween 20, 40, 60 or 80, Emasol.
Commercially available as 4115 or 4130.

Among these, polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether (POELE) can be mentioned as particularly preferable surfactants. Extracting an enzyme from bacterial cells using these surfactants not only enhances the extraction efficiency of the enzyme, but also reduces contamination with non-enzymatic contaminating proteins, nucleic acids or proteases as compared with other extraction methods. An enzyme extract can be obtained. According to the conventional extraction method using DYNO (registered trademark) MILL, the amount of enzyme extracted per 1 g of wet cells is about 150.
Although the amount was about 200 to about 200 units, at least about 500 to 600 units of enzyme can be extracted per 1 g of wet microbial cell when extracted with a surfactant. By performing chromatographic treatment with a weak cation exchange resin and a strong cation exchange resin from the extract with a surfactant, the endotoxin content is so small that it can be used for pharmaceuticals.
A highly active chondroitinase ABC that electrophoretically exhibits a single band can be easily obtained in a high yield.

Extraction is performed by using wet bacterial cells in the amount of 2 to 7% of the surfactant.
In addition to the buffer solution containing the microbial cell suspension, at 15 to 45 ° C,
It is preferably heated to around 37 ° C. and stirred for about 1 to 10 hours, preferably 2 to 6 hours, and the suspension is cooled to about room temperature.
This is separated into bacterial cell residue and extract by a separation means such as centrifugation. Since the extract thus obtained mainly contains chondroitinase ABC, other enzymes, proteins and nucleic acids, the following chondroitinase ABC purification step is performed.

Proteins, nucleic acids, etc. are removed from the bacterial cell extract thus extracted. Removal of proteins and nucleic acids may be carried out by any of the commonly used methods. In particular, in order to remove nucleic acids, addition of protamine sulfate should be taken into consideration when the enzyme is used as a drug. preferable. Protamine treatment was applied to the above-mentioned cell extract 3
~ 7% aqueous solution of protamine sulfate at a final concentration of about 0.25 to 1
The mixture is added so as to have a concentration of 10% and stirred for about 10 to 30 minutes at about 4 ° C. to room temperature to cause precipitation of nucleic acid and the like, which is separated and removed by centrifugation or another method.

The resulting supernatant is chondroitinase A.
Since it contains BC, protease and other enzymes, it is chromatographed using a cation exchange resin. In the method for producing the purified chondroitinase ABC of the present invention, a weak cation exchange resin and a strong cation exchange resin are used in combination and subjected to a chromatography treatment. Examples of the weak cation exchange resin used here include cation exchange resins whose exchange group is a carboxyalkyl group such as a carboxymethyl group. Specific examples include polysaccharide derivatives having a carboxymethyl group as an exchange group (agarose derivatives, cross-linked dextran derivatives, etc.), and commercially available products include CM Sepharose, CM Sephadex (all are trade names, Pharmacia). To be As the strong cation exchange resin, a cation exchange resin whose exchange group is a sulfoalkyl group can be exemplified. Specific examples thereof include polysaccharide derivatives having a sulfoethyl group, a sulfopropyl group or the like as an exchange group (agarose derivative, cross-linked dextran derivative, etc.), and commercially available products include S Sepharose or SP Sepharose (trade name,
Pharmacia), SP Sephadex (trade name, Pharmacia), SP Toyopearl (trade name, Tosoh Corporation) and the like. As a chromatographic treatment in which the above two kinds of cation exchange resins are combined, the following method can be given as an example.

The first chromatographic treatment is carried out by using the same buffer as that used for the extraction of the cells using the weak cation exchange resin, for example, a buffer having a pH of 6.5 to 7.5 (for example, 1 to 50 mM phosphate buffer, (Tris-HCl buffer, acetate buffer, etc.)
Equilibrate with, contact the supernatant containing the enzyme,
The enzyme is adsorbed, and the cation exchange resin is washed with a salt solution (for example, 20 to 25 mM NaCl solution and / or the surfactant solution (for example, 0.5% POELE solution)) as needed. Prepare an eluate in which about 0.1 μM of sodium chloride is dissolved, and contact with the resin to elute the fractions having enzyme activity.The elution method can be performed by the concentration gradient method (gradient method) or the stepwise method. Such a chromatographic treatment may be carried out by a column method or a batch method, and the obtained fraction is contacted with a strong cation exchange resin equilibrated with the same buffer to adsorb chondroitinase ABC, This cation exchange resin is washed with a salt solution (for example, 20 to 50 mM NaCl solution and / or water) if necessary, and then the same buffer solution (for example, phosphoric acid) containing 0 to about 0.5 M sodium chloride is used. The chondroitinase ABC is eluted and isolated by a concentration gradient method using a buffer solution, Tris-hydrochloric acid buffer solution, acetate buffer solution, etc. Such a chromatographic treatment is preferably carried out by a column method. It is also possible to carry out the chromatographic treatment with the exchange resin in the reverse order.

When the cells are extracted with a buffer containing a surfactant, the amount of impurities in the extract is small as described above. Therefore, a procedure for removing nucleic acids from the extract, such as protamine treatment, is performed. Alternatively, the enzyme can be purified by a simple operation in which the chondroitinase ABC is directly adsorbed on the CM sepharose column, the column is washed, and then the enzyme is eluted from the column by a concentration gradient method (gradient method) or the like. . The purified enzyme solution subjected to chromatography may be concentrated and desalted by a conventional method, and may be directly used as a purified enzyme solution as a drug, a reagent, etc. Alternatively, the concentrated and desalted enzyme solution may be denatured or lost of the enzyme. It may be pulverized by a general drying method (freeze-drying method etc.) under a condition that it is not activated. Further, the purified enzyme solution is treated with a polyether having a structure having hydroxyl groups at both ends (eg, polyethylene glycol, polypropylene glycol, etc.)
It is also possible to crystallize chondroitinase ABC by mixing and contacting with chondroitinase ABC. For example, chondroitinase ABC
Polyethylene glycol (molecular weight 4,000, 6,000
Etc.) to adjust the enzyme concentration to 250 to 500 U / ml and the polyethylene glycol concentration to 5 to 20%, preferably 10 to 15%, and leave at room temperature to 4 ° C. until crystals are formed. This crystal is an orthorhombic or monoclinic needle-like or columnar crystal and exhibits the crystal parameters as shown in the examples.

The chondroitinase ABC thus obtained is free from impurities such as endotoxin, nucleic acid, protease, and other proteins, and shows a single band by electrophoresis (SDS-PAGE), and HPLC (Gel filtration,
(Cation exchange) also shows a single peak, and the specific activity is about 3 times higher than that of conventional chondroitinase ABC. Moreover, in the method of the present invention, it is not necessary to carry out a concentrated desalting operation in the middle of the ammonium sulfate fractionation or the manufacturing process as in the conventional method, so that the manufacturing time of chondroitinase ABC is shortened and the yield is improved. Therefore, the manufacturing cost can be reduced. Furthermore, even a small amount of chondroitinase ABC or a large amount of chondroitinase ABC can be freely produced regardless of the production amount. Moreover, chondroitinase ABC crystals obtained by crystallization by the above method were needle-like or columnar crystals (see FIG.
See also), the quality is stable, the specific activity is high, and the storage stability is excellent.

The properties of chondroitinase ABC obtained by the above method of the present invention are shown below. (1) Action hyaluronic acid, chondroitin sulfate, chondroitin,
It acts on dermatan sulfate and produces a small amount of large unsaturated oligosaccharides in the early stage of the reaction, and finally, unsaturated disaccharide (Δ ⁴ -glucuronyl-N-acetylhexosamine and its 4-
Or 6-sulfate) and unsaturated oligosaccharide mixtures. (2) Optimum pH and stable pH When chondroitin sulfate C is used as a substrate (in Tris-HCl buffer), the optimum pH is 8.0 to 8.2 (Fig. 2). pH 5
-9 shows a residual activity of about 80% or more when left at 25 ° C. for 24 hours (FIG. 3).

(3) Method for measuring titer It is based on the fact that an unsaturated disaccharide having a remarkable absorption in the ultraviolet is produced by an enzymatic reaction. Chondroitin sulfate C (substrate) 1.2 mg, 50 mM Tris-hydrochloric acid buffer solution (pH 8 to 8.5) containing 50 mM sodium acetate, an enzyme reaction solution obtained by adding an enzyme to 10 μg of casein were reacted by incubating at 37 ° C. for 10 minutes, pH
Stop the reaction by adding 1.8M 0.05M hydrochloric acid and measure the absorption at 232nm. On the other hand, as a control, the heat-denatured enzyme solution was held in the substrate solution having the above composition, and the same treatment was performed.
Measure the absorption at 232 nm. The amount of unsaturated disaccharide in the product is calculated from the increase in absorption over the control. In addition, 2-
The millimolar molecular extinction coefficient of acetamido-2-deoxy-3-0- (β-D-gluco-4-empyranosyluronic acid) -6-0-sulfo-D-galactose is 5.5. As a result, 1 unit (U) of enzyme is defined as the amount which catalyzes the reaction of liberating 1 micromol of unsaturated disaccharide in 1 minute under the above reaction conditions.

(4) Optimum temperature of action and temperature stability The optimum temperature of action is 37 ° C, and approximately 90% or more of the activity is shown at 30 to 37 ° C (Fig. 4). When Tris-hydrochloric acid buffer of pH 7.0 was used and each temperature was maintained for 1 hour and the residual activity was measured, it was stable between 2 ° C and 30 ° C and inactivated at 50 ° C (Fig. 5).

(5) Inhibition As shown in Table 1, the activity is inhibited by zinc (Zn ²⁺ ), nickel (Ni ²⁺ ), iron (Fe ³⁺ ), and copper (Cu ²⁺ ).

[0023]

[Table 1] ────────────────────────── Compound concentration (mM) Relative activity (%) ───────── ────────────────── Additive ─── 100 ZnCl ₂ 2 1.2 CuCl ₂・ 2H ₂ O 2 1.2 NiCl ₂・ 6H ₂ O 2 4.2 FeCl ₃・ 6H ₂ O 0.2 5 ──────────────────────────

(6) Molecular weight Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results in a single band, which is about 100,000 daltons in both reduced and non-reduced states. In addition, about 10
It is 0,000 daltons (see Figure 6; conditions are described below). (7) Isoelectric point about 8.2 and about 8.5 [Phast Gel IEF pH3-9]
And pI calibration kit 3 as standard
~ 10 and measured using the Phast System instrument
(All reagents and equipment are manufactured by Pharmacia)

(8) Amino acid analysis As shown in Table 2.

[0026]

[Table 2] ────────────────────── Amino acid residue number / molecule ─────────────────── ──── Asp 106 Thr 51 Ser 63 Glu 87 Gly 53 Ala 50 Val 37 Met 16 Ile 45 Leu 76 Tyr 32 Phe 34 Lys 54 His 18 Arg 28 Pro 34 ─────────────── ────────

This analysis is based on chondroitinase AB
C was decompressed in 6N hydrochloric acid and hydrolyzed at 110 ° C. for 24 hours. The sample was measured three times and the average value was shown. The total molecular weight was calculated as 100,000. Trp and Cys have not been measured.

(9) Terminal Amino Acid The N-terminal amino acid sequence is Edman degradation (“Biochemistry Experimental Course 1-Protein Chemistry II, Primary Structure Determination” 13
2 to 142, August 28, 1976, Tokyo Chemical Co., Ltd. (published by Doujin), it was alanine. The N-terminal amino acid sequence was alanine-threonine-X-asparagine-proline-alanine-phenylalanine-aspartic acid-proline- (where X is unidentified). In addition, the C-terminal amino acid sequence was determined by the carboxypeptidase method using carboxypeptidase Y (“Biochemistry Laboratory 1-Protein Chemistry II, Primary Structure Determination”).
˜Page 211) and was -serine-leucine-proline. As a result, the terminal of chondroitinase ABC of the present invention consists of the following amino acid residues. Ala-Thr-X-Asn-Pro-Ala-Phe-Asp-Pro- ……… Ser-Leu-P
ro X: unidentified

(10) Stability In the presence of a phosphate buffer (pH 6 to 8), it is stable at room temperature for 3 months or longer in both solution and dry conditions. (11) Specific activity 300 U / mg protein or more (12) Other HPLC (cation exchange chromatography) and HPLC (GP
C; gel filtration) showed a single peak. In addition,
The conditions and results of gel filtration are as follows.

Condition column: TSK G3000SW _xl (manufactured by Tosoh Corporation) _Eluent : 0.1M phosphate buffer containing 0.2M sodium chloride
(pH7.0) Elution rate: 0.5ml / min Operating temperature: 35 ℃ Detection wavelength: 280nm Injection volume: 20μl Molecular weight marker: Cytochrome C (12.4 kilodalton) Adenylate kinase (32 kilodalton) Enolase (67 kilodalton) Lactate Dehydrogenase (142 kilodalton) Glutamate dehydrogenase (290 kilodalton) (These are all manufactured by Oriental Yeast Co., Ltd.)

Results The chondroitinase ABC of the present invention had a retention time of 18.43.
It eluted as a single peak at the minute position (Figure 6). This retention time was compared with the above-mentioned molecular weight markers to obtain a molecular weight of about 10
Decided to be 0 kilodaltons. It contained virtually no endotoxin, and DNA and protease were below the detection limit.
When a commercially available chondroitinase ABC (Seikagaku Corporation, Catalog No. 100332) was measured in the same manner, DN
A is 5,000 times the detection limit, and protease is 2 times the detection limit.
It was 00 times.

The chondroitinase ABC of the present invention
When compared with the most highly purified chondroitinase ABC (Seikagaku Corporation, Chondroitinase ABC protease free, Catalog No. 100332), the molecular weight of the former was SDS-PAGE and gel filtration. In both cases, the latter is about 100,000 daltons, while the latter is about 80,000 daltons by SDS-PAGE and about 120,000-145,000 daltons by gel filtration, which is a big difference. Moreover, the specific activity of the former is 300 U / mg protein or more, whereas the latter is around 110 U / mg protein, and the stability is stable at room temperature for 3 months or more, while the latter is -70 ° C. It is stable for more than 3 months, which is another point. Further, the former chondroitinase ABC can be singly crystallized by electrophoresis and HPLC, and thus the terminal amino acid, isoelectric point and the like can be measured, while the latter can be identified as a substance. Since it was not a pure enzyme preparation, the terminal amino acid, isoelectric point, etc. could not be measured, and this point also differs.

The chondroitinase ABC of the present invention is a highly purified enzyme as described above, and can be used not only as a reagent but also as a pharmaceutical. As a medicine, for example, if necessary, a suitable pH adjusting agent (eg, phosphate buffer) is added to the purified chondroitinase ABC of the present invention or chondroitinase ABC crystals to adjust the pH to a stable pH range, and then purified water , Physiological saline, etc. to make a liquid formulation as it is, or dextran,
As a solid preparation mixed with sucrose, lactose, maltose, mannitol, xylitol, sorbitol, serum albumin or the like, a therapeutic agent for disc herniation containing chondroitinase ABC as an active ingredient can be used. This therapeutic agent can be injected into the intervertebral disc space of a herniated patient and used for intervertebral disc lysis therapy in which the hernia is dissolved and treated. The amount used varies depending on symptoms, age, etc. and cannot be specified in a general manner, but usually 10 to 100U
Inject the degree.

[0034]

Example 1 Next, the chondroitinase ABC of the present invention
The manufacturing method will be specifically described with reference to examples. Proteus bulgaris (NCTC 4636, ATCC 6896, IFO 39
88) and the conventional method (J. Biol. Chem., 243) .
(7), 1523-1535 (1968)) to obtain wet bacterial cells. To 200 g of the wet cells, 600 ml of 5 mM phosphate buffer (pH 6.5 to 7.0) was added and suspended, the cells were crushed by DYNO (registered trademark) MILL and centrifuged to obtain an enzyme extract. (Step 1). Protamine sulfate was used to remove nucleic acids from the cell extract. Add 5% to the final concentration of 0.5% in the enzyme extract.
% Protamine sulfate solution was added and stirred at 4 ° C for about 30 minutes,
The resulting precipitate was removed by centrifugation to obtain a supernatant (step 2). About 5 times the amount of water was added to the obtained supernatant and the mixture was purified by column chromatography. That is, a column filled with CM sepharose was added with 5 mM phosphate buffer (pH 6.5 to 7.
After 0) was flowed to reach an equilibrium state, the supernatant was flowed and adsorbed. The column is then washed with the same buffer and then 0.025
The extract was washed with the same buffer containing M sodium chloride and then eluted with the above buffer containing 0.1 M sodium chloride to obtain a fraction having enzyme activity (step 3).

About 5 times the amount of water was added to this enzyme active fraction,
It was adsorbed on an S-Sepharose column previously equilibrated with 5 mM phosphate buffer (pH 6.5 to 7.0). The column was then washed with the above buffer, then with the above buffer containing 0.025 M sodium chloride, and then eluted by the gradient method with the above buffer containing 0.025 to about 0.35 M sodium chloride. The eluted chondroitinase ABC showed a single band (SDS-PAGE) and
(DNA), protease, etc. are removed, and the specific activity is 38
It was 0 U / mg, and the specific activity was about 3 times higher than that of the conventional chondroitinase ABC, and the enzyme was highly pure. Polyethylene glycol (molecular weight 4,000) was added to this enzyme solution (phosphate buffer solution (pH 7.0)) at a concentration of 15%, and the mixture was allowed to stand at room temperature for about 1 week. Chondroitinase ABC
Crystals formed. A micrograph (2.5 times) of the obtained crystal is shown in FIG. As a result of X-ray crystallography, the obtained crystal was an orthorhombic or monoclinic acicular or columnar crystal, and exhibited the following crystal parameters.

In the case of the orthorhombic system; In the case of the monoclinic system; Space group P222 ₁ Space group P2 ₁ Lattice constant a = 214Å Lattice constant a = 214Å b = 92Å b = 56Å c = 56Å c = 92Å α = 90 ° α = 90 ° β = 90 ° β ≧ 90 ° (about 108 °) γ = 90 ° γ = 90 °

The results of SDS-PAGE in each purification step are shown in FIG. SDS-PAGE uses 10% gel and is
mli, UK, Nature, 227 , 680-685 (1970)). Treatment with S-Sepharose results in a distinct single band in reduced and non-reduced states (FIG. 7,
(See C and D). The degree of purification of chondroitinase ABC in each step of the present invention is shown in Table 3.

[0038]

[Table 3]

Table 4 shows the endotoxin content and the residual amount of protease in each step.

[0040]

[Table 4] * Endotoxin content per 100 units (U) of chondroitinase ABC; measured using Toxicolor System (Seikagaku Corporation). EU is endotoxin unit (End
otoxin Unit). ** Measurement using FITC-casein as a substrate The endotoxin content in the enzyme solution obtained in step 4 was substantially 5.0 pg / 100U, as described above, and was substantially free of nucleic acid (DNA). Was measured by the threshold method [DNA measuring device: threshold (manufactured by Molecular Devices)].
Was not detected (below the detection limit).

Table 5 shows the stability of the above chondroitinase ABC obtained in step 4 of the present invention when it is left in a solution state at each temperature. It was stored at a concentration of 350 U / ml for 5 days, and the activity when stored at -40 ° C was defined as 100% and expressed as a relative activity.

[0042]

[Table 5]

[0043]

Example 2 7.5 kg of wet cells of Proteus vulgaris (NCTC4636 ATCC 6896, IFO 3988) used in Example 1 was
0% polyoxyethylene lauryl ether [(POELE; BL-9
EX (trade name)] added 40 mM phosphate buffer (pH 7.0 ± 0.
5) About 15 liters and about 7.5 liters of purified water were put into a tank and stirred to obtain a cell suspension. This bacterial cell suspension, 35 ℃ ± 3
The mixture was heated to ℃ and stirred at the same temperature for about 2 hours. After about 2 hours, about 30 l of a 20 mM phosphate buffer solution (pH 7.0 ± 0.5) was added to dilute the solution, and the suspension was cooled until the solution temperature reached 20 ° C. This suspension was centrifuged with a Sharpless centrifuge to obtain the centrifugation supernatant. The liquid temperature was measured before and after centrifugation so as not to exceed 20 ° C.

The centrifugal supernatant obtained was diluted by adding 30 liters of purified water cooled to 2 to 15 ° C., and this was passed through a CM-Sepharose column (gel amount about 5 liters) to obtain chondroitinase ABC.
Was adsorbed on the column. The column was washed with 1 L of purified water, and 10 L of 0.5% POELE aqueous solution was added.
The column was washed with 5 L of 04M phosphate buffer (pH 6.2 ± 0.2). Next, about 10 L each of 0.04 M phosphate buffer (pH 6.2 ± 0.2) containing OM and 0.25 M NaCl was applied to this column by a linear gradient to flow the eluent to elute chondroitinase ABC. The eluate was passed through the pores.
The solution was filtered through a 22 μm filter-sterilized felt and collected in a sterile container to obtain a chondroitinase ABC enzyme solution. The relationship between the bacterial cell extraction time (hr) by POELE and the enzyme activity is shown in Table 6 below.

[0045]

[Table 6]

When the cells were extracted, the enzyme concentration reached equilibrium within 1 to 2 hours. In addition, the total amount of nucleic acid in the extract reached the maximum within 0.25 hours after stirring (about 25 g / bacteria), and then decreased to about 1
After time, equilibrium (about 2-3 g / bacillus) was reached.

[0047]

Example 3 5% of 5 kg of wet cells of Prolaus vulgaris (NCTC4636, ATCC6896, IFO3988) used in Example 1 was added to 5%.
5 volumes of 20 mM phosphate buffer (pH 7.0) with the addition of POELE
In addition, the mixture was extracted at 37 ° C for 3 hours. This extract was used in Example 1.
Protamine treatment, CM- Sepharose treatment and S-
After treatment with Sepharose (these conditions are shown in Table 7), chondronacetinase ABC was eluted by elution with Glaciendo. This solution showed a single band of chondroitinase ABC on electrophoresis (SDS-PAGE). The enzyme solution was passed through a 0.22 μm filter sterilization filter using a liquid feed pump, and the filtrate was collected in a sterilization container to give a chondroinase ABC enzyme solution. As a result, when calculated based on the table, about 500 units of chondoitinase A per 1 g of bacterial cells
BC could be extracted. Next, the degree of purification of chondroitinase ABC in each step of Example 3 is shown in Table 7.

[0048]

[Table 7]

Extraction time and chondroitinase A in the extraction of chondroitinase ABC from the cells
The relationship with the activity of BC is shown in FIG.

[0050]

Example 4 A surfactant was used in the same manner as in Example 3.
Chondroitinase ABC was extracted using Triton, Brij, Nonidet or POELE at a concentration of 2% or 5% and a suspension temperature of 25 ° C or 37 ° C.
The results are shown in FIGS. In each case, the activity of the extract was higher when the mixture was heated at 37 ° C. and the surfactant concentration was 5%, and most of the activity was extracted in about 1 to 2 hours.

[0051]

EFFECT OF THE INVENTION Purified chondroitinase ABC of the present invention
Alternatively, the crystal thereof is extremely highly purified and substantially free of protease, nucleic acid and endotoxin, and therefore has extremely high utility as a pharmaceutical and a research reagent. That is, the purified chondroitinase ABC of the present invention
Alternatively, when the crystal is used as a medicine, the storage stability is excellent and the activity hardly decreases. In addition, since it has a high specific activity and does not contain impurities, the dose can be reduced and no side effects are exhibited. Further, when used as a research reagent, for example, in an experiment using animal cells, the enzyme of the present invention does not contain endotoxin and protease, so that the reproducibility of the experimental result is enhanced. Furthermore, since the purification method of the present invention omits the conventionally required ammonium sulfate fractionation and concentration desalting operation during purification, the number of purification steps and time are shortened, the yield is improved, and the production cost is increased. Could be reduced.

[Brief description of drawings]

FIG. 1 shows a micrograph of a chondroitinase ABC crystal of the present invention.

FIG. 2 shows the relationship between the activity of chondroitinase ABC of the present invention and the reaction pH.

FIG. 3 shows the chondroitinase ABC of the present invention at a predetermined pH.
Shows the relationship between pH and residual activity when left at 25 ° C for 24 hours.

[Explanation of symbols]

○ ── ○ Acetate buffer × ── × Tris-acetate buffer △ ── △ Tris-HCl buffer ● ── ● Residual activity of glycine buffer when kept in each buffer

FIG. 4 shows the relationship between the activity of chondroitinase ABC of the present invention and the reaction temperature.

FIG. 5 shows the relationship between temperature and residual activity when the chondroitinase ABC of the present invention was kept at a predetermined temperature for 1 hour.

FIG. 6 shows the chondroitinase ABC of the present invention,
The chromatogram when gel filtration by HPLC is performed is shown.

FIG. 7: Chondroitinase ABC of Example 1 of the present invention
The band of SDS-PAGE in each stage of the purification process of is shown.

[Explanation of symbols]

 A: Protamine-treated supernatant B: CM-Sepharose treated solution C: S-Sepharose treated solution (non-reduced) D: S-Sepharose treated solution (reduced)

FIG. 8 shows the relationship between chondroitinase ABC extraction time and chondroitinase ABC activity from the cells of Example 3 of the present invention.

FIG. 9: Triton with different concentrations of the bacterial cells of Example 4 of the present invention
The relationship between the extraction time and the chondroitinase ABC activity of the X-100-containing buffer extracted under the conditions of 25 ° C and 37 ° C is shown.

[Explanation of symbols]

1 Surfactant concentration 2%, extraction temperature 25 ℃: (── □ ─
─) 2 Surfactant concentration 2%, extraction temperature 37 ℃: (── ＋ ─
─) 3 Surfactant concentration 5%, extraction temperature 25 ℃: (── ◇ ─
─) 4 Surfactant concentration 5%, extraction temperature 37 ℃: (── △ ─
-)

[Fig. 10] Brij of Example 4 of the present invention with different concentrations of Brij
The relation between the extraction time and the chondroitinase ABC activity of the sample extracted with -35-containing buffer at 25 ° C and 37 ° C is shown.

[Explanation of symbols]

1 Surfactant concentration 2%, extraction temperature 25 ℃: (── □ ─
─) 2 Surfactant concentration 2%, extraction temperature 37 ℃: (── ＋ ─
─) 3 Surfactant concentration 5%, extraction temperature 25 ℃: (── ◇ ─
─) 4 Surfactant concentration 5%, extraction temperature 37 ℃: (── △ ─
-)

FIG. 11 shows that the cells of Example 4 of the present invention were treated with Noni at different concentrations.
The relation between the extraction time and the chondroitinase ABC activity of the extract extracted with the det P-40-containing buffer under the conditions of 25 ° C and 37 ° C is shown.

[Explanation of symbols]

1 Surfactant concentration 2%, extraction temperature 25 ℃: (── □ ─
─) 2 Surfactant concentration 2%, extraction temperature 37 ℃: (── ＋ ─
─) 3 Surfactant concentration 5%, extraction temperature 25 ℃: (── ◇ ─
─) 4 Surfactant concentration 5%, extraction temperature 37 ℃: (── △ ─
-)

FIG. 12: POEL with different concentrations of the bacterial cells of Example 4 of the present invention
The relationship between the extraction time and the chondroitinase ABC activity of the E-containing buffer extracted at 25 ° C. and 37 ° C. is shown.

[Explanation of symbols]

1 Surfactant concentration 2%, extraction temperature 25 ℃: (── □ ─
─) 2 Surfactant concentration 2%, extraction temperature 37 ℃: (── ＋ ─
─) 3 Surfactant concentration 5%, extraction temperature 25 ℃: (── ◇ ─
─) 4 Surfactant concentration 5%, extraction temperature 37 ℃: (── △ ─
-)

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 13, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

FIG. 1 is a micrograph showing a crystal structure of chondroitinase ABC of the present invention.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

Claims (11)

[Claims] 1. A purified chondroitinase ABC having the following characteristics. (i) SDS-polyacrylamide gel electrophoresis (S
Approximately 100,000 daltons by DS-PAGE) (both reduced and non-reduced) and gel filtration. (ii) The isoelectric points are about pH 8.2 and about pH 8.5. (iii) The optimum pH is 8.0 to 8.2 (substrate: chondroitin sulfate C, buffer: Tris-hydrochloric acid buffer), and the optimum temperature is 37 ° C. (iv) The activity is inhibited by Zn ²⁺ , Ni ²⁺ , Fe ³⁺ , and Cu ²⁺ . (v) The N-terminal amino acid is alanine and the C-terminal amino acid is proline. (vi) SDS-PAGE shows a single band, and high performance liquid chromatography (gel filtration and cation exchange) also shows a single peak. (vii) Endotoxin is not substantially contained, and nucleic acid and protease contents are below the detection limit. (viii) Crystallizable. (ix) The specific activity is 300 U / mg or more. 2. The method for producing the purified chondroitinase ABC according to claim 1, wherein the chondroitinase ABC is extracted from the chondroitinase ABC-producing bacterial cells and the extract is purified. 3. The method for producing purified chondroitinase ABC according to claim 2, wherein the extraction is performed using a surfactant solution. 4. The method for producing purified chondroitinase ABC according to claim 1, which comprises the following steps. (i) obtaining an enzyme extract from chondroitinase ABC-producing cells (step 1), (ii) removing nucleic acid from the obtained enzyme extract (step 2), (iii) removing nucleic acid The enzyme extract is chromatographed using (a) a weak cation exchange resin to adsorb chondroitinase ABC, the adsorbed enzyme is eluted, and the eluate is chromatographed using a strong cation exchange resin. Treated with chondroitinase ABC
Of adsorbing and eluting (step 3-1), or (b)
Chromatographic treatment using strong cation exchange resin to adsorb and elute chondroitinase ABC, and eluate is subjected to chromatographic treatment using weak cation exchange resin to adsorb and elute chondroitinase ABC ( Step 3-2). 5. In step 3-1 or 3-2, a resin whose exchange group is a carboxyalkyl group is used as the weak cation exchange resin, and a resin whose exchange group is a sulfoalkyl group is used as the strong cation exchange resin. The method for producing the purified chondroitinase ABC according to claim 4. 6. The chondroitinase AB in step 1
The method for producing purified chondroitinase ABC according to claim 4, wherein the C-producing microbial cell is extracted with a surfactant solution to obtain an enzyme extract. 7. The method for producing purified chondroitinase ABC according to claim 1, which comprises the following steps. (i) a step of obtaining an enzyme extract by extracting from chondroitinase ABC-producing cells with a surfactant solution, (ii) the obtained enzyme extract is a weak cation exchange resin or a strong cation exchange resin A step of adsorbing the chondroitinase ABC onto the resin by performing a chromatographic treatment using (iii) eluting the chondroitinase ABC adsorbed to the resin by continuously changing the ionic strength of the eluent Process. 8. A chondroitinase ABC crystal having the characteristics (i) to (ix) of claim 1 and being a needle-like or columnar crystal. 9. The chondroitinase ABC crystal according to claim 8, which is crystallized using a polyether having a structure in which both ends are hydroxyl groups. 10. An orthorhombic or monoclinic chondroitinase ABC needle-like or columnar crystal having the characteristics of claim 1 and having the following crystal parameters. In case of orthorhombic system; space group P222 ₁ lattice constant a = 214Å b = 92Å c = 56Å α = 90 ° β = 90 ° γ = 90 ° In case of monoclinic system; space group P2 ₁ lattice constant a = 214Å b = 56Å c = 92Å α = 90 ° β ≧ 90 ° γ = 90 ° 11. A pharmaceutical composition containing chondroitinase ABC, wherein the purified chondroitinase ABC according to claim 1 or the crystalline chondroitinase ABC according to claim 8 or 10 is used as chondroitinase ABC. A pharmaceutical composition containing chondroitinase ABC, characterized in that