JPH10262660A - New chondroitin sulfate lyase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new chondroitin sulfate lyase useful for the analysis of the sequence of a saccharide chain and the treatment of hernia of the intervertebral disk, etc. SOLUTION: This chondroitin sulfate has the hollowing physicochemical properties: activity, decomposing an N-acetylhexosaminide bond of a glucosaminoglucan and producing only an unsaturated glucosaminoglucan disaccharide by the complete decomposition, and having an exo-type enzymatic activity; substrate specificity, active on a sulfate chain part of chondroitin (4-sulfate), dermatan sulfate, etc., and indicative on keratan sulfate, heparin, etc.; molecular weight, 105 kDa determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis under reductive condition; amino acid composition, detecting cystine by the amino acid analysis of an hydrolysate of the enzyme; amino acid at the N-terminal, leucine; isoelectric point, pH8.45; and optimum temperature, around 40 deg.C. Further, the objective chondroitin sulfate lyase is obtained by cultivating a cell (e.g. Proteus vulgaris, strain NCTC 4636) having a productivity of a chondroitin sulfate lyase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel chondroitin sulfate degrading enzyme, a crystal of the enzyme, an antibody recognizing the enzyme, a method for producing the enzyme, and an unsaturated glycosaminoglycan disaccharide using the enzyme. And a method for analyzing glycosaminoglycan, a composition comprising the enzyme and a carrier, and a therapeutic agent for a herniated disc comprising the composition.

[0002]

2. Description of the Related Art Chondroitin sulfate degrading enzyme (hereinafter also referred to as chondroitinase) is a general term for enzymes capable of degrading chondroitin sulfate, a kind of glycosaminoglycan, and is produced by certain microorganisms. It has been known.

[0003] In particular, Proteus vulgaris
chondroitinase ABC (Chondoroitinase ABC) [EC 4.
2.2.4], and this enzyme has been reported to act on chondroitin sulfate, dermatan sulfate, chondroitin, and hyaluronic acid to produce unsaturated disaccharides. However, this chondroitinase A
When BC is purified to an extremely high purity (hereinafter, the ultra-highly purified chondroitinase ABC is also referred to as “purified chondroitinase ABC”), the purified chondroitinase ABC completely eliminates glycosaminoglycan. It was found that the mixture did not decompose to a saturated disaccharide, but produced a mixture of an unsaturated disaccharide and an unsaturated oligosaccharide as a final decomposition product (Japanese Patent Laid-Open No. 153947/1994). Japanese Patent Application Laid-Open No. 6-153947 discloses that this purified chondroitinase ABC produces a small amount of large unsaturated oligosaccharide in the early stage of the reaction.

The enzymes produced by Proteus vulgaris are described in WO95 / 29256 and WO95 / 29232 as "chondroitinase I" and "chondroitinase II". Kind is disclosed. In these publications, (1) chondroitinase I acts on chondroitin sulfate and chondroitin sulfate chain of chondroitin sulfate proteoglycan but not on chondroitin sulfate tetrasaccharide, (2) chondroitinase II It is disclosed that it acts on tetrasulfate sulfate but not on chondroitin sulfate and the chondroitin sulfate chain of chondroitin sulfate proteoglycans.

However, chondroitin sulfate, a chondroitin sulfate degrading enzyme that acts on both chondroitin sulfate chains of chondroitin sulfate proteoglycans and chondroitin sulfate tetrasaccharide has not been known.

At present, various glycosaminoglycan-degrading enzymes are known, but they act exo-type on glycosaminoglycans and glycosaminoglycan chains of proteoglycans, and are successively unsaturated from the non-reducing terminal side. No enzyme that liberates disaccharides was known.

[0007]

SUMMARY OF THE INVENTION Chondroitin sulfate of various molecular sizes, from chondroitin sulfate chains of chondroitin sulfate and chondroitin sulfate proteoglycans (high molecular weight chondroitin sulfate) to chondroitin sulfate tetrasaccharide (low molecular weight chondroitin sulfate). If a chondroitin sulfate degrading enzyme with a broad substrate specificity and an exo-type degradation pattern can be obtained, it is extremely useful for the production of chondroitin sulfate disaccharides and the analysis of chondroitin sulfate, especially for sugar chain sequence analysis. Is expected. Furthermore, like purified chondroitinase ABC, which is currently expected to be used as a therapeutic agent for herniated discs, it is expected to be used as a pharmaceutical.

[0008] That is, an object of the present invention is to provide a novel chondroitin sulfate-degrading enzyme, particularly an exo-type chondroitin sulfate-degrading enzyme that acts on chondroitin sulfate having various molecular sizes. An antibody recognizing the enzyme, a method for producing the enzyme, a method for producing an unsaturated glycosaminoglycan disaccharide using the enzyme, a method for analyzing glycosaminoglycan, a composition containing the enzyme, and the composition. It is to provide a therapeutic agent for herniated disc.

[0009]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, purified chondroitinase ABC is an endo-type enzyme, and purified chondroitinase ABC acts on unsaturated tetrasaccharide. It was confirmed that it did not decompose into unsaturated disaccharides,
Proteus vulgaris suggested that Bulgaris also produces a chondroitin sulfate degrading enzyme capable of degrading unsaturated oligosaccharides into unsaturated glycosaminoglycan disaccharides in addition to purified chondroitinase ABC. The present inventors attempted to separate a novel chondroitin sulfate degrading enzyme of the exo type, which acts on chondroitin sulfate of various molecular sizes and on the chondroitin sulfate chain of chondroitin sulfate proteoglycan to generate an unsaturated disaccharide, thereby completing the present invention. Was.

That is, the present invention provides a chondroitin sulfate degrading enzyme having the following physicochemical properties (hereinafter, also referred to as “enzyme of the present invention”). Action: (1) Decomposes the N-acetylhexosaminide bond of glycosaminoglycan, and when completely decomposed, substantially produces only unsaturated glycosaminoglycan disaccharide. (2) It is an exo-type enzyme. Substrate specificity: chondroitin, chondroitin 4-
Sulfate, chondroitin 6-sulfate, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin 6-sulfate hexasaccharide, chondroitin 6-sulfate tetrasaccharide,
It acts on both the chondroitin sulfate proteoglycan and the chondroitin sulfate chain portion. It has no effect on keratan sulfate, heparin and heparan sulfate. Molecular weight: about 105 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions
kDa. Amino acid composition: Cystine is detected by amino acid analysis of the acid hydrolyzate of the enzyme.

Preferably, in addition to the above physicochemical properties,
A chondroitin sulfate degrading enzyme having the following physicochemical properties is provided. N-terminal amino acid: The N-terminal amino acid is leucine. Isoelectric point: pH about 8.45 (isoelectric focusing) Optimum temperature: around 40 ° C (substrate: chondroitin 6-sulfate, buffer: Tris-HCl buffer, pH:
8.0) Optimum reaction pH: around pH 8 (substrate: chondroitin 6)
(Sulfuric acid, buffer: Tris-HCl buffer, temperature: 37 ° C.) The present invention also relates to a crystal of the above chondroitin sulfate degrading enzyme, which is a plate-like crystal. (Hereinafter, also referred to as “crystal of the present invention”).

The present invention further provides an antibody that selectively recognizes the above-mentioned chondroitin sulfate degrading enzyme (hereinafter, also referred to as “antibody of the present invention”). Furthermore, the present invention provides a method for producing the above-mentioned chondroitin sulfate-degrading enzyme, which comprises culturing cells having a chondroitin sulfate-degrading enzyme-producing ability and collecting the above-mentioned chondroitin sulfate-degrading enzyme from a culture of the cells (hereinafter referred to as chondroitin sulfate-degrading enzyme; , "Also referred to as the" enzyme production method of the present invention ").

Further, the present invention provides a glycosaminoglycan which can be a substrate for the chondroitin sulfate-degrading enzyme,
Degrading with the chondroitin sulfate degrading enzyme, and fractionating an unsaturated glycosaminoglycan disaccharide from the degradation product, characterized in that the unsaturated glycosaminoglycan disaccharide is characterized by the following steps: (Hereinafter, also referred to as “the method for producing an unsaturated disaccharide of the present invention”).

Further, the present invention provides a glycosaminoglycan which can be a substrate for the chondroitin sulfate degrading enzyme,
A method for analyzing glycosaminoglycans (hereinafter, also referred to as "the analysis method of the present invention"), comprising at least a step of decomposing with the chondroitin sulfate degrading enzyme and a step of analyzing the decomposition product. provide.

Further, the present invention provides a chondroitin sulfate degrading enzyme-containing composition (hereinafter referred to as "the composition of the present invention") comprising the above chondroitin sulfate degrading enzyme and the above-mentioned carrier which does not affect the chondroitin sulfate degrading enzyme activity. To provide).

The present invention further provides a therapeutic agent for herniated disc comprising the above composition (hereinafter, also referred to as "the therapeutic agent of the present invention").
I will provide a. In addition, in this specification, the "unsaturated type" means a sugar chain having an unsaturated bond such as a double bond in the non-reducing terminal sugar of the sugar chain.

In the present specification, “saturated type” means
It means a sugar chain having no unsaturated bond such as a double bond in the non-reducing terminal sugar of the sugar chain. In the present specification, the term “glycosaminoglycan” includes, in addition to glycosaminoglycans (polysaccharides) used in a usual meaning, low molecular weight products of the glycosaminoglycans (eg, glycosaminoglycan oligosaccharides). Is done. Examples of glycosaminoglycan oligosaccharides include, for example, glycosaminoglycan octasaccharide, glycosaminoglycan hexasaccharide, glycosaminoglycan tetrasaccharide and the like.

Similarly, the term “chondroitin sulfate” as used herein includes chondroitin 4-sulfate (also called chondroitin sulfate A), chondroitin 6
-Sulfuric acid (also referred to as chondroitin sulfate C), chondroitin sulfate D, chondroitin sulfate E, chondroitin sulfate K, etc., which are usually classified as chondroitin sulfate, and dermatan sulfate (formerly also referred to as "chondroitin sulfate B"). ), And their low molecular weight compounds (eg, chondroitin sulfate oligosaccharides).
Examples of the chondroitin sulfate oligosaccharide include chondroitin sulfate octasaccharide, chondroitin sulfate hexasaccharide, and chondroitin sulfate tetrasaccharide.

[0019]

Embodiments of the present invention will be described below. <1> Enzyme of the Present Invention The chondroitin sulfate-degrading enzyme of the present invention acts on chondroitin sulfate having a wide range of molecular sizes from high to low molecules and on the chondroitin sulfate chain of chondroitin sulfate proteoglycan to produce unsaturated disaccharides. Is a novel exo-type chondroitin sulfate degrading enzyme having the following physicochemical properties.

Action: (1) It decomposes the N-acetylhexosaminide bond of glycosaminoglycan and, when completely decomposed, produces substantially only unsaturated glycosaminoglycan disaccharide. That is, when glycosaminoglycan serving as a substrate of the enzyme of the present invention is completely decomposed by the action of the enzyme of the present invention, the product substantially consists only of unsaturated glycosaminoglycan disaccharide. This point is clearly different from the purified chondroitinase ABC (which produces unsaturated disaccharides and unsaturated oligosaccharides) disclosed in, for example, JP-A-6-153947.

When the enzyme of the present invention is allowed to act on glycosaminoglycan serving as a substrate of the enzyme of the present invention, and the enzyme reaction is stopped before the glycosaminoglycan is completely degraded, the molecular weight is reduced to a limited extent. A mixture consisting essentially of glycosaminoglycan and unsaturated glycosaminoglycan disaccharide is obtained.

(2) An exo-type enzyme. Specifically, it is an exo-type lyase that sequentially cleaves glycosaminoglycan disaccharide from the non-reducing end of glycosaminoglycan.
This is a point clearly different from purified chondroitinase ABC which is an endo-type enzyme (JP-A-6-153947).

An enzyme (exolyase) that degrades glycosaminoglycan from the non-reducing end in an exo-type, such as the enzyme of the present invention, has not been known so far, and has been found for the first time by the enzyme of the present invention. This is an important feature of the enzyme of the present invention.

Substrate specificity: chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin 6-sulfate hexasaccharide, chondroitin 6
-It acts on both tetrasaccharide sulfate and the chondroitin sulfate chain portion of chondroitin sulfate proteoglycan.
It has also been confirmed that it acts on hyaluronic acid.

It has no effect on keratan sulfate, heparin and heparan sulfate. This point is based on the fact that purified chondroitinase ABC which does not act on chondroitin sulfate tetrasaccharide (JP-A-6-153947) and chondroitinase I (WO
95/29256). It is also distinctly different from chondroitinase II (WO95 / 29256 and WO95 / 29232) which does not act on the chondroitin sulfate chains of chondroitin 6-sulfate and chondroitin sulfate proteoglycans. Thus, the enzyme of the present invention is clearly distinguished from these known enzymes in terms of substrate specificity.
In addition, the polysaccharide which does not indicate the number of sugar residues among the substrates is generally a polysaccharide having ten or more saccharides.

Molecular weight: about 105 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions.

Amino acid composition: Cystine is detected by amino acid analysis of the acid hydrolyzate of the enzyme of the present invention. In the case of amino acid analysis of an acid hydrolyzate, the detected cystine is
It cannot be distinguished from cysteine residues or cystine. That is, the reason that cystine is detected by this amino acid analysis may be that (1) a cysteine residue is present in the enzyme of the present invention, and cystine is generated by oxidation of the cysteine residue during acid hydrolysis of the enzyme of the present invention. ,
(2) The possibility that cystine is present in the enzyme of the present invention, and (3) the possibility that both cysteine residue and cystine are present in the enzyme of the present invention are considered. This indicates that at least cysteine residues and / or cystine are present in the enzyme of the present invention.

Purified chondroitinase ABC (Japanese Unexamined Patent Publication No.
No. 153947) has been confirmed by the present inventor to contain neither cysteine residues nor cystine, and also chondroitinase I and II (WO95 / 292).
No. 56) contains neither cysteine residues nor cystine, and thus the enzyme of the present invention is clearly distinguished from these known enzymes also in terms of structure.

A cysteine residue is a residue whose SH group contributes as a component of an active center or which is involved in maintaining a steric structure by oxidizing to form cystine. Is thought to greatly affect the properties of the enzyme. In addition, cystine plays an important role in maintaining the higher-order structure of the enzyme, and is thought to greatly affect the properties of the enzyme like cysteine residues.

The enzyme of the present invention is preferably a chondroitin sulfate degrading enzyme having the following physicochemical properties in addition to the above physicochemical properties. N-terminal amino acid: The N-terminal amino acid is leucine. Isoelectric point: pH about 8.45 (isoelectric focusing) Optimum temperature: around 40 ° C (substrate: chondroitin 6-
Sulfuric acid, buffer: Tris-HCl buffer, pH: 8.0) Optimal reaction pH: around pH 8 (substrate: chondroitin)
6-sulfuric acid, buffer: Tris-HCl buffer, temperature: 37 ° C)

The enzyme of the present invention is more preferably a chondroitin sulfate degrading enzyme having the following physicochemical properties in addition to the above physicochemical properties. Amino acid composition: Asx 125 Met 25 Thr 75 Ile 50 Ser 58 Leu 106 Glx 125 Tyr 41 Pro 41 Phe 38 Gly 61 Lys 62 Ala 86 His 29 1/2 Cys 2 Arg 35 Val 41 Trp Not measured (composition is 1000 amino acid residues It was shown by the numerical value per.
The numerical values are actual measurement values, and no correction was performed. The method for measuring the amino acid composition will be described in detail in Examples.)

Temperature stability: (1) By incubating at 37 ° C. for 30 minutes,
Approximately 10% inactivated (enzyme concentration: 0.1 mg / ml, buffer: T
ris-HCl buffer, pH: 8.0, substrate: chondroitin
6-tetrasulfate) (2) By incubating at 40 ° C. for 30 minutes,
Approximately 23% inactivated (enzyme concentration: 0.1 mg / ml, buffer: T
ris-HCl buffer, pH: 8.0, substrate: chondroitin
6-tetrasaccharide sulfate) (3) By incubating at 100 ° C. for 1 minute,
Inactivates about 100% (enzyme concentration: 0.1 mg / ml, buffer: Tris-HCl buffer, pH: 8.0, substrate: chondroitin 6-sulfate tetrasaccharide)

PH stability: Enzyme activity of about 70% or more is maintained even after 2 hours of incubation at a pH of about 4.5 to about 8.5 (enzyme concentration: 0.5 mg / ml, buffer : Phosphate buffer, temperature: 37 ° C, substrate: chondroitin 6-sulfate tetrasaccharide)

Inhibition: The enzyme activity is almost completely inhibited by 1 mM Zn ²⁺ . Enzyme activity is inhibited by 50% or more by 1 mM Ni ²⁺ .

Michaelis constant (Km): Approximately 80 μM (substrate: chondroitin 6-sulfate) Approx. 33 μM (substrate: chondroitin 6-sulfate tetrasaccharide) (Km value is expressed in molar concentration based on hexuronic acid residue)

Specific activity: about 40 units / mg (substrate: chondroitin 6-sulfate,
Reaction temperature; 37 ° C, pH: 8.0) About 150 units / mg (substrate: chondroitin 6-sulfate hexasaccharide, reaction temperature: 37 ° C, pH: 8.0) About 200 units / mg or more (substrate: chondroitin 6) −
Tetrasaccharide sulfate, reaction temperature; 37 ° C, pH: 8.0)

As used herein, the term "1 unit" refers to an amount of an enzyme capable of decomposing 1 μmol of a substrate per minute, ie, an amount of an enzyme capable of releasing 1 μmol of unsaturated chondroitin sulfate disaccharide per minute from the substrate. .

Crystal form when crystallized: plate-like crystal (crystallized using polyethylene glycol)

The origin of the enzyme of the present invention is not particularly limited as long as it has the above-mentioned physicochemical properties. The enzyme is derived from natural products such as cells (for example, microbial cells) or produced by genetic engineering techniques. Can be used. When a cell-derived enzyme is used as a natural product-derived enzyme, it is preferably an enzyme derived from a microbial cell belonging to the genus Proteus, more preferably an enzyme derived from Proteus vulgaris. Proteus
More specifically, Bulgaris is exemplified and preferred by Proteus vulgaris NCTC 4636 strain (same as ATCC 6896 strain or IFO 3988 strain). The method for producing the enzyme of the present invention using cells will be described in detail in "<4> Method for producing enzyme of the present invention" described later.

The enzyme of the present invention can be obtained by cloning the gene of the enzyme of the present invention according to a known method, introducing the gene into an appropriate host, and expressing it. For example, using the specific glycosaminoglycan degrading activity of the enzyme of the present invention as an indicator, a DNA encoding the enzyme is isolated from a DNA library of Proteus vulgaris, and this is inserted into a vector by a gene recombination technique. The enzyme of the present invention can be obtained by introducing it into a host cell and expressing it there. In addition, cloning can also be performed by preparing an antibody specific to the enzyme of the present invention and using the same (for a method for preparing an antibody specific to the enzyme of the present invention, refer to “<3> Antibody of the present invention” described later). explain in detail). Alternatively, a DNA having a nucleotide sequence deduced from the N-terminal amino acid sequence of the enzyme of the present invention is determined.
Can be used as a probe for cloning. The expressed enzyme can be recovered using the extraction / purification method described in “<4> Method for producing enzyme of the present invention”.

<2> Crystal of the Present Invention The crystal of the present invention is a crystal of the chondroitin sulfate-degrading enzyme of the present invention, and is characterized in that it is a plate-like crystal. An example of a specific shape of the crystal of the present invention is shown in FIG. The method for producing the crystal of the present invention is not particularly limited, but a purified enzyme solution of the enzyme of the present invention (detailed in “<4> Method for producing enzyme of the present invention” described later) is mixed with a polyether having a structure in which both ends are hydroxyl groups. It is preferable to produce by crystallization by mixing and contacting. Examples of the polyether having a structure in which both terminals are hydroxyl groups include polyethylene glycol and polypropylene glycol, but polyethylene glycol is preferred, and polyethylene glycol having a molecular weight of about 4,000 to 6,000 is more preferred. For example, a polyethylene glycol 4000 solution is added to a purified enzyme solution of the enzyme of the present invention to a degree that causes slight turbidity, and the mixture is allowed to stand at room temperature to about 4 ° C. until crystals are formed. it can.

<3> Antibody of the Present Invention The antibody of the present invention is an antibody that selectively recognizes the enzyme of the present invention. The antibody of the present invention can be prepared using the enzyme of the present invention as an antigen and according to a conventional method, for example, as follows.

The method for producing the enzyme of the present invention used as an antigen will be described in detail in "<4> Method for producing enzyme of the present invention" described later. The polyclonal antibody of the present invention can be obtained, for example, by immunizing an animal to be immunized, such as a rabbit, mouse, rat, guinea pig, goat, or sheep, with the above antigen, and collecting serum from these animals. When immunizing an animal to be immunized, it is desirable to use an auxiliary agent (adjuvant) in combination, since it activates antibody-producing cells. From the obtained antiserum, the immunoglobulin fraction may be purified by a conventional method.

The monoclonal antibody of the present invention can be obtained, for example, as follows. That is, the antigen is a rabbit,
A spleen or popliteal lymph node is removed after intraperitoneal, subcutaneous or footpad administration of immunized animals such as mice, rats, guinea pigs, goats, sheep, etc. A hybridoma is established by cell fusion with myeloma cells, the obtained hybridoma is continuously grown, and a cell line that continuously produces a specific antibody against the antigen is selected from the obtained hybridoma. By culturing the selected strain in a suitable medium, the monoclonal antibody of the present invention can be obtained in the medium. Alternatively, a large amount of the monoclonal antibody of the present invention can be produced by culturing the hybridoma in a living body such as the abdominal cavity of a mouse.

As the cells used for cell fusion, lymph node cells and lymphocytes in peripheral blood can be used in addition to spleen cells. Further, the myeloma cell line is preferably derived from the same cell line as compared with that derived from a heterologous cell type, and a stable antibody-producing hybridoma can be obtained.

The polyclonal and monoclonal antibodies obtained can be purified by salting out with sodium sulfate, ammonium sulfate, etc., selective precipitation fractionation using low-temperature alcohol, polyethylene glycol, etc., DEAE
(Diethylaminoethyl) -derivative, ion-exchange chromatography using an ion exchanger such as CM (carboxymethyl) -derivative, affinity chromatography using protein A, protein G, etc., hydroxyapatite chromatography, and antigen immobilized Examples include immunoadsorption chromatography using a carrier, gel filtration, electrophoresis, and ultracentrifugation.

The antibody of the present invention may be a fragmented one. However, it is necessary that the antigen-binding site (Fab) is preserved in the fragmented antibody from the viewpoint of binding between the antigen and the antibody. Specific examples of the fragmented antibody of the present invention include Fab-containing fragments obtained by treating the antibody of the present invention with a protease that does not degrade the antigen-binding site (eg, plasmin, pepsin, papain, etc.).

Furthermore, once the nucleotide sequence of the gene encoding the antibody of the present invention or the amino acid sequence of the antibody is determined, fragments or chimeric antibodies (for example, chimeric antibodies containing the Fab portion of the antibody of the present invention, etc.) can be genetically engineered. )
Can be produced.

[0049] In the present specification, these fragments and chimeric antibodies containing the Fab portion of the antibody of the present invention are also included in the term "antibody of the present invention". The antibody of the present invention may be labeled by binding to a labeling substance.
Labeling substances that can be used for labeling antibodies include enzymes (peroxidase, alkaline phosphatase, β-
Galactosidase, luciferase, acetylcholinesterase, etc.), isotopes ( ¹²⁵ I, ¹³¹ I, ³ H)
Etc.), fluorescent dyes (luminol, fluorescein isothiocyanate (FITC), umbelliferone, 7-amino-4-methylcoumarin-3-acetic acid, etc.), chemiluminescent substances, haptens, biotin, avidin (eg, streptavidin, etc.) However, it is not particularly limited as long as it can be generally used for labeling a protein. In addition,
Here, the labeling substance is used in a method in which a substance having a specific binding ability (eg, avidin) combined with a detectable label is used in combination without directly detecting the substance itself, such as biotin. Substances are also included. Metal colloids, latexes, and the like are also included in the labeling substance because they can be detected by an optical method such as a turbidity method or a turbidimetric method.

The fact that the antibody of the present invention selectively recognizes the enzyme of the present invention can be confirmed by an ordinary immunological assay using the enzyme of the present invention and a substance other than the enzyme of the present invention as an antigen. Specific examples of the immunoassay include octalony diffusion, immunoblotting (eg, Western blotting), labeled immunoassay (eg, EIA, enzyme-linked immunosorbent assay (ELISA), radio Immunoassay, fluorescence immunoassay, etc.), flow cytometry and the like. These methods are known methods [Irie
Minoru, "Continuing Radio Immunoassay", Kodansha Co., Ltd., 19
Published on May 1, 1979, edited by Eiji Ishikawa et al., "Enzyme Immunoassay", 2nd edition, Medical Shoin, December 15, 1982, Method in Enzymology, Vol. 92 (1983), Immunoc.
chemical Techniques, Part E: Monoclonal Antibodies
and General Immunoassay Methods, published by Academic Press].

<4> Method for Producing the Enzyme of the Present Invention The method for producing the enzyme of the present invention is characterized in that cells having the ability to produce chondroitin sulfate-degrading enzyme are cultured, and the enzyme of the present invention is collected from a culture of the cells.

The cell capable of producing chondroitin sulfate-degrading enzyme is not particularly limited as long as it produces the enzyme of the present invention. Microbial cells belonging to the genus Proteus are preferable, Proteus vulgaris is more preferable, and Proteus vulgaris is more preferable. NCTC 4636 shares (ATCC 6896 shares, IFO 3988
The same is true for strains). In addition, as a cell having the ability to produce chondroitin sulfate-degrading enzyme, a transformed cell obtained by cloning the gene of the enzyme of the present invention according to a known method and introducing the gene into an appropriate host cell can also be used.

The cells having the ability to produce chondroitin sulfate-degrading enzyme are subjected to physical stimulation (irradiation treatment with X-rays, γ-rays, ultraviolet rays, etc.) to such an extent that the cells do not die and the expression of the enzyme of the present invention is not lost. Chemical stimulation (for example, treatment with an alkylating agent such as ethyl methanesulfonate or nitrosoguanidine), transformation, transduction, conjugation, treatment by a commonly used cell mutation treatment method such as genetic manipulation,
Cells having an enhanced ability to produce the enzyme of the present invention may be used.

The cells having the ability to produce chondroitin sulfate-degrading enzyme can be cultured according to a usual cell culture method according to the type and characteristics of the cells. The culture medium, culture conditions, and the like to be used are items that can be appropriately determined by those skilled in the art, and are not particularly limited.

For example, when Proteus vulgaris is used as a cell capable of producing chondroitin sulfate-degrading enzyme, it can be cultured as follows. As a culture method, a shaking or stirring culture method using a liquid medium is preferable.

The medium used for the culture may be any medium containing a medium component that Proteus vulgaris can use as a nutrient source. For example, glucose, mannose, starch, molasses, liquefied starch, glycerin and the like can be used as a carbon source. As a nitrogen source, for example, meat extract, yeast extract, peptone, casein hydrolyzate, gelatin, corn meal, soybean powder, ammonium phosphate, ammonium sulfate, urea and the like can be used. Various inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, calcium carbonate, manganese chloride, magnesium chloride, and sodium sulfate can also be added as needed.

Further, chondroitin sulfate, dermatan sulfate,
By adding hyaluronic acid, chondroitin, etc., alone or in combination of two or more, to the medium, the ability to produce chondroitin sulfate degrading enzyme can be increased.
Such medium components may be appropriately combined or added during culturing.

The culturing temperature is preferably about 37 ° C., and the culturing time is preferably 10 to 30 hours. The culture conditions described above can be appropriately selected according to the characteristics of the cells to be used.

The cells are cultured as described above, and the cells are collected from the resulting culture by a general cell collection method such as a centrifugation method or a filtration method. The enzyme of the present invention can be obtained, for example, from such cells by ordinary methods for extracting and purifying the enzyme.

Specifically, the method for extracting the enzyme of the present invention is as follows:
Processing operations such as homogenization, glass bead milling, sonication, osmotic shock, freeze-thaw extraction, etc., by cell crushing, surfactant extraction, or a combination thereof are mentioned.

For example, when extracting the enzyme of the present invention from Proteus vulgaris, it is preferable to use a surfactant. As the surfactant used for the extraction, a nonionic surfactant is preferable, and for example, polyoxyethylene alkyl ether, polyoxyethylene pt-octylphenyl ether, polysorbate and the like are preferable. Among these, a particularly preferred surfactant is polyoxyethylene lauryl ether.
ther; also called POELE. For example, Nikkol BL-9EX (product name)
And the like). When enzymes are extracted from Proteus vulgaris using these surfactants, not only can the extraction efficiency of the enzymes be increased, but also compared to other extraction methods, the amount of contaminating proteins other than enzymes, nucleic acids, or proteases can be reduced. An enzyme extract with less contamination can be obtained.

More specifically, a buffer containing about 2 to 10% of the above-mentioned surfactant is added to the wet cells (cells) of Proteus vulgaris to form a cell suspension at about 15 to 45 ° C. Preferably, the mixture is heated at about 37 ° C. for about 0.5 to 10 hours, and then the suspension is cooled, and extraction is performed by separating into a cell residue and a supernatant (extract) by a separation means such as centrifugation. It can be carried out.

Specific examples of the method for purifying the enzyme of the present invention from the extract include salting out with ammonium sulfate (ammonium sulfate) and sodium sulfate, centrifugation, dialysis, ultrafiltration, and the like.
Adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, zymography (Anal. Biochem. 225, 333-340 (1995 ))
Processing operations such as a combination of these are exemplified.

Of these, ion exchange chromatography is preferably used, and cation exchange chromatography is more preferably used. As a carrier for cation exchange chromatography, a carrier having a carboxymethyl group as an exchange group (a commercially available product such as C
M Sephadex, CM Sepharose (all trade names, manufactured by Pharmacia), CM Cellulofine (trade names,
Seikagaku Co., Ltd.), CM Toyopearl (trade name, manufactured by Tosoh Corporation), and carriers having a sulfoalkyl group as an exchange group (commercially available products include S Sepharose, S
Examples include P Sephadex, Mono S (both trade names, manufactured by Pharmacia), SP Cellulofine (trade name, sold by Seikagaku Corporation), SP Toyopearl (trade name, manufactured by Tosoh Corporation), and the like. Among them, it is preferable to use a carrier having a carboxymethyl group as an exchange group.

The enzyme of the present invention can be purified by performing ion exchange chromatography using a buffer solution having a pH around neutrality and a buffer solution containing salts with the above-mentioned cation exchange carrier. . The fractions from which the enzyme of the present invention is eluted are collected by such chromatography and desalted to obtain a purified enzyme solution of the enzyme of the present invention. Using the purified enzyme solution, the crystal of the present invention can be produced (see the above “<2> Crystal of the present invention”). Also, by freeze-drying the purified enzyme solution, a freeze-dried product of the purified enzyme of the present invention can be obtained. The most convenient and preferable method for producing the enzyme of the present invention is as follows. Proteus vulgaris wet cells 3
About 10% of polyoxyethylene lauryl ether (P
OELE) and a buffer of about neutral pH (preferably about 20 mM phosphate buffer) containing about 3 to 5 volumes are added, and the mixture is stirred well to suspend the suspension. 0.5-3
Heat for about an hour. At this time, the suspension is stirred occasionally or continuously so that the cells do not precipitate. Thereafter, the suspension is diluted to about 2 to 4 volumes with a buffer having a near neutral pH (preferably about 10 to 50 mM phosphate buffer) and centrifuged at a low temperature to obtain a clean suspension. Collect the supernatant. The supernatant is diluted with water, if necessary, to lower the ionic strength, passed through a column packed with a carrier having a carboxymethyl group, and the enzyme of the present invention is adsorbed on the column carrier. After washing the column with water or a buffer having a pH around neutrality (preferably about 40 mM phosphate buffer), use a buffer containing sodium chloride at a pH near neutrality (preferably about 40 mM phosphate buffer). The enzyme of the present invention is eluted with a linear gradient of the salt concentration. The pH of the buffer during this linear concentration gradient was 6
The range of ~ 7 is preferred. In this chromatography,
First, the conventionally known chondroitinase ABC
(JP-A-6-153947; corresponding to the above-mentioned "purified chondroitinase ABC"). After the elution of the known chondroitinase ABC is completed, the enzyme of the present invention is eluted with a delay. The fractions are collected and desalted using an ultrafiltration device or a dialysis membrane having a molecular weight of about 30 kDa to obtain a purified enzyme solution of the enzyme of the present invention. By freeze-drying with a general freeze dryer,
A freeze-dried product of the enzyme of the present invention can also be obtained.

According to such a method, a known chondroitinase ABC (JP-A-6-15) can be obtained from a microbial cell belonging to the genus Proteus (preferably Proteus vulgaris).
3947) and the enzyme of the present invention. That is, according to the present invention, a microorganism belonging to the genus Proteus is treated with a surfactant (preferably polyoxyethylene lauryl ether) to extract the enzyme, and the extract is subjected to ion exchange chromatography to obtain a known cell culture. A method for separating the enzyme of the present invention from itinase ABC (JP-A-6-153947) is also included.

<5> Method for Producing Unsaturated Disaccharide of the Present Invention The method for producing unsaturated disaccharide of the present invention comprises the steps of decomposing glycosaminoglycan, which can be a substrate of the enzyme of the present invention, with the enzyme of the present invention; Fractionating the unsaturated glycosaminoglycan disaccharide.

The glycosaminoglycan that can serve as a substrate of the enzyme of the present invention is not particularly limited as long as it is a glycosaminoglycan on which the enzyme of the present invention can act. Chondroitin, chondroitin 4-sulfate, chondroitin 6
-Sulfuric acid, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin sulfate proteoglycan (chondroitin sulfate chain portion), hyaluronic acid, and oligosaccharides thereof (for example, chondroitin 6-sulfate octasaccharide, chondroitin 6-sulfate hexasaccharide, chondroitin 6) -Tetrasulfate sulfate).

Since the composition of the unsaturated glycosaminoglycan disaccharide obtained by the method for producing an unsaturated disaccharide of the present invention reflects the disaccharide composition of glycosaminoglycan as a raw material, a desired unsaturated glycosaminoglycan can be obtained. Those skilled in the art can appropriately select glycosaminoglycan as a raw material according to the noglycan disaccharide. By completely decomposing glycosaminoglycan as a raw material with the enzyme of the present invention, substantially only an unsaturated glycosaminoglycan disaccharide derived from each glycosaminoglycan can be produced.

For example, when chondroitin is used as a raw material, Δ
Di-0S (2-acetamido-2-deoxy-3-O- (β-D-gluco-4-e
nopyranosyluronic acid) -D-galactose), and using chondroitin 4-sulfate as a raw material, ΔDi-
4S (2-acetamido-2-deoxy-3-O- (β-D-gluco-4-enopyra
nosyluronic acid) -4-O-sulfo-D-galactose), and using chondroitin 6-sulfate as a raw material, ΔD
i-6S (2-acetamido-2-deoxy-3-O- (β-D-gluco-4-eno
pyranosyluronic acid) -6-O-sulfo-D-galactose).
_{i-diS D (2-acetamido} -2-deoxy-3-O- (2-O-sulfo-β-
D-gluco-4-enopyranosyluronic acid) -6-O-sulfo-D-gal
actin) can be obtained. When chondroitin sulfate E is used as a raw material, ΔDi-diS _E (2-acetamido-2-deoxy-3-O-
(β-D-gluco-4-enopyranosyluronicacid) -4,6-bis-O-su
lfo-D-galactose), and ΔDi-diS _B (2-acetamido-2-deoxy-3-
O- (2-O-sulfo-β-D-gluco-4-enopyranosyluronic acid)
-4-O-sulfo-D-galactose), and when using hyaluronic acid as a raw material, ΔDi-HA (2-acetamido-2-deoxy
-3-O- (β-D-gluco-4-enopyranosyluronic acid) -D-gluc
ose).

Further, a saturated glycosaminoglycan oligosaccharide is prepared from the above-mentioned glycosaminoglycan (for example, chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate or hyaluronic acid, and bovine or sheep-derived hyaluronidase is sufficiently added thereto). By acting, a saturated glycosaminoglycan tetrasaccharide or a saturated glycosaminoglycan hexasaccharide can be prepared), and by reacting this with the enzyme of the present invention, a saturated glycosaminoglycan disaccharide can be prepared. A mixture with unsaturated glycosaminoglycan disaccharide can be obtained. This mixture can be separated into a saturated glycosaminoglycan disaccharide and an unsaturated glycosaminoglycan disaccharide by a fractionation method described below. That is, the present invention comprises a step of decomposing a saturated glycosaminoglycan oligosaccharide (preferably a saturated glycosaminoglycan tetrasaccharide or hexasaccharide) with the enzyme of the present invention, and a step of decomposing a saturated glycosaminoglycan from the decomposition product. A method for producing a saturated and / or unsaturated glycosaminoglycan disaccharide, which comprises at least a step of fractionating the disaccharide and the unsaturated glycosaminoglycan disaccharide.

Further, the enzyme of the present invention is allowed to act on the glycosaminoglycan described above, and the action of the enzyme of the present invention is eliminated before the enzyme of the present invention completely decomposes the glycosaminoglycan, thereby reducing the molecular weight. A mixture consisting essentially of the glycosaminoglycan thus obtained and the unsaturated glycosaminoglycan disaccharide can also be obtained. Examples of the method for removing the action of the enzyme of the present invention include a method for inactivating the enzyme, a method for inhibiting the enzyme activity, and a method for separating the enzyme from the substrate.
Specific methods for inactivating the enzyme of the present invention and inhibiting the enzyme activity are as follows:
It will be easily understood by those skilled in the art by referring to the physicochemical properties of the enzyme of the present invention. The timing of removing the action of the enzyme of the present invention is when glycosaminoglycan is reduced to a desired molecular size. This timing depends on the temperature and pH of the enzyme reaction, the concentration of the enzyme of the present invention, the type of glycosaminoglycan used as the substrate,
It depends on the concentration and molecular size, the desired molecular size of glycosaminoglycan, and the like, but can be appropriately determined by those skilled in the art through preliminary experiments and the like.

A mixture of a low-molecular-weight glycosaminoglycan and an unsaturated glycosaminoglycan disaccharide is subjected to a low-molecular-weight glycosaminoglycan and an unsaturated glycosaminoglycan disaccharide by a fractionation method described later. Can be separated into sugar. That is, the present invention includes a step of decomposing glycosaminoglycan which can be a substrate of the enzyme of the present invention with the enzyme of the present invention, a glycosaminoglycan whose molecular weight has been reduced from the decomposition product, and an unsaturated glycosaminoglycan disaccharide. And a method for producing a low-molecular-weight glycosaminoglycan and / or unsaturated glycosaminoglycan disaccharide, which comprises at least a step of fractionating the following. Since the enzyme of the present invention used in this method has an exo-type decomposition mode, a low-molecular-weight glycosaminoglycan having a desired molecular size can be produced, and fine adjustment of the molecular size is also possible.

In these methods, the step of decomposing with the enzyme of the present invention is not particularly limited as long as it is carried out under conditions that maintain the activity of the enzyme of the present invention. Preferably,
It is preferable to carry out the reaction in a buffer having a buffering action at the pH.

For small-scale production, the enzyme of the present invention may be allowed to act in the presence of a glycosaminoglycan solution.
In the case of mass production, an immobilized enzyme in which the enzyme of the present invention is bound (adsorbed) to an appropriate solid phase (beads or the like) or the enzyme of the present invention is included in a gel, an ultrafiltration membrane, a dialysis membrane, or the like is used. Enzymes can also be made to act continuously using a membrane reactor or the like.

In the step of fractionating the unsaturated glycosaminoglycan disaccharide from the decomposition product generated by decomposing the glycosaminoglycan with the enzyme of the present invention, ordinary steps for separating and purifying sugar chains are carried out. Techniques can be used. Examples of the fractionation method include ultrafiltration, dialysis, adsorption chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration, gel permeation chromatography, and the like, and combinations thereof. However, the present invention is not limited to these.

The unsaturated glycosaminoglycan disaccharide thus fractionated was subjected to HPLC (refer to “<6>
By strictly fractionating and identifying by the same method as in the “Analysis method of the present invention”), high-purity ΔDi-0S, ΔD
i-4S, ΔDi-6S, ΔDi-diS D, ΔDi-
diS _E , ΔDi-diS _B , ΔDi-HA and the like can be obtained.

By the fractionation method as described above, the purified fraction of unsaturated glycosaminoglycan disaccharide (if necessary, saturated glycosaminoglycan disaccharide or low molecular weight glycosaminoglycan) Is obtained. If necessary, this fraction is desalted and freeze-dried to obtain a freeze-dried product.

<6> Analysis Method of the Present Invention The analysis method of the present invention comprises a step of decomposing glycosaminoglycan, which can be a substrate of the enzyme of the present invention, with the enzyme of the present invention;
And analyzing the decomposition product.

The glycosaminoglycan that can be a substrate of the enzyme of the present invention and the step of decomposing the glycosaminoglycan with the enzyme of the present invention are described in the above “<
5> Method for producing unsaturated disaccharide of the present invention ".

The decomposition product generated by decomposing the glycosaminoglycan with the enzyme of the present invention contains an unsaturated glycosaminoglycan disaccharide. Since the composition of the unsaturated glycosaminoglycan disaccharide in the decomposition product reflects the composition of the disaccharide unit of glycosaminoglycan decomposed by the enzyme of the present invention, the decomposition product, particularly the decomposition product By analyzing the unsaturated glycosaminoglycan disaccharide in the sugar chain in consideration of the fact that unsaturation is caused by the enzyme of the present invention at the sugar terminal on the reducing end side of the cleavage site, the analysis of glycosaminoglycan is performed. It can be carried out.

The analysis of unsaturated glycosaminoglycan disaccharide in the decomposition product can be carried out by a method usually used in the field of sugar chain engineering. Examples include disaccharide analysis using high performance liquid chromatography (HPLC), analysis using an antibody against unsaturated glycosaminoglycan disaccharide, and the like. Particularly, disaccharide analysis using HPLC is preferable,
It is not limited to these.

In particular, the disaccharide analysis method using HPLC is a well-known and commonly used method for those skilled in the art, and a mixture of various unsaturated glycosaminoglycan disaccharides having different numbers and positions of sulfate groups is analyzed by HPLC. To elute the unsaturated glycosaminoglycan disaccharide, and compare the elution position with the elution position of the standard unsaturated disaccharide. Elution position of HPLC is usually unsaturated glycosaminoglycan disaccharide (delta ⁴ of said two in the sugar - hexuronic acid (delta ⁴ -
hexuronate) residue) (e.g., wavelength 23
2 nm). The content of the disaccharide unit derived from each unsaturated glycosaminoglycan disaccharide in the glycosaminoglycan is determined by the integrated value (area) of the elution pattern and the elution pattern of the standard unsaturated disaccharide with a known concentration. By comparing with the integral value (area) of ΔDi-UA2S (2-ace
tamido-2-deoxy-3-O- (2-O-sulfo-β-D-gluco-4-enopyra
nosyluronic acid) -D-galactose), distinction between [Delta] Di-4S, and [Delta] Di-6S, and [Delta] Di-diS _B and [Delta] Di
Strict distinction -dis _E can be carried out by digestion by specific sulfatase (e.g. chondro-6-sulfatase, etc.). For example unsaturated glycosaminoglycan disaccharide result of processing by chondro-6-sulfatase, and amount of elution position of HPLC is shifted to the position of [Delta] Di-4S is identified as a [Delta] Di-diS _E. In addition, an HPLC column capable of separating unsaturated glycosaminoglycan disaccharides whose elution positions are close to each other (eg, Zorbax SAX column; Rockland TeX)
These compounds can be strictly distinguished from each other by using unsaturated glycosaminoglycan disaccharides.

Further, the enzyme of the present invention used in the analysis method of the present invention is an exo-type enzyme which acts to sequentially excise glycosaminoglycan disaccharide from the non-reducing terminal side of the glycosaminoglycan. The sequence of the disaccharide unit of glycosaminoglycan can also be determined by the analysis method of the present invention.

Further, according to the analysis method of the present invention, the side chain of glycosaminoglycan can be analyzed.

<7> Composition of the Present Invention The composition of the present invention comprises the enzyme of the present invention and a carrier which does not affect the activity of the enzyme of the present invention. The enzyme of the present invention may be in the form of the crystal of the present invention.

The enzyme of the present invention and the crystal of the present invention used in the composition of the present invention are described in detail in “<1> Enzyme of the present invention”, “<2> Crystal of the present invention” and “<4> Method for producing enzyme of the present invention”. I mentioned.

The carrier that can be used in the composition of the present invention is not particularly limited as long as it does not affect the activity of the enzyme of the present invention. Substances that affect the activity of the enzyme of the present invention include, for example, substances that generate Zn ²⁺ and Ni ²⁺ . For example, when about 1 mM of Zn ²⁺ or Ni ²⁺ is present in the solution of the composition of the present invention, the activity of the enzyme of the present invention is inhibited by at least 50% or more. It is preferable not to use it as a carrier. In addition, it is also preferable that a substance (for example, an organic solvent or the like) which may generally deactivate an enzyme is not used as a carrier in the composition of the present invention.

Substances that can be used as a carrier in the composition of the present invention include, for example, albumin (for example, bovine serum albumin), glycerin, pH regulator (for example, buffer having a pH around neutrality), and pharmaceuticals. Among the carriers (for details, see "<8> Therapeutic agent of the present invention" described later), carriers that do not affect the activity of the enzyme of the present invention and the like, and combinations thereof are exemplified. The composition of the present invention can be used as a reagent composition, a pharmaceutical composition, and the like. When used as a pharmaceutical composition, a carrier that does not affect the activity of the enzyme of the present invention and a target animal ( Adverse effects (eg, antigenicity or toxicity) on humans). The carrier used in the composition of the present invention can be appropriately determined by those skilled in the art in consideration of the above-mentioned conditions, the provision form, purpose of use, and method of use of the composition of the present invention. Also in the composition of the present invention, the enzyme of the present invention,
The mixing ratio with the carrier that does not affect the enzyme activity of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art.

The composition of the present invention may be in any of a solution state, a frozen state, and a dried state (for example, a lyophilized state).

<8> Therapeutic Agent of the Present Invention The therapeutic agent of the present invention is a therapeutic agent for a herniated disc comprising the composition of the present invention. The enzyme of the present invention and the crystal of the present invention used in the therapeutic agent of the present invention have been described in detail in the above “<1> Enzyme of the present invention”, “<2> Crystal of the present invention” and “<4> Method for producing enzyme of the present invention”. The purified enzyme solution of the enzyme of the present invention and the crystal of the present invention are purified to high purity and do not substantially contain proteins other than the enzyme of the present invention. However, in consideration of administration to animals such as humans as pharmaceuticals, it is preferable to further remove substances that are not preferred as pharmaceuticals from the purified enzyme solution of the enzyme of the present invention or the fraction containing the crystals of the present invention.

For example, to remove microorganisms from the fraction containing the enzyme of the present invention, a purified enzyme solution of the enzyme of the present invention is passed through a hydrophilic filtration membrane having a pore size of 0.22 μm for filtration and sterilization, and the filtrate is collected under aseptic conditions. Can be done by

The removal of the pyrogen (endotoxin) from the fraction containing the enzyme of the present invention can be carried out, for example, by bringing an anion exchange carrier such as Q Sepharose (manufactured by Pharmacia) into contact with a purified enzyme solution of the enzyme of the present invention. It can be carried out. Endotoxin is adsorbed on the carrier, and the enzyme of the present invention is not adsorbed on the carrier, so that endotoxin can be easily removed. Similarly, a purified enzyme solution of the enzyme of the present invention can be removed by contacting activated carbon with adsorbed endotoxin.

The carrier which can be used in the therapeutic agent of the present invention does not affect the activity of the enzyme of the present invention as in the above <7> Composition of the present invention, and at the same time, the animal to be administered (human, etc.) Should not adversely affect (eg, antigenicity, toxicity, etc.). Specific examples include carriers commonly used in the pharmaceutical field, for example, conventional excipients, binders, lubricants, coloring agents, disintegrants, buffers, isotonic agents, preservatives, soothing agents and the like. A carrier that does not affect the activity of the enzyme of the present invention can be appropriately selected and used. It is preferable that these carriers have such a purity that they can be used as a medicament, and substantially do not contain a substance that is not allowed to be mixed as a medicament.

More specifically, carriers used in the therapeutic agent of the present invention include, for example, dextrans, saccharose,
Lactose, maltose, xylose, trehalose,
Mannitol, xylitol, sorbitol, inositol, serum albumin, gelatin, creatinine, polyethylene glycol, nonionic surfactant (polyoxyethylene sorbitan fatty acid ester (polysorbate), polyoxyethylene hydrogenated castor oil, sucrose fatty acid ester or polyoxyethylene Examples of the polyoxyethylene sorbitan fatty acid ester include polylauthylene monolaurate, monopalmitate, monooleate, monostearate, and trioleate of polyoxyethylene sorbitan (degree of polymerization: about 20). Commercially available products include polysorbate 80 (Tween 80) (polyoxyethylene sorbitan monooleate (20 EO)), polysorbate
60 (polyoxyethylene sorbitan monostearate (2
0 EO)), polysorbate 40 (polyoxyethylene sorbitan monopalmitate (20 EO)), Tween 21, 81,
65, 85, etc. can be exemplified. As polyoxyethylene hydrogenated castor oil, commercially available HCO-10 and HCO-
50 and HCO-60 (both are trade names). Examples of the sucrose fatty acid ester include commercially available DK ester F-160 (trade name) and the like. As polyoxyethylene polyoxypropylene glycol, commercially available Pluronic F-68 (trade name)
Etc. can be exemplified.

The buffer is not particularly limited as long as it is physiologically acceptable. Hydrochloric acid, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, phosphoric acid, potassium dihydrogen phosphate, hydrogen phosphate Dipotassium,
At least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, aminoacetic acid, sodium benzoate, citric acid, sodium citrate, acetic acid, sodium acetate, tartaric acid, sodium tartrate, lactic acid, sodium lactate, ethanolamine, arginine or ethylenediamine And the like.

In addition to these, the composition may contain components necessary for isotonicity (salts such as sodium chloride; saccharides and the like), preservatives and soothing agents, and the like. In the therapeutic agent of the present invention, these carriers can be appropriately used in combination.

The mixing ratio of the enzyme of the present invention and the carrier in the therapeutic agent of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art. The therapeutic agent of the present invention can be used as a therapeutic agent for a herniated disc containing the enzyme of the present invention as an active ingredient, and can be injected into the disc space of a patient with herniosis, and can be used for intervertebral disc lysis therapy for dissolving and treating a hernia. . Therefore, the therapeutic agent of the present invention is mainly provided as an injectable preparation.

The therapeutic agent of the present invention may be in any of a solution state, a frozen state, and a dry state (eg, a lyophilized state). Depending on the form of the therapeutic agent of the present invention, ampules, vials, It can be filled and sealed in an appropriate container such as a syringe for injection, distributed or stored as it is, and provided for administration as an injection.

The dose to a patient with herniation should be set individually according to the patient's condition, age and the like, and is not particularly limited. Usually, the amount of the enzyme of the present invention is 5 to 1 times.
Inject about 00 units.

[0101]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, which are merely examples of the present invention.
It is not limited to this. 1. Materials The following glycosaminoglycans were manufactured by Seikagaku Corporation; hyaluronic acid (derived from human umbilical cord), chondroitin (produced by desulfating chondroitin 4-sulfate), chondroitin 4-sulfate (whale cartilage) Origin: 75%
4-sulfuric acid, 20% 6-sulfuric acid, chondroitin 6-sulfuric acid (derived from shark cartilage; molecular weight 20,000-80,000, 15% 4-sulfuric acid, 75% 6
-Sulfuric acid, 10% 2,6-disulfate, chondroitin sulfate D (from shark cartilage; 20% 2,6-disulfate, 25% 4-sulfate, 45% 6-sulfate), chondroitin sulfate E (from squid cartilage) ; 60% 4,6-
Disulfuric acid, 25% 4-sulfuric acid, 10% 6-sulfuric acid), dermatan sulfate
(From pig skin; 90% 4-sulfuric acid, 2% 6-sulfuric acid, 7% 2,4-disulfuric acid), keratan sulfate (from bovine cornea), heparan sulfate (from bovine kidney). Chondroitin sulfate tetrasaccharide and hexasaccharide are R
According to the method of Oden et al. (Methods Enzymol. 28, 638-676 (1972)), chondroitin 6-sulfate was digested with hyaluronidase from bovine testes, and then cellulofine (Cellulofin).
e) It was prepared by performing chromatography using GCL-90sf gel (available from Seikagaku Corporation). Chondroitin sulfate proteoglycans were prepared according to the method of Oegema et al. (J. Bi.
ol. Chem. 250, 6156-6159 (1975)) from bovine nasal cartilage. Heparan sulfate (derived from pig small intestine) and bovine serum albumin were obtained from Sigma. CM-Sepharose FF was obtained from Pharmacia Biotech Inc.

2. Enzyme activity measurement method Enzyme activity was basically determined by the method of Yamagata et al. (J. Biol. Che.
m. 243, 1523-1535 (1968)). Enzyme reaction solution is enzyme, 150μg
Substrate, 10 μmol sodium acetate, 5 μg casein and 250 μl of 40 mM Tris-HCl, pH 8.0. As a control enzyme reaction solution, a heat-inactivated enzyme was used in place of the enzyme in the enzyme reaction solution. After incubation at 37 ° C. for 20 minutes, the enzyme reaction was stopped by heating for 1 minute in a boiling water bath. After that, add 10 minutes
After dilution with twice the volume of 50 mM HCl, the UV absorption at ₂₃₂ nm (A ₂₃₂ ) of the control enzyme reaction solution was measured. The amount of increase in _A232 due to the enzymatic reaction was measured, the molecular extinction coefficient was calculated as 5,500, and 1 μmol of unsaturated glycosaminoglycan disaccharide (Δ ⁴ -hexuronic acid (Δ ⁴ − hexuro
nate) residue) was determined as “1 unit”.

3. Protein concentration measurement method The protein concentration was determined by the Lowry method (J. Biol. Chem. 193, 265-275 (195) using bovine serum albumin as a standard.
1)).

[0104]

Example 1 Production of the enzyme of the present invention and the crystal of the present invention (Production Example 1) Proteus vulgari
s) The NCTC 4636 strain was prepared by a known method (J. Biol. Chem. 243, 15
23-1535 (1968)), and cultured at 30 ° C. using a medium containing peptone, a meat extract, a yeast extract, NaCl, and an inducer (chondroitin 6-sulfate; concentration 5 g / L).

The cells at the end of logarithmic growth are centrifuged.
(16,000 xg for 30 minutes at 4 ° C). Approximately 150 g (wet weight) of cells were obtained from the 20 L of culture. This cell 10
0g containing 5% (w / v) POELE (Nikkol BL-9EX (brand name)) 2
Suspend in 400 mL of 0 mM phosphate buffer, pH 7.0, and
The mixture was stirred gently for a time to extract the enzyme. The suspension is diluted with an equal volume of 20 mM phosphate buffer, pH 7.0, and
It was then centrifuged at 16,000 xg for 45 minutes. To the supernatant (enzyme extract), cold purified water was added to adjust the POELE to a final concentration of 1.5% (w / v), and this solution was added to a 10 mM phosphate buffer, pH
CM-Sepharose FF column equilibrated in 7.0 (3 x 15c
m), and the enzyme was adsorbed. This column is 10
Washing was performed in the order of 0 mL of a 0.5% (w / v) aqueous POELE solution, 100 mL of a 40 mM phosphate buffer, and pH 6.2. The column was eluted with a linear gradient (0-0.2 M) of NaCl in 40 mM phosphate buffer, pH 6.2. The total eluted amount by the linear concentration gradient was 320 mL. The flow rate was 1.6 mL / min. The eluate was fractionated by 5 mL, and the enzyme activity (using chondroitin 6-sulfate and chondroitin sulfate tetrasaccharide as a substrate) and the protein concentration of each fraction were measured. FIG. 1 shows the elution pattern of the CM-Sepharose FF column.

The fraction indicated by "I" in FIG.
153947 (identified by the present inventors as being the same as the known enzyme) was eluted first, and then the fraction indicated by "II" in FIG. 1 (the enzyme of the present invention)
Was eluted. Hereinafter, the fraction indicated by “I” in FIG. 1 (known enzyme) may be referred to as “enzyme I”, and the fraction indicated by “II” in FIG. 1 (the enzyme of the present invention) may be referred to as “enzyme II”.

For each of enzyme I and enzyme II,
Fractions containing the major part of the elution peak were pooled, dialyzed against cold purified water and desalted. 30% (w / v) in the solution after desalting
10 mM phosphate buffer, pH 7.0, containing polyethylene glycol 4000, was added until slight turbidity occurred.
It was left in the cold room until crystals formed. FIG. 2 shows a microscopic image of the generated crystal. A in FIG. 2 shows a crystal of enzyme I, and B shows a crystal of enzyme II.

FIG. 2 shows that the enzyme I is a needle-like or columnar crystal and the enzyme II is a plate-like crystal. From this, it was found that enzyme II can be clearly distinguished from enzyme I in crystallography.

Table 1 shows the amounts of protein and enzyme activities in the enzyme extract, CM-Sepharose elution fraction and crystals (repeated crystallization twice) (using chondroitin 6-sulfate and chondroitin sulfate tetrasaccharide as substrates). Is shown. In Table 1, ND indicates that it was below the detection limit.

[0110]

[Table 1] 質量 Total protein mass Controitin 6-sulfate Controitin sulfate 4 Sugar Total activity Specific activity Total activity Specific activity ──────────────────────────────────── (mg) (Unit ) (Unit / mg) (Unit) (Unit / mg) Enzyme extract 8,200 43,900 5.4 8,850 1.1 Enzyme I CM-Sepharose fraction 115 37,500 326 ND ND crystal 76 25,700 338 ND ND Enzyme II (Enzyme of the present invention) CM-Sepharose Fraction 40 1,240 31 5,880 147 Crystal 32 1,010 32 4,760 149 ────────────────────────────────────

According to Table 1, the enzyme I was chondroitin 6-
It acts on sulfuric acid but not on chondroitin sulfate tetrasaccharide, indicating that enzyme II acts on both chondroitin 6-sulfate and chondroitin sulfate tetrasaccharide.
This indicated that enzyme II was clearly distinguishable from enzyme I in terms of substrate specificity.

In addition, no significant decrease in the enzyme activity was observed even when crystallization was repeated. This suggested that enzyme I and enzyme II were highly purified.

(Production Example 2) Proteus vulgaris NCT
The C4636 strain was cultured in the same manner as in Production Example 1 above, and 7.5 kg
(Wet weight) was obtained. 6.6% (w / v) POELE (Nikkol
BL-9EX (trade name)), 20 mM phosphate buffer, pH 7.0, about 2
Suspend in 2.5L, gently stir at 35 ± 3 ° C for 2 hours,
The enzyme was extracted. This suspension was diluted with about 30 L of 20 mM phosphate buffer, pH 7.0, cooled to 5 to 15 ° C., and centrifuged with a Sharpless centrifuge. About 30 L of cold purified water was added to the supernatant (enzyme extract), and this solution was added to 10 mM phosphate buffer,
The mixture was applied to a CM-Sepharose column (5 L) equilibrated at pH 7.0 to adsorb the enzyme. The column was washed with 3 L of purified water in the order of 10 L of 40 mM phosphate buffer, pH 6.2. The column was eluted with a linear gradient (0-0.25 M) of NaCl in 40 mM phosphate buffer, pH 6.2. The total amount eluted by the linear concentration gradient was 20 L.

In this chromatography, first, enzyme I
Was eluted between about 0.05M and about 0.09M salt concentration. Next, enzyme II was eluted between about 0.11 M and about 0.13 M salt concentration. The front and back of the elution fraction of enzyme II were cut, and the peak portions were collected to obtain about 0.8 L of the elution fraction of enzyme II. 2 of this fraction
The absorbance at 80 nm was about 2.02. This fraction is dialyzed against purified water and freeze-dried to give a white cotton-like enzyme.
About 1.2 g of a freeze-dried product of II was obtained. Enzyme II thus obtained
Was measured by an enzyme activity measuring method to be about 202 units / mg.

The results of Production Example 2 suggested that the enzyme production method of the present invention is applicable to mass production on an industrial scale.

[0116]

Example 2 Physicochemical Properties of Enzyme II (Enzyme of the Present Invention) The physicochemical properties of the enzymes I and II produced in "Production Example 1" in "Example 1" were examined. (A) Operation: This will be described in a fourth embodiment described later. (B) Substrate specificity: chondroitin, chondroitin 4-
Sulfuric acid, chondroitin 6-sulfate, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin sulfate hexasaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate proteoglycan, hyaluronic acid, keratan sulfate, heparin and heparan sulfate as enzymes I
And the enzyme activity of enzyme II was measured. Table 2 shows the results. In Table 2, NR indicates that the enzyme did not act.

[0117]

TABLE 2 ──────────────────────────────────── specific activity (the A ₂₃₂ variation / min / mg protein) Substrate Enzyme I Enzyme II (enzyme of the present invention) ─────────────────────────────────── ─ Chondroitin 105 29 Chondroitin 4-sulfate 400 25 Chondroitin 6-sulfate 330 30 Chondroitin sulfate D 230 28 Chondroitin sulfate E 75 27 Dermatan sulfate 180 23 Chondroitin sulfate proteoglycan 230 20 Chondroitin sulfate hexasaccharide 75 110 Chondroitin sulfate tetrasaccharide NR 150 Hyaluronic acid 3 5 Keratan sulfate / heparin / heparan sulfate NR NR ────────────────────────────────────

In the enzyme activity measurement method, when 40 mM acetate buffer, pH 6.0 was used as the buffer, the specific activity for hyaluronic acid increased to 18 for enzyme I and 9 for enzyme II. Table 2 shows that enzyme I did not act on chondroitin sulfate tetrasaccharide, and enzyme II also acted on chondroitin sulfate tetrasaccharide. This confirms that enzyme II can be clearly distinguished from enzyme I in terms of substrate specificity. Also from this result, enzyme II is a known chondroitinase I that does not act on chondroitin sulfate tetrasaccharide (WO95 / 29256).
) And known chondroitinase II (WO95 / 29256 and WO95 / 29232) which does not act on the chondroitin sulfate chain portion of chondroitin 6-sulfate and chondroitin sulfate proteoglycans. It has been shown.

As described above, it was shown that the enzyme II was clearly different from these known enzymes in terms of substrate specificity. Enzyme II was also shown to have a broad substrate specificity for chondroitin sulfate of various molecular sizes.

(C) Molecular weight: To estimate the molecular weight of enzyme I and enzyme II, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed.

The SDS-PAGE was carried out on a 10% (w / v) polyacrylamide gel under reducing conditions. Before electrophoresis,
Samples (Enzyme I and Enzyme II) are transferred to sample buffer (1.2%
(w / v) sodium dodecyl sulfate (SDS), 1.0% (w / v) dithiothreitol, 50 mM Tris-HCl, pH 6.8 and 10% (w / v) glycerol) and diluted at 100 ° C. Treated for 2 minutes.
As molecular weight markers for SDS-PAGE also myosin (molecular weight 212,000), alpha ₂ - macroglobulin (molecular weight 170,000),
β-galactosidase (MW 116,000), transferrin (MW 76,000), and glutamate dehydrogenase (MW 53,000) were used. After electrophoresis, the proteins in the gel were detected by Coomassie brilliant blue staining.

As a result, the enzyme I was located near 100 kDa,
In each case, a single band was detected at around 105 kDa. This suggested that both enzymes were uniformly purified as proteins.

Further, both enzyme I and enzyme II were analyzed by HPLC.
Showed a single peak on gel filtration, further suggesting that all enzymes were uniformly purified as proteins.

(D) Amino acid composition: The amino acid composition was analyzed using a Hitachi L-8500 amino acid analyzer. The samples (Enzyme I and Enzyme II) were hydrolyzed with 6M HCl gas at 110 ° C. for 24 hours under a reduced pressure of nitrogen for analysis. An analysis chart is shown in FIG. 3 and an amino acid composition analysis result is shown in Table 3. In Table 3, the amino acid composition is shown in numerical values per 1000 amino acid residues. The numerical values are actual measurement values, and no correction was performed.

[0125]

[Table 3] ア ミ ノ 酸 Amino acid Enzyme I Enzyme II (enzyme of the present invention) ──────────────────────────────────── Asx 135 125 Thr 64 75 Ser 74 58 Glx 109 125 Pro 44 41 Gly 66 61 Ala 69 86 1 / 2Cys 0 2 Val 45 41 Met 23 25 Ile 57 50 Leu 98 106 Tyr 42 41 Phe 42 38 Lys 71 62 His 24 29 Arg 37 35 Trp Not measured Not measured ──── ────────────────────────────────

From Table 3, it can be seen that enzyme I contains neither cysteine residues nor its oxidized form of cystine and enzyme II
Has been shown to contain cysteine residues and / or cystine. From this, the structure of enzyme I and enzyme II is
(Amino acid residues). In addition, known chondroitinase I and II (WO95
/ 29256) shows that the enzyme II is clearly distinguished from these known enzymes in terms of structure because neither contains a cysteine residue or cystine.

Further, the N-terminal amino acid residues of enzyme I and enzyme II were replaced with Tosoh phenylthiohydantoin-derivative a
Determined by the Edman degradation method using a nalytical HPLC system. As a result, the N-terminal amino acid residue of enzyme I was an alanine residue, and the N-terminal amino acid residue of enzyme II was a leucine residue.

(E) Isoelectric point: The isoelectric point (pI) of enzyme II was determined by isoelectric focusing (IEF) pH3-9. As a result, pI = 8.
A single band was detected around 45. This suggested that enzyme II was uniformly purified as a protein.

(F) Optimum temperature: chondroitin 6-sulfate as a substrate and Tri as a buffer in the enzyme activity measurement method
As a result of measuring the enzyme activity at various temperatures using an s-HCl buffer, pH 8.0, the optimum temperature of enzyme II was around 40 ° C.

(G) Optimal reaction pH: Enzyme activity was measured at various pHs under conditions of 37 ° C. using chondroitin 6-sulfate as a substrate and Tris-HCl buffer as a buffer in the enzyme activity measurement method. As a result, the optimal reaction pH was around pH 8 for both enzyme I and enzyme II.

For enzyme II, chondroitin sulfate tetrasaccharide was further used as a substrate, and the enzyme activity at various pH values was examined in the same manner. These results are shown in FIG.

(H) Temperature stability: Enzyme II at a concentration of 0.1 mg / mL
Was incubated in a Tris-HCl buffer, pH 8.0, at various temperatures for a certain period of time, and then the enzyme activity was measured using chondroitin sulfate tetrasaccharide as a substrate in the enzyme activity measurement method. The results are shown below. (1) By incubating at 37 ° C for 30 minutes, about 10
% Deactivated. (2) By incubating at 40 ° C for 30 minutes, about 23
% Deactivated. (3) By incubating at 100 ° C for 1 minute,
0% deactivated.

(I) pH stability: 0.5 mg / mL enzyme II
Was incubated at 37 ° C. for a certain period of time in phosphate buffers at various pHs, and then the enzyme activity was measured using chondroitin sulfate tetrasaccharide as a substrate in the enzyme activity measurement method. The results are shown below.

Even when incubated at a pH of about 4.5 to 8.5 for 2 hours, about 70% or more of the activity was maintained.

(J) Inhibition: 1 mM metal salt (ZnCl ₂ and NiCl ₂ )
As a result of an experiment using a chelating agent (ethylenediaminetetraacetic acid), it was shown that the enzyme activity of enzyme II was almost completely inhibited by 1 mM Zn ²⁺ . Enzyme II is 1
It was shown that the enzyme activity was inhibited by 50% or more by mM Ni ²⁺ .

(K) Michaelis constant (Km): The initial reaction rate of enzyme II was determined using various concentrations of the substrate, and the
A k-plot was created and the Michaelis constant (Km) was determined. The results are shown below. The Km value was expressed as a molar concentration based on the hexuronic acid residue. Approx. 80 μM (substrate: chondroitin 6-sulfate) Approx. 33 μM (substrate: chondroitin 6-sulfate tetrasaccharide)

(L) Specific activity: Specific activity of enzyme II was determined using various substrates. The results are shown below. Approx. 40 units / mg (substrate: chondroitin 6-sulfate, reaction temperature: 37 ° C., pH: 8.0) Approx. 150 units / mg (substrate: chondroitin 6-sulfate hexasaccharide, reaction temperature: 37 ° C., pH: 8.0) Approx. 200 Unit / mg or more (substrate: chondroitin 6-sulfate tetrasaccharide, reaction temperature: 37 ° C, pH: 8.0)

(M) Crystal form when crystallized: As can be seen from “Preparation Example 1” in “Example 1” and FIG. 2B, the enzyme II was crystallized using polyethylene glycol. To give plate-like crystals.

[0139]

Example 3 Preparation of Antibody Against Enzyme II (Enzyme of the Present Invention)
(1) Preparation of Antibodies to Enzyme I and Enzyme II 1 mg of Enzyme I and Enzyme II produced in “Production Example 1” of “Example 1” described above were each completely Freund's adjuvant ( complete Freund's adjuvant; manufactured by Yatron) and emulsified. This was immunized by injecting a white rabbit (18-week-old, male) three times subcutaneously at weekly intervals. After breeding for 1 week, whole blood was collected, and serum was separated and tested against enzymes I and II. Serum was obtained. Ammonium sulfate was added to these antisera, and the precipitate formed in the range of 20 to 50% saturation was collected, and 50 mM sodium phosphate buffer, p
Dissolved in H7.2. This was dialyzed against 2 L of 50 mM sodium phosphate buffer (pH 7.2) for 20 hours while exchanging the buffer three times to give an antibody against enzyme I (anti-enzyme I antibody) and enzyme II (enzyme of the present invention). An antibody (anti-enzyme II antibody) was obtained.

(2) Immunochemical Analysis of Enzyme II Using the anti-enzyme I and anti-enzyme II antibodies prepared in (1) above and enzymes I and II, the Ouchterlony diffusion method (A
rk. Kemi Mineral. Geol. 26, 1-9 (1948)). As a result, the enzyme II and the anti-enzyme II antibody showed a clear sedimentation line as a result of the antigen-antibody reaction. However, no precipitation line was observed between enzyme II and anti-enzyme I antibody. On the other hand, a clear sedimentation line was observed for the enzyme I and the anti-enzyme I antibody, but no reaction was observed between the enzyme I and the anti-enzyme II antibody, and no sedimentation line was observed. From these results, it was confirmed that the enzyme I and the enzyme II are enzymes having completely different antigenicities in immunochemistry. In addition, it was confirmed that the anti-enzyme I antibody selectively recognizes enzyme I and the anti-enzyme II antibody is an antibody that selectively recognizes enzyme II.

[0141]

Example 4 Analysis of Physicochemical Properties (Action) of Enzyme II (Enzyme of the Present Invention) and Degradation Products of Glycosaminoglycan In Example 4, the enzymatic reaction was carried out as follows. 50mM
1m of 50mM Tris-HCl buffer containing sodium acetate, pH 8.0
Prepare the reaction solution to contain 10 mg of substrate per L,
After equilibrating this in a 37 ° C water bath for 10 minutes,
0.015 units of enzyme were added to 1 mg of substrate and incubated at 37 ° C. After a certain period of time, a part of the enzyme reaction solution was taken out and heated for 1 minute in a boiling water bath, and then the following items were analyzed as necessary.

(A) Equivalent amount of 0.4 M NaC
Then, the viscosity was measured at 30 ° C. using an Ubbelohde type automatic viscometer VMC-052 (manufactured by Rigosha). This change in viscosity is indicative of a change in the average length of glycosaminoglycan chains.

(B) A 50-fold amount of 50 mM HC
and diluted for _A232 measurement. This change in _A232
It is an indicator of the change in the number of parts degraded by the enzyme. (c) The enzyme reaction solution after heating was diluted with 40 times the volume of 0.2 M NaCl, and then subjected to gel permeation high performance liquid chromatography (HPLC).
Applied. Columns are TSKgel G3000PW _XL and TSKg
A connection column in which el G2500PW _XL (all manufactured by Tosoh) were connected in this order was used. HPLC at 35 ° C, 0.2
M NaCl was used at a flow rate of 0.5 ml / min. Elute the solution with Tosoh UV (232nm) monitor UV8010 and Tosoh differential refractometer
By monitoring in this order using RI8010,
A ₂₃₂ (unsaturated glycosaminoglycan oligosaccharide)
And the size distribution of glycosaminoglycans was examined.

(1) Analysis of Degradation Mode of Enzyme II Chondroitin 6-sulfate or dermatan sulfate was used as a substrate.
(a) and (b) were analyzed. The viscosity of the above (a),
When the enzyme reaction time is 0, the viscosity of each substrate (chondroitin 6-sulfate is 0.85, dermatan sulfate is 0.22) is 10
It was calculated as 0%. FIG. 5 shows the results. In addition, a and c in FIG. 5 are the results for the enzyme I, and b and d are the results for the enzyme II. Further, a and b in FIG. 5 show the results when chondroitin 6-sulfate was used as the substrate, and c and d show the results when dermatan sulfate was used as the substrate.

From a in FIG. 5, _A232 (ie, enzyme I
It can be seen that the viscosity (that is, the average length of glycosaminoglycan) sharply decreases as compared with the increase in the number of parts decomposed by the above. On the other hand, from b in FIG. 5,
Reduction in viscosity is slow, this decrease in viscosity can be read that is substantially proportional to the increase in A _232. Also, c and d in FIG. 5 can be read in the same manner as a and b in FIG. 5, respectively. Similar results were obtained when chondroitin 4-sulfate was used as the substrate. from these things,
Enzyme I was an endo-type enzyme and Enzyme II was an exo-type enzyme. From this, enzyme II is replaced with enzyme I
Was also shown to be a completely different enzyme in terms of action.

When the enzyme II is allowed to act on chondroitin sulfate proteoglycan (all of the reducing ends of the chondroitin sulfate chain are bound to the core protein), the enzyme acts on the chondroitin sulfate chain at almost the same initial rate as free chondroitin sulfate. As a result, an unsaturated glycosaminoglycan disaccharide is produced (see Table 2). Therefore, the enzyme II is an exo-type lyase that cleaves glycosaminoglycan disaccharide sequentially from the non-reducing end of glycosaminoglycan. It was shown that there is.

(2) Degradation product of glycosaminoglycan by enzyme II Chondroitin 6-sulfate, dermatan sulfate or chondroitin sulfate hexasaccharide was used as a substrate, and the above-mentioned (c) was analyzed for enzyme I and enzyme II, respectively. .

As for the substrate using chondroitin 6-sulfate or dermatan sulfate as the substrate, the enzyme reaction time is 0 minutes, 20 minutes, 60 minutes and 5 hours (the time is sufficient for completely decomposing the substrate). Was analyzed. In the case of using chondroitin sulfate hexasaccharide as a substrate, the enzyme reaction time was analyzed for 0 minutes and 5 hours (a time sufficient for completely decomposing the substrate).

FIG. 6 shows the results when chondroitin 6-sulfate was used as the substrate, FIG. 7 shows the results when dermatan sulfate was used, and FIG. 8 shows the results when chondroitin sulfate hexasaccharide was used.

In FIGS. 6 and 7, a to d and a 'to d' are results for enzyme I, and eh and e 'to h' are results for enzyme II. Further, in FIG. 6 and FIG. 7, to h is the relative refractive index (size of the glycosaminoglycans distribution), A'～h 'the result of examining the A ₂₃₂ (unsaturated glycosaminoglycan oligosaccharides) is there. 6 and 7, aa 'and e-e', b-b 'and f-f',
c-c 'and g-g', d-d 'and h-h' each have an enzyme reaction time of 0 minutes,
The results are for 20 minutes, 60 minutes, and 5 hours. In the figure, the dotted line on the left (retention time 30.3 minutes) indicates the elution position of the tetrasaccharide, and the dotted line on the right (retention time 32.4 minutes) indicates the elution position of the disaccharide.

In FIG. 8, ab and a′-b ′ are enzymes I,
cd and c'-d 'are results for enzyme II. Further, in FIG. 8, to d relative refractive index (size distribution of the glycosaminoglycan), a'-d 'is the result of examining the A ₂₃₂ (unsaturated glycosaminoglycan oligosaccharides). In FIG. 8, a-
a 'and c-c', bb 'and d-d' are respectively enzyme reaction time 0 min, 5
It is a result about time. In the figure, the dotted line on the left (retention time 30.3 minutes) indicates the elution position of the tetrasaccharide, and the dotted line on the right (retention time 32.4 minutes) indicates the elution position of the disaccharide.

From the results shown in FIGS. 6 and 7, when the enzyme I was allowed to act on chondroitin 6-sulfate and dermatan sulfate, the size of these glycosaminoglycans decreased markedly with the lapse of the enzyme reaction time. enzyme
It is suggested that I is an endo-type enzyme), and that a mixture of unsaturated glycosaminoglycan tetrasaccharide and disaccharide is finally obtained.

On the other hand, enzyme II was replaced with chondroitin 6
-When acted on sulfate and dermatan sulfate, the size of these glycosaminoglycans does not decrease much over the course of the enzyme reaction time (this suggests that enzyme II is an exo-type enzyme). It was shown that substantially only the unsaturated glycosaminoglycan disaccharide was produced with the lapse of the enzyme reaction time, and that substantially only the unsaturated glycosaminoglycan disaccharide was finally obtained. Was. Similar results were obtained when chondroitin 4-sulfate was used as the substrate.

From the results shown in FIG. 8, when enzyme I was allowed to act on chondroitin sulfate hexasaccharide, a mixture of unsaturated glycosaminoglycan tetrasaccharide and saturated glycosaminoglycan disaccharide was finally obtained. It has been shown.

On the other hand, it was shown that when the enzyme II was allowed to act on chondroitin sulfate hexasaccharide, substantially only an unsaturated glycosaminoglycan disaccharide was finally obtained. From the above results, it was shown that the enzyme I decomposes the N-acetylhexosaminide bond of glycosaminoglycan to produce a mixture of glycosaminoglycan tetrasaccharide and glycosaminoglycan disaccharide.

On the other hand, it was shown that the enzyme II decomposes the N-acetylhexosaminide bond of glycosaminoglycan and, when completely decomposed, generates substantially only unsaturated glycosaminoglycan disaccharide. Was done. From this, enzyme I
It was shown that I can be clearly distinguished from enzyme I also in terms of its enzyme reaction product.

(3) Production of unsaturated glycosaminoglycan disaccharide using enzyme II One liter of 10 g of chondroitin sulfate prepared from bovine cartilage was used.
Was dissolved in 25 mM sodium phosphate buffer, pH 7.6, 100 units of enzyme II was added, and the mixture was incubated for 15 hours in a thermostatic water bath adjusted to 37 ° C. This liquid was filtered through an ultrafiltration membrane having a molecular weight of 10,000 cuts, and the liquid passing through the membrane was collected. This solution was freeze-dried to obtain about 10 g of bovine cartilage-derived unsaturated glycosaminoglycan disaccharide. Since the unsaturated glycosaminoglycan disaccharide contained some phosphate derived from a buffer solution, 5 g of the lyophilized product was dissolved in purified water, and Cellulofine GCL-50m (Seikagaku Corporation) (Company sales)
Was desalted by passing through a column packed with, and fractions eluted with unsaturated glycosaminoglycan disaccharide were collected and lyophilized to obtain about 4.2 g of a lyophilized powder containing no salt. The lyophilized product was subjected to gel permeation chromatography using a TSKgel-G2500 _XL column. As a result, a peak was observed only at the position where the unsaturated glycosaminoglycan disaccharide was eluted.

[0158]

The enzyme of the present invention acts on glycosaminoglycans of various sizes from macromolecules to tetrasaccharides, in particular, chondroitin sulfate, and when completely decomposed, substantially unsaturated glycosaminoglycan disaccharide. A novel exo-type chondroitin sulfate degrading enzyme that produces only sugar,
It is useful as a reagent, an antigen for producing the antibody of the present invention, or the like. In particular, the enzyme of the present invention has an action of decomposing disaccharides one by one from the non-reducing terminal side of glycosaminoglycan, and is therefore extremely useful as a sugar chain analysis reagent.

The crystals of the present invention can be used as pharmaceuticals,
Very useful as a reagent. The antibody of the present invention can be used for specific detection of the enzyme of the present invention. It is particularly useful for tracking the enzyme of the present invention administered to animals as a pharmaceutical, searching for the enzyme of the present invention in natural products, and the like. Further, the antibody of the present invention can be bound to an insoluble carrier and used for affinity purification of the enzyme of the present invention.

The enzyme production method of the present invention is a method that can efficiently produce the enzyme of the present invention in large quantities, and is an extremely useful method because it can be carried out on an industrial scale. The unsaturated disaccharide production method of the present invention is a method for efficiently producing a large amount of unsaturated glycosaminoglycan disaccharide using the enzyme of the present invention. Very useful as a reagent.

The analysis method of the present invention utilizes the characteristics of the enzyme of the present invention and is extremely useful as a sugar chain analysis method. In particular, the fact that it can be used for the sequencing of disaccharide units of glycosaminoglycans is a very useful point not found in conventional enzymes.

The composition of the present invention can be used as a pharmaceutical composition or a reagent composition containing the enzyme of the present invention, and is useful. The therapeutic agent of the present invention can be used as a therapeutic agent for herniated disc containing the enzyme of the present invention, and is extremely useful.

[Brief description of the drawings]

FIG. 1 shows an elution pattern of CM-Sepharose FF column chromatography of an enzyme extract from Proteus vulgaris cells. ● indicates the enzyme activity using chondroitin 6-sulfate as a substrate. ○ indicates enzyme activity when chondroitin sulfate tetrasaccharide is used as a substrate. X indicates the protein concentration.

FIG. 2 is a micrograph of crystals obtained by crystallizing enzyme I and enzyme II (enzyme of the present invention) with polyethylene glycol (crystal structure). A is a crystal of enzyme I, B is enzyme II
1 shows crystals of (enzyme of the present invention).

FIG. 3 shows an analysis chart of amino acid composition.

FIG. 4. Various pH values of enzyme I and enzyme II (enzyme of the present invention)
Shows the enzyme activity of ○ indicates the enzyme activity of enzyme I using chondroitin 6-sulfate as a substrate. ● indicates the enzyme activity of enzyme II using chondroitin 6-sulfate as a substrate. X indicates the enzyme activity of enzyme II when chondroitin sulfate tetrasaccharide is used as a substrate.

FIG. 5 shows changes in viscosity (○) and changes in _A232 (●) when Enzyme I and Enzyme II (enzyme of the present invention) act on chondroitin 6-sulfate and dermatan sulfate.

FIG. 6 shows elution patterns of reaction products obtained by reacting enzyme I and enzyme II (the enzyme of the present invention) with chondroitin 6-sulfate for various periods of time by gel permeation high performance liquid chromatography. a to h are relative refractive indices, a 'to h' are A
₂₃₂ is shown.

FIG. 7 shows the reaction products obtained by allowing enzyme I and enzyme II (the enzyme of the present invention) to act on dermatan sulfate for various times.
Fig. 3 shows an elution pattern obtained by gel permeation high performance liquid chromatography. a~h is a relative refractive index, a'~h 'denotes the A _232.

FIG. 8 shows an elution pattern by gel permeation high performance liquid chromatography of a reaction product obtained by allowing enzyme I and enzyme II (the enzyme of the present invention) to act on chondroitin sulfate hexasaccharide. a~d is a relative refractive index, a'-d 'denotes the A _232.

Claims (12)

[Claims] 1. A chondroitin sulfate degrading enzyme having the following physicochemical properties. Action: (1) Decomposes the N-acetylhexosaminide bond of glycosaminoglycan, and when completely decomposed, substantially produces only unsaturated glycosaminoglycan disaccharide. (2) It is an exo-type enzyme. Substrate specificity: chondroitin, chondroitin 4-
Sulfate, chondroitin 6-sulfate, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin 6-sulfate hexasaccharide, chondroitin 6-sulfate tetrasaccharide,
It acts on both the chondroitin sulfate proteoglycan and the chondroitin sulfate chain portion. It has no effect on keratan sulfate, heparin and heparan sulfate. Molecular weight: about 105 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions
kDa. Amino acid composition: Cystine is detected by amino acid analysis of the acid hydrolyzate of the enzyme. 2. The chondroitin sulfate degrading enzyme according to claim 1, which has the following physicochemical properties. N-terminal amino acid: The N-terminal amino acid is leucine. Isoelectric point: pH about 8.45 (isoelectric focusing) Optimum temperature: around 40 ° C (substrate: chondroitin 6-sulfate, buffer: Tris-HCl buffer, pH: 8.0) Optimum reaction pH: around pH 8 (substrate: chondroitin 6)
-Sulfuric acid, buffer: Tris-HCl buffer, temperature: 37 ° C) 3. A crystal of the chondroitin sulfate degrading enzyme according to claim 1 or 2, which is a plate-like crystal. 4. The crystal of the chondroitin sulfate-degrading enzyme according to claim 3, wherein the crystal is crystallized using a polyether having a structure in which both terminals are hydroxyl groups. 5. An antibody which selectively recognizes the chondroitin sulfate degrading enzyme according to claim 1 or 2. 6. A cell having the ability to produce chondroitin sulfate degrading enzyme, wherein the chondroitin sulfate degrading enzyme according to claim 1 or 2 is collected from a culture of the cell. 3. The method for producing a chondroitin sulfate degrading enzyme according to item 1. 7. A step of degrading glycosaminoglycan, which can be a substrate for the chondroitin sulfate degrading enzyme according to claim 1 or 2, with the chondroitin sulfate degrading enzyme according to claim 1 or 2; And a step of fractionating the unsaturated glycosaminoglycan disaccharide. A method for producing an unsaturated glycosaminoglycan disaccharide. 8. A step of degrading a glycosaminoglycan that can be a substrate for the chondroitin sulfate degrading enzyme according to claim 1 or 2 with the chondroitin sulfate degrading enzyme according to claim 1 or 2, and treating the degradation product. Analyzing the glycosaminoglycan at least. 9. The method for analyzing glycosaminoglycan according to claim 8, wherein the decomposition product is analyzed by liquid chromatography. 10. A chondroitin sulfate degrading enzyme-containing composition comprising the chondroitin sulfate degrading enzyme according to claim 1 and a carrier that does not affect the activity of the chondroitin sulfate degrading enzyme. 11. The composition according to claim 10, wherein the chondroitin sulfate degrading enzyme is in the form of a crystal according to claim 3 or 4. 12. An agent for treating a herniated disc, comprising the composition according to claim 10 or 11.