KR20020046293A - Novel heparinases derived from Bacteroides stercoris HJ-15 which hydrolyze glycosaminoglycan, a preparing method and a use thereof - Google Patents

Abstract

PURPOSE: A novel heparinase capable of decomposing glycosaminoglycans(GAG), derived from human intestinal microorganism, a producing method thereof and the use thereof are provided, therefore the heparinase having improved substrate specificity and stability can be produced. CONSTITUTION: The heparinase capable of decomposing glycosaminoglycans(GAG) is isolated from Bacteroides stercoris HJ-15(KCCM-10096), wherein glycosaminoglycans(GAG) are heparin, heparan sulfate and acharan sulfate; the heparinase has 45 or 70 kDa of molecular weight and optimal NaCl concentration of 200 to 900 mM, optimal pH of 6.0 to 7.5 and optimal temperature of 35 to 55 deg. C or 40 to 45 deg. C; and the activity of heparinase is catalyzed by EDTA, Co2+, Cu2+, Ni2+, Ba2+ and Zn2+. The method for producing heparinase comprises the steps of: culturing Bacteroides stercoris in an appropriate medium; recovering the cultured cells and preparing cell extract; and subjecting the cell extract to chromatography, wherein the chromatography is selected from QAE-cellulose, DEAE-cellulose, CM-sephadex C-50, hydroxyapatite, CM-sephadex C-25 and Hi-Trap SP.

Description

Novel heparinases derived from Bacteroides stercoris HJ-15 which hydrolyze glycosaminoglycan, a preparing method and a use according to human intestinal microorganisms that degrade glycosaminoglycans

The present invention is a novel heparinase derived from bacteroids Stechoris HJ-15, which produces glycosaminoglycans (abbreviated as "GAG") degrading enzymes. , And a method for using the same, and specifically, two novel heparines that are isolated and purified from human intestinal microorganisms, bacteroids Sterchoris HJ-15, and specifically decompose heparin and heparan sulfuric acid, respectively. It relates to an ease, a production method and a use thereof.

Glycosaminoglycans are structurally diverse and complex through partial changes in the biosynthesis process. GAGs have a basic backbone that is partially sulfated with uronic acid and glucosamine, and these complex chemical structures enable them to exhibit a variety of biological activities. As is known to date, these various biological activities appear to be due to the presence of specific sequences of their own.

Animal GAGs include hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, heparin, and the like. Akaran sulfuric acid was found and patented (Korean Patent No. 1995-46690). Among these GAGs, heparin is isolated in mast cells along the arteries in the liver, lungs, and skin mucosa, and the other GAGs are distributed in connective tissue. Chondroitin sulfate, used as a medicine, is obtained from shark cartilage and bovine bronchus. In addition, GAGs are also found in molluscs, which are inferior, such as the presence of GAGs with antithrombin binding sites with 3-0-sulfuric acid groups in shellfish (Pejler et al., J. Biol. Chem ., 262; 11413-11421, 1986), chondroitin sulfates substituted with sulfuric acid have also been found in sea cucumbers and sea urchins (Mourao et al., TIGG , 7; 235-246, 1995). It is also known that chondroitin sulfate is present in the cornea of squid (Karamanos et al ., Int. J. Biochem ., 23; 67-72, 1991), and acaran sulfate is found to be abundant in African king snails ( Kim et al. , J. Biol. Chem., 271; 170-175, 1996) .

GAGs exist in the form of proteoglycans in vivo, and these proteoglycans are cleaved by protease and glycosidase to release glycosides. The physiological role of GAGs is not known yet, but it seems to show physiological activity by binding to proteins in vivo. Proteins that bind GAGs in vivo can be largely classified into five types, such as protease inhibitors, plasma lipoproteins, growth factors, lipolytic enzymes, and extracelluar matrix proteins. These proteins bind to GAGs and act on their specific biological activities.

The first GAGs exhibit physiological activity in combination with protease inhibitors. Representative protease inhibitors that bind GAGs include antithrombin III and heparin cofactor II. Heparin binds with antithrombin III to inhibit the action of blood factors Xa and thrombin, and binds to heparin cofactor II to inhibit the action of thrombin to prevent blood coagulation.

The second GAG class binds to lipoproteins of plasma. Representative plasma lipoproteins that bind to GAG class are apolipoprotein E and apolipoprotein B. GAGs that bind to plasma lipoproteins include heparin and chondroitin, and these heparin and chondroitin interact with sites in which the acid and basic amino acids of apolipoprotein E and apolipoprotein B are concentrated to regulate the occurrence of atherosclerosis.

The third GAG class binds to the growth factor, and the growth factor that binds to the GAG class is basic fibroblast growth factor (bFGF), and the GAG class that binds to the growth factor includes heparin and heparan sulfate. Heparin and heparan sulfate play an important role in cell growth and differentiation by binding to bFGF and preventing the degradation of bGFG.

The fourth GAG binds to lipolytic enzymes. Intravenous injection of heparin and heparan sulfate releases lipolytic low density lipoproteinases and triglycerides into plasma cells. Likely, free lipolytic enzymes break down fat in the blood and play a major role in preventing atherosclerosis.

Fifth GAGs bind to enveloped cell conjugates, and proteins of the enveloped cell conjugates that interact with GAGs include fibronectin, vitronectin, laminin, and thrombospondin. . Proteins that bind to these GAGs are important for cell adhesion, information transfer between cells, cell differentiation, and cell growth.

As described above, GAGs can be used for various purposes because they show various physiological activities in combination with proteins in vivo. As an example in which GAGs are used as drugs, chondroitin sulfate is also used in eye drops, arthritis treatment agents, and recently, tonic drinks. Heparin is widely used as a glycan drug in the clinic, and heparin is an anticoagulant drug. Various biological activities, including antiviral action, have been reported. Heparin is extracted from animal tissues as a biological substance of GAG class, and is used for such use by using their anticoagulant and antithrombinability, and heparin is particularly useful for the treatment of postoperative venous thrombosis. However, heparin in its natural form has drawbacks with very limited conditions of use, in particular anticoagulant activity can cause bleeding and sensitivity to serum factors such as PF4 leads to the use of relatively large doses. Therefore, in the absence of anticoagulant activity due to antithrombin effect, in particular antithrombin activity due to antithrombinase activity must be promoted.

On the other hand, acaran sulfate exhibits various physiological activities including the physiological activities of the aforementioned GAGs. Acaran sulfate is likely to be used as an anticoagulant with less side effects due to structural modifications such as deacetylation, sulfate, and new oligo GAG. Among these, sulfated oligosaccharides of acaran sulfate bind to fibroblast growth factors. It also plays an important role in cell growth and differentiation as it provides the structural features necessary for. In fact, the desulfated compound -NH ₂ after deacetylation promotes cell growth about 6 times as compared to the control group, and thus the wound healing effect of sulfated acaran sulfate can be expected (Wang et al. , Biochem. Biophy. Res. Commun ., 235; 369-373). Acaran sulfate is also ideal as a model compound for reactions using sulfate group transferase because of its simple structure of repeating disaccharides. In addition, since acaran sulfate is derived from a snail used as a health food, it is very effective for liver deterioration and menopausal disorders including osteoporosis.

Although GAGs exhibit various pharmacological effects as described above, their molecular weight is too large, so it is not easy to deal with them, and there is a problem of causing side effects.

In order to achieve the above object, it has been proposed to fragment heparin into molecules of low average molecular weight. In particular, European Patent Application No. 40,122 discloses a mixture of sulfuric acid and polysaccharides, consisting of the general structure of the polysaccharides constituting heparin, having an ethylenic double bond at its non-reducing end and having a weight average molecular weight of 2,000-10,000 Daltons. These mixtures are obtained by polymerization and saponification of heparin esters, which have been reported to show lower antithrombin activity and total anticoagulant activity than heparin. However, the main problem faced by this technique is that the heparin thus obtained is a very heterogeneous product.

In addition, the method described in European Patent Application No. 40,144 raises the problem that it is not possible to obtain compounds with the essential pharmacological properties for improved action, namely half-life in sufficiently long plasma, significantly high absorption rate. In addition, recent studies on the mechanism of heparin action in thrombin formation have demonstrated the effect of heparin's average molecular weight on heparin activity in in vitro experiments (Beguin et al . , Thromb. Haemost., 61:30, 1989). It has been reported that low molecular weight heparin has a decreased thrombin inhibitory activity, and high molecular weight heparin has a tendency to have thrombin inhibitory activity.

At the same time, European Patent Application No. 337,327 proposed a method for fractionating heparin to extract mixtures of more homogeneous average molecular weight, which makes it possible to obtain mixtures with dispersions with much lower average molecular weight, from heparin Described are methods for preparing the derived small sugar fragments. According to this method, an aliquot of molecular weight of 3,000 Daltons or less is first removed, followed by the removal of fragments containing 10-16 sugars or less from the final product, leaving only heparin aliquots of molecular weight of 7,000 Daltons or more. Homogenization of this final mixture produced heparin with reduced anticoagulant activity while preferred antithrombotic activity was preserved.

There are two methods for producing low molecular weight GAGs, chemical methods and methods using enzymes. Enzymes that decompose GAGs include lyases and eliminases, which are hydrolyzed to release water and double-bond compounds, and hydrolyases, which hydrolyze to form compounds of single bonds. Is I, II, III and chondroitin degrading enzymes A, B, C and the like. Heparinase I specifically cleaves and degrades heparin's main binding structure-> 4) -α-D-GlcNS (6S or OH) (1-> 4) -α-L-IdoA2S (1->). Recently, clinical routines using Heparinase I have been underway for the purpose of neutralizing excess heparin in heparin therapy (Zimmerman, U.S. Patent Application No. 5,262,325). It was licensed for use (Tejidor et al., Thrombo. Haemost ., 69: 866, 1993). Heparinase II is -4) -α-D-GlcNS (6S or OH) (1-> 4) -α- Decomposes heparin and acaran sulfate by acting on L-IdoA (2S or OH) or -β-D-GlcA (-> 1. Heparinase III is the main bond of heparan sulfate-> 4) -α- Acts specifically on D-GlcNS (or Ac) (1-> 4) -α-L-GlcA (or-> IdoA) (1->) to degrade heparin sulfate (Steffen E., Critic. Rev. in Biochem. And Mol. Biol. , 30 (5): 387-444, 1995) .

Most of the above heparinases are Flavobacterium heparinum (Flavobacterium heparinum)Isolated from Flavobacterium spp.Bacillus sp(Bellamy et al., U.S. Patent Application No. 5,145,778) and the genus of the anaerobic bacteroid genus (Bacteroides sp.)Heparin degrading enzyme has also been found in. For the first time in the genus anaerobic bacteroid, Gesner et al. (Gesner et al.,J. Bacteriol, 81: 595-604, 1961), the bacteroid Heparinoraiticus found in the oral cavity (Bacteroides heparinolyticus)(Nakamura et al.,J. Clin. Microbiol.,26: 1070-1071, 1988), the bacteroid obata in the gut (Bacteroides ovata)And bacteroid thetaotaomicron (Bacteroides thetaiotaomicron)Has been reported (Riley, T. V. et al.,Microbios Lett.,25: 141-148, 1984; Riley,J. Clin. Pathol.,40: 384-386, 1987). Thus, bacteroid thetaotao micron (inhabiting intestine of person)B. thetaiotaomicron),Bacteroid Obata(B. ovata)And Bacterial Uniformis(B. uniformis)Heparinase has been reported to exist in strains such as (Riley, T.V.,Microb. Lett.,25: 141-149, 1984; Riley,J. Clin. Path.40: 384-386, 1987) There have been no reports of purified bacteroid heparinase. Therefore, the study of the new heparinase will be useful for the structural analysis of GAGs such as heparin and heparan sulfuric acid as well as acaran sulfuric acid and the production of low molecular weight heparin.

The present inventors have also recently isolated, identified and patented Bacteroides stercoris HJ-15, a microorganism that synthesizes GAG degrading enzymes in the human intestine (Korean Patent Application No. 10- 0257168). The strain degrades heparin, heparan sulfuric acid, chondroitin sulfate A and chondroitin sulfate C.

Therefore, the present inventors have identified and identified bacteroids stecoris which have been identified in the human intestine by the present inventors as microorganisms producing GAG-like enzymes. Purification of heparinase from HJ-15 (KCCM-10096) to decompose heparin, heparan sulfuric acid and acaranic acid, respectively, to reveal its biochemical properties The present invention has been completed by revealing that it can be usefully used.

An object of the present invention is to provide a novel heparinase digested GAGs isolated from human intestinal microorganisms, which can be usefully used for structural analysis of GAGs and production of low molecular weight heparin.

1A is an elution profile obtained by purifying the cell extract of bacteroid Stercoris HJ-15 according to CM-Sephadex C-50 ion exchange column chromatography. (profile),

●: Acaran Sulfate Heparinase Activity

△: heparin degrading heparinase activity

-Absorbance at 280 nm

Figure 1b shows the elution profile of the heparin decomposed heparinase active fraction obtained in Figure 1a purified by hydroxyapatite column chromatography,

Figure 1c shows the elution profile of the heparin decomposed heparinase active fraction obtained in Figure 1a purified by CM-Sephadex C-25 ion exchange chromatography,

FIG. 2A shows the elution profile of the heparan sulfate-dissolved heparinase active fraction obtained in FIG. 1A purified by CM-Sephadex C-50 ion exchange column chromatography.

FIG. 2B shows the elution profile of the heparan sulfate decomposed heparinase active fraction obtained in FIG. 2A purified by hydroxyapatite column chromatography.

FIG. 2C shows the elution profile of the active fraction of heparan sulfate decomposed heparinase obtained from FIG. 2B subjected to SP Hi-Trap column chromatography.

●: Activity of Heparan Sulfate Heparinase

-Absorbance at 280 nm

Figure 3 shows the substrate specificity for acaran sulfate, heparin and heparan sulfuric acid of the enzyme active fraction obtained in Figure 1 ,

Figure 4 shows the SDS-PAGE results of heparin decomposed heparinase isolated and purified in the present invention,

Lane 1: standard molecular weight

Lane 2: enzyme solution eluted from CM-Sephadex C-25 column

Figure 5 shows the SDS-PAGE results of the purification step of heparan sulfate decomposition heparinase of the present invention,

Lane 1: enzyme solution eluted from a CM-Sepadex C-50 column

Lane 2: enzyme solution eluted from the hydroxyapatite column

Lane 3: enzyme solution eluted from the high trap SP column

Lane 4: standard molecular weight

Figure 6 shows the optimal pH of heparin decomposed heparinase isolated and purified in the present invention,

Figure 7 shows the optimal pH of heparan sulfate disintegrated heparinase isolated and purified in the present invention,

Figure 8 shows the stability to the temperature of heparin decomposed heparinase isolated and purified in the present invention,

Figure 9 shows the effect of NaCl concentration on heparin decomposed heparinase isolated and purified in the present invention,

FIG. 10 shows a Hanes plot for determining kinetic constants of heparan sulfate decomposed heparinase isolated and purified in the present invention.

a: heparin b: heparan sulfate

In order to achieve the above object, the present invention is a novel heparinase that decomposes heparin, heparan sulfate and acaran sulfate, isolated and purified from Bacteroides stercoris HJ-15, which is a human intestinal microorganism. And a method for producing the same.

In addition, the present invention provides the use of the novel heparinase in the production and structural analysis of GAGs or the production of low molecular weight heparin.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel heparinase for decomposing heparin, heparan sulfuric acid and acaran sulfuric acid separated and purified from Bacteroides stercoris HJ-15, and a method for producing the same.

Used in the present invention The bacteroids were identified and identified as follows. After culturing human and rat intestinal bacterial guns in anaerobic condition, they obtained a cell solution, and then selected a strain having the activity of decomposing heparin, acaran sulfate and chondroitin from the cell solution, and identified according to the general strain identification method and Burgy's Manual (Bergey's). manual of systemic bacteriology, 1991). As a result, the strain is a bacteroid (BacteroidIt was identified as Bacterial Stechoris HJ-15, and it was deposited on February 19, 1997 with the Korean spawn association (accession number KCCM-10096). GAG degrading enzymes of bacteroids Stechoris HJ-15 show heparinase I, II and chondroitinase activity as lyases and can be used for the preparation of low molecular weight heparin, moreover, derived from Flavobacterium In addition to exhibiting similar properties to GAG degrading enzymes, it is produced by microorganisms in the intestine, and thus can be administered orally as well as injections when administering GAGs.

Therefore, the present inventors isolated and purified a novel heparinase, a GAG degrading enzyme, from the bacteroid Stercoris HJ-15 which is the intestinal microorganism as follows.

The method for producing a novel heparinase of the present invention

1) culturing the bacteroids stericis in a suitable medium;

2) recovering the cultured cells and preparing a cell extract; And

3) purification of the cell extract by chromatography to obtain enzymatic activity.

In more detail, in the step 1, in order to separate the heparinase from the bacteroid Stercoris HJ-15 strain, the strain was first used in a tryptic shoot medium containing heparin (heparin) instead of glucose (glucose). culture in anaerobic conditions using tryptic soy broth ( Can. J. Microbiol., 44: 423-429, 1998). Cells cultured in step 2 are recovered by centrifugation, washed with buffer and suspended. Sonication is performed on the suspended cells to remove cell debris by centrifugation. In step 3, only the cell extract of step 2 was purified by a DEAE-cellulose column, a QAE-cellulose column, and a CM-Sephadex C-50 column to obtain a fraction showing enzymatic activity. Enzyme activity to degrade heparin and heparan sulfate for all fractions is measured. Through this process, the present inventors secured three (a, b, c) fractions showing enzymatic activity, and examined the substrate specificity for GAGs. As a result, fraction a preferentially decomposes acaran sulfate and fraction b is heparin. Rather, heparan sulfuric acid was preferentially decomposed and fraction c was found to act preferentially on heparin rather than heparan sulfuric acid (see FIG. 3 ).

Heparinase which specifically decomposes heparin by dialysis of fraction c showing heparin degrading activity and then purified by hydroxyapatite column and CM-Sephadex C-25 column (hereinafter referred to as "heparin decomposed heparinase"). Fractions showing “abbreviated” activity were obtained (see FIGS. 1A, 1B, 1C, Table 1 and FIG. 4 ).

In addition, heparanese (hereinafter referred to as "hepa") is used to purify fraction b showing heparan sulfate activity using a hydroxyapatite column, and then dialyzed and purified by a Hi-Trap SP column. Fractions exhibiting activity (hereinafter abbreviated as "lan sulfate sulfate heparinase") were obtained ( see FIGS. 2A, 2B, 2C, Table 2 and FIG. 5 ).

In addition, the present invention provides the biochemical and physicochemical properties of heparin decomposed heparinase and heparan sulfate decomposed heparinase isolated and purified as described above.

1) Biochemical Properties of Heparin-Degraded Heparinase

The molecular weight of heparin degraded heparinase was measured to be 48 kDa (4) Showed optimal enzyme activity at pH 7.0 (see6Reference). The enzyme showed a stable enzyme activity up to 50 ℃, the enzyme activity was sharply reduced above 50 ℃ (8And optimal activity at 500 mM sodium chloride (NaCl, sodium chloride).9Reference). As a result of examining the substrate specificity of the heparin-degraded heparinase of the present invention, the heparinase was more specific for heparin than heparan sulfate (Table 5Co) in divalent cations²⁺, Ba²⁺, EDTA promoted enzymatic activity (TABLE 3Reference).

2) Biochemical Properties of Heparan Sulfate Decomposed Heparinase

The molecular weight of heparan sulfated heparinase was 70 kDa, showing a molecular weight similar to that of Flavobacterium heparinase III (see FIG. 5 ), and the optimum pH was 7.2 (see FIG. 7 ). The heparinase stably increased the enzyme activity up to 40 ℃ and decreased activity above that temperature, the optimum reaction temperature was 45 ℃. Heparan sulfate decomposed heparinase of the present invention, Mg ²⁺ , Ca ²⁺ , Ba ²⁺ among divalent cations slightly increased the enzyme activity and Cu ²⁺ , Pb ²⁺ inhibited the enzyme activity (see Table 4 ). ), Heparin and heparan sulfuric acid showed high substrate specificity, while acaran sulfate derivatives showed very low activity or no activity at all (see Table 6 ). In addition, the heparinase enzyme activity was increased by a reducing agent such as dithiothreitol or 2-mercaptoethanol (see Table 7 ), and amino acid composition analysis showed lysine (lysine). A large proportion was included, which was consistent with the measured pI value of 8.7 (see Table 8 ). As a result of analyzing the internal amino acid sequence, the internal amino acid sequence of the heparan sulfated heparinase of the present invention showed 73% higher homology with Flavobacterium heparinase III than Flavobacterium heparinase II. (See Table 9 ). In addition, as a result of determining the Michaelis-Menten constant by measuring the initial rate of the heparinase enzyme activity, the Vm and Kmax values for heparin were 9.05 × 10 ^-5 M and 38.2 μmol, respectively. / min / mg and Vm and Km for heparan sulfuric acid were measured at 1.53 × 10 ⁻⁵ M and 58.4 μmol / min / mg, respectively (see Table 10 and FIG. 10 ).

In addition, the present invention provides a use of the novel heparinase in the production and structural analysis of GAGs or the production of low molecular weight heparin.

As described in the prior art, GAGs can be used in various applications because they bind to proteins in vivo and exhibit various physiological activities. In particular, chondroitin sulfate is also used in eye drops, arthritis treatment agents, and recently, tonic drinks. Is an anticoagulant, and various biological activities have been reported, such as thrombolytic agents, artificial blood vessels, and antiviral action. However, it has been pointed out that natural type heparin may cause bleeding when used clinically and its sensitivity to certain serum factors requires relatively large doses. Therefore, a method for producing a low average molecular weight heparin has been developed, but heparin produced by the method for preparing a mixture of low molecular weight sulfuric acid and polysaccharide described in European Patent Application No. 40,122 has the disadvantage that it is a very non-uniform product, the European patent application The manufacturing method described in No. 40,144 has a problem in that a mixture having high absorption and biocompatibility cannot be obtained.

However, the novel heparinase of the present invention can be usefully used for the preparation and structural analysis of GAGs or for the preparation of low molecular weight heparin, because the enzyme activity is more stable and substrate specificity is solved while solving the above problems.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Novel Heparinase Isolated from Enterobacteriaceae Bacterial Stecoris

<1-1> Cell Culture and Preparation of Cell Extract

First, bacteroid Bacteroides stercoris HJ-15 strain (Accession No .: KCCM-10096) was substituted for glucose (heparin) 0.15 g / L and sodium thioglycolate (sodium thioglycolate) 0.01 w inoculated in 100 l of tryptic soy broth (pH 7.2) containing / v% and ascorbic acid 0.1 w / v%, 37 ° C., N ₂ (g) 90% and CO ₂ (g) incubated in anaerobic conditions of 10%. The culture solution was collected by centrifugation at 5,000 rpm for 30 minutes at 4 rpm, and then washed with 50 mM sodium phosphate buffer (pH 7.0). After the cells were suspended in the same buffer solution, cells were disrupted by applying heat for 1 minute at 80% power for 30 minutes using an ultrasonic processor (Col-Parmer Instruments, II.USA). Cell debris was removed by centrifugation at 18,000 rpm for 60 minutes at 4 ° C. and only cell extracts were separated.

<1-2> Purification of Cell Extract

The cell extract was passed through a DEAE-cellulose column (DEAE-cellulose column, 5 x 30 cm) equilibrated with 50 mM sodium phosphate buffer and the column was washed with the same buffer until the activity of heparinase was not measured. . The fractions passed through the column were collected and passed through a QAE-column (QAE-column, 5 × 40 cm) equilibrated with the same buffer, followed by washing the column with the same buffer. The fractions passed through the column were collected and passed through a CM-Sephadex C-50 column (CM-Sephadex C-50, 3 × 30 cm), the column was washed with the same buffer solution, and KCl was dissolved in the same buffer solution. Enzyme was eluted at a flow rate of 105 ml / h with a linear concentration of 0.6 M.

As a result of measuring the enzymatic activity of decomposing heparin and heparan sulfuric acid on all the eluted fractions, three fractions a, b, and c representing enzymatic activity were obtained ( FIGS. 1A, 1B and 1C ). Enzyme activity to degrade sulfuric acid was measured.

Enzyme activity was measured by the following method (Linhardt, RJ, Current Protocols in Molecular Biology., 17.13.17-17.13.32, 1994). 700 μl of 50 mM sodium phosphate buffer (pH 7.0) containing 1 mg of a substrate such as heparin substrate or heparan sulfuric acid and 500 mM NaCl was added to a 1 ml quartz cuvette, and the reaction mixture was equilibrated to 40 ° C. Then, 50 μl of the fraction sample eluted as described above was added to the mixture and immediately mixed into an ultraviolet spectrophotometer to measure the change in absorbance at 232 nm. Enzyme activity was calculated using a molar absorption coefficient of 3,800 ⁻¹ · cm ⁻¹ for unsaturated oligosaccharides produced by heparinase (1 U = 1 μmole of ΔUA containing product formed / min).

As a result , as shown in FIG . 3 , fraction a preferentially decomposes acaran sulfate, fraction b preferentially decomposes heparan sulfuric acid rather than heparin, and fraction c preferentially acts on heparin rather than heparan sulfuric acid. .

<1-3> Purification of heparin-decomposed heparinase

Fraction which specifically degrades heparin obtained from Example <1-2> (86.5) Ml) was dialyzed with 50 mM sodium phosphate buffer (pH 7.0) and passed through a hydroxyapatite column (2.5 x 10 cm) equilibrated with the same buffer. After washing the column with the same buffer solution, NaCl was dissolved in the same buffer solution, and the enzyme was eluted at a flow rate of 120 mL / h by giving a linear concentration tool at a concentration of 0-0.5 M (Figure 2a). After collecting and dialysis the fraction showing the enzyme activity through a CM-Sephadex C-25 column (CM-Sephadex C-25, 3 × 30 cm), wash the column with the same buffer as above and NaCl 0-1.0 M The enzyme was eluted at 100 mL / h by using a linear thickener. Fractions showing enzymatic activity were collected and SDS-PAGE was performed to confirm protein purity. The protein concentration, enzyme activity, substrate specificity, and the like of the purified heparin-decomposed heparinase were as follows.Table 1Shown in

Purification of Heparin Degraded Heparinase step Total protein (mg) Total activity (U) Specific activity (U / mg) Production degree Drainage Cell extract CM-Sephadex C-50HydroxyapatiteCM-Sephadex C-25 7636.819.045.480.0834 223.417.2212.344.83 0.0290.92.2557.97 1007.75.52.2 131781999

It can be seen from Table 1 that the specific activity of heparin decomposed heparinase was purified about 2000-fold from 0.013 U / mg to 57.97 U / mg. In particular, there was a decrease in total protein in cation chromatography after passing through the hydroxyapatite column, but the specific activity increased by about 25 times or more. As described above, it is possible to predict that the isoelectric point of heparin decomposed heparinase is basic from the fact that purification is performed by two kinds of cation chromatography. Moreover, since heparin is an acidic sugar, it is fully possible.

<1-4> Purification of Heparan Sulfate Heparinase

The fraction b (149.5 ml) that specifically degrades heparan sulfuric acid among the fractions obtained in Example <1-2> was equilibrated with 50 mM sodium phosphate buffer, and then passed through a hydroxyapatite column (2.5 × 10 cm). I was. KCl was dissolved in 0.5 M in the same buffer, and the column was washed with 300 ml. KCl was dissolved in 0.5 M and 1.3 M in the same buffer, and 800 ml of linear thickener was added to elute the enzyme at a flow rate of 120 ml / hr. ( FIG. 2B ). Collect the fractions showing the enzyme activity and pass the Hi-Trap SP column equilibrated with the same buffer as above, wash the column with the same buffer as above, and dissolve KCl in the same buffer to 300 with 0-0.5 M. The enzyme was eluted with mL of linear thickener ( FIG. 2C ). Fractions showing enzymatic activity were collected and SDS-PAGE was performed to investigate protein purity. The protein concentration, enzyme activity, substrate specificity, and the like of the purified heparan sulfate decomposed heparinase are shown in Table 2 below.

Purification of Heparan Sulfate Heparinase step Total protein (mg) Total activity (U / min) Specific activity (U / mg) Production degree Drainage Cell extract CM-Sephadex C-50Hydroxyapatite HiTrap SP (SP-Sepharose) 7,63732.98.52.4 223.483.558.646.7 0.0292.546.8919.5 ^a 54.1 ^b 10037.426.220.9 188238672

a: Heparinase activity measured using heparin as a substrate

b: Heparinase activity measured using heparan sulfuric acid as a substrate

It can be seen from Table 2 that the specific activity of heparan sulfate decomposed heparinase was 672-fold purified from 0.029 U / mg to 54.1 U / mg.

The heparinase activity seen in fraction b is very similar to Flavobacterium heparinase II and is present in much more purified form after passing through hydroxyapatite column and Hytrap trap column.

Example 2 Investigation of Biochemical Properties of Novel Heparinase

<2-1> molecular weight measurement

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by Laemmli method ( Nature, 227: 680-685, 1970) to determine the molecular weight of the novel heparinase of the present invention isolated and purified from Example 1 And stained with Coomassie brilliant blue R-250.

The molecular weight of heparin decomposed heparinase was measured at 48 kDa and the isoelectric point measured at IEF was 9.0 ( FIG. 4 ). In addition, the molecular weight of heparan sulfated heparinase was measured to be 70 kDa, and the isoelectric point measured by IEF was 8.7, which was much smaller than Flavobacterium heparinase II of 84.1 kDa, and Flavobacterium hepa of 71 kDa. Very similar to the molecular weight of Linease III ( FIG. 5 ).

Heparin degrading enzyme has a higher molecular weight and slightly higher isoelectric point than Flavobacterium heparinase I. Heparan sulfate degrading enzyme has a similar molecular weight to flavobacterium heparinase III but shows a significant difference in isoelectric point. Indicates that heparin degrading enzyme and heparan sulfate degrading enzyme are completely different from known enzymes.

<2-2> Optimal reaction pH investigation

In order to investigate the optimum reaction pH of the novel heparinase of the present invention, the following experiment was performed. Specifically, heparin-degraded heparinase used 50 mM sodium phosphate buffer at various pH values ranging from pH 5.8 to 8.0, and heparan sulfate-dissolved heparinase used 50 mM sodium phosphate buffer solution at pH 6.0 to pH 8.5. The enzyme reaction was measured in the same manner as in Example <1-2> to determine the optimum pH.

As a result, heparin-degraded heparinase gradually increased the enzyme activity between pH 6.0 and pH 7.0, and showed the maximum enzyme activity at pH 7.0, and gradually decreased the pH at higher pH ( Fig. 6 ). The optimum pH of the heparin degrading heparinese was slightly lower than the previously reported enzymes, and these properties were also different from the existing enzymes.

In addition, heparan sulfate-decomposed heparinase showed an increase in enzyme activity gradually between pH 6.0 and pH 7.25, but showed maximum enzyme activity at pH 7.0 to 7.25, whereas enzyme activity was the maximum enzyme activity at pH 6.0 and above pH 8.4. Decreased to below 10% ( FIG. 7 ). The optimal pH of the heparan sulfate decomposed heparinase was similar to that of the conventional Flavobacterium heparinase II but was completely different from heparinase III. These properties also indicate that the enzyme is different from the existing enzyme.

<2-3> Stability investigation with temperature

In order to investigate the stability of the novel heparinase of the present invention to the temperature, the enzyme was preliminarily 3 minutes in 50 mM sodium phosphate buffer (pH 7.0) at each temperature without substrate at various temperatures from 25 ℃ to 60 ℃ After the reaction, the substrate was added to determine the optimum temperature by measuring the enzyme reaction in the same manner as in Example <1-2>.

As a result, heparin decomposed heparinase was found to be very stable enzyme activity from 25 ℃ to 50 ℃ and the activity was rapidly reduced at higher temperatures ( Fig. 8 ). The optimum temperature of the heparin-decomposed heparinase was about 10-15 ° C higher than that of Flavobacterium heparin heparinase, which is 35 ° C.

In addition, heparan sulfate decomposed heparinase exhibited the highest activity at 40 ° C and no activity above 50 ° C. The optimum reaction temperature of the enzyme was 45 ° C., which was slightly higher than the optimum reaction temperature of F. heparinum heparinase II, 40 ° C., and was different from Flavobacterium heparinase III. It was a similar temperature. This feature is thought to be similar to Heparinase III, and because it is reactive even at relatively high concentrations, it is thought that the reaction product can be easily produced in large quantities.

<2-4> Effect of Divalent Cation

In order to investigate the effect of divalent cations on the enzyme activity of the novel heparinase of the present invention, various divalent cations were added to the enzyme at a final concentration of 100 μM and enzymes were prepared in the same manner as in Example <1-2>. Activity was measured and the results are shown in Tables 3 and 4 below.

Effect of Divalent Cation on Enzyme Activity of Heparin-Degraded Heparinase Divalent cation Concentration (mM) Relative activity ^a (%) EDTACo ²⁺ Cu ²⁺ Ni ²⁺ Ba ²⁺ Zn ²⁺ Pb ²⁺ Mg ²⁺ Ca ²⁺ Cd ²⁺ Mn ²⁺ 0.10.10.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 100203.5222.8 114.1 106.3 111.1 108.5 93.9 83.3 99 77.7 90.5

a: 0.03 unit of enzyme activity was considered 100%.

Effect of Divalent Cation on Enzyme Activity of Heparan Sulfate Heparinase Divalent cation Concentration (mM) Relative activity ^a (%) Mg ²⁺ Ca ²⁺ Ni ²⁺ Co ²⁺ Cu ²⁺ Pb ²⁺ Mn ²⁺ Zn ²⁺ Cd ²⁺ Ba ²⁺ EDTA 0.10.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 100106 111 88 104 8 22 91 73 93 107 96

a: 0.03 unit of enzyme activity was considered 100%.

remindTABLE 3As shown in the figure, the enzymatic activity of heparin decomposed heparinase was determined by EDTA, Co²⁺, Cu²⁺, Ni²⁺, Ba²⁺And Zn²⁺Increased by Pb²⁺, Mg²⁺, Ca²⁺, Cd²⁺And Mn²⁺Decreased by. From these results, divalent cations seem to affect enzyme activity.

Also, theTable 4As shown in, heparan sulfate decomposed heparinase is a Ca²⁺, Mg²⁺And Ba²⁺Slightly increased enzyme activity²⁺And Pb²⁺Enzyme activity was inhibited by strong. Cu²⁺Has been reported to inhibit the activity of all heparinases (J. Biol. Chem, 267: 24347-24355, 1992), the enzymatic activity of the heparan sulfate decomposed heparinase of the present invention²⁺Inhibited by These results indicate that amino acids such as cysteine are present in the active site of the enzyme, so that chelate binding of divalent metal ions is easy and the size of the active site is Cu.²⁺It is considered that it is because is suitable to combine. Ca²⁺and Ba²⁺Although it has been reported to inhibit the activity of Flavobacterium heparinase II and III, it promoted the activity of heparan sulfate decomposition heparinase of the present invention.

<2-5> Substrate Specificity Investigation

In order to investigate the substrate specificity of the novel heparinase of the present invention, it is carried out after the reaction of heparinase and various substrates under the optimum reaction conditions of the enzymes obtained in Examples <2-2> to <2-4>. Example Enzyme activity was measured in the same manner as in <1-2>, and the results are shown in Tables 5 and 6 below. At this time, the substrates used to investigate the substrate specificity of sulfated oligosaccharides of heparin-decomposed heparinase were heparin (pork intestinal mucosa, 12 kDa), heparan sulfate (pork intestinal mucosa, 11.7kDa), chondroitin sulfate A (Bronchial bronchus), chondroitin sulfate B (bovine intestinal mucosa), chondroitin sulfate C (shark cartilage), and acaran sulfate (snail) were used. In addition, the substrates used to investigate the specificity of heparan sulfated heparinase for sulfidated polysaccharides include heparin, heparan sulfate, acaran sulfate, desulfurized acaran sulfate, and N-sulfoacharan sulfate. , Chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C were used. Acharan sulfate, de-O-sulfated acharan sulfate and N-sulfoacharan sulfate in the substrate were used for measuring the enzyme activity because of low solubility. The remaining substrates were used in 1 mg increments.

Substrate Specificity of Heparin Degrading Heparinase temperament Relative activity ^a (%) Heparin Degradation Heparinase of the Invention Rhine Flavobacterium derived HEPA Izu ^b (Flavobacterial heparinase) I II III Heparin heparan sulfate chondroitin sulfate A chondroitin sulfate B chondroitin sulfate C acharan sulfate 10017.10000 100 100 10025 167 900 0 00 0 00 0 00 100 ^c 0

a: The activity for heparin (or heparan sulfate in Flavobacterium-derived Heparinase III) was set to 100% by substrate.

b: J. Biol. Chem ., 267: 24347-24355, 1992; Crit. Rev. Biochem. Mol. Biol., 30: 387-444, 1995; J. Biol. Chem ., 271: 11750-11755, 1996; Results from Glycobiology, 8: 869-877, 1998

c: unpublished results

Substrate Specificity of Heparan Sulfate Heparinase temperament Relative activity ^a (%) Of the present invention Heparan Sulfate Decomposition Flavobacterium-derived heparinaseFlavobacterialheparinase)^b I II III Heparin Heparan Sulfate (porcine) Heparan Sulfate (bovine) Desulfurized Acaran Sulfate N-Sulfocaran Sulfate Chondroitin Sulfate A Chondroitin Sulfate B Chondroitin Sulfate C 100262 6100 32000 100 100 0nd ^c nd nd30 172 1000 100 ^d 0nd nd ndnd nd nd0 0 00 0 00 0 0

a: Activity for heparin (or heparan sulfate in Flavobacterium-derived heparinase III) was set to 100% as a substrate.

b: J. Bio. Chem ., 267: 24347-24355, 1992; Crit. Rev. Biochem. Mol. Biol., 30: 387-444, 1995, J. Biol. Chem ., 271: 11750-11755, 1996; Data from Glycobiology, 8: 869-877, 1998)

c: not determined.

d: unpublished data

As a result, as shown in Table 5 , heparin-decomposed heparinase did not show activity against substrates other than heparin and heparan sulfate, and showed very high substrate specificity for heparin rather than heparin sulfate. eliminase). The heparin-decomposed heparinase is a heparin containing a trisulfated sequence-> 4) -α-D-GlcNS6S (1-> 4) -α-L-IdoA2S (1->) which is a basic structure of heparin. Heparan sulfate was also degraded by 38% compared to heparin-degrading activity, because some of the structure of heparan sulfate was similar to heparin, whereas the heparin-degraded heparin Nezu hardly decomposed acaran sulfate, therefore, the bacteroid-derived heparinase of the present invention has a much higher substrate specificity for heparin than heparan sulfate or acaran sulfate.

In addition, as shown in Table 6 , heparan sulfate decomposed heparinase contains aca containing de-2-O-sulfated acharan sulfate and N-sulfoacharan sulfate. Reacted slowly to the lan sulfate derivatives. On the other hand, the enzyme is an unsulfated sequence comprising heparan sulfuric acid, which provides unsaturated unsulfated disaccharides (ΔUA-GlcNAc) and monosulfated disaccharides (ΔUA-GlcNS and ΔUA-GlcNAc6S). -> 4) -α-D-GlcNAc (1-> 4) -β-D-GlcA (1->,-> 4) -α-D-GlcNS (1-> 4) -β-D-GlcA ( Better acting on 1->and-> 4) -α-D-GlcNAc6S (1-> 4) -β-D-GlcA (1-> Heparinase of the present invention is trisulfide which provides unsaturated disaccharides The substrate specificity was also shown for heparin including the trisulfated sequences-> 4) -α-D-GlcNS6S (1-> 4) -α-L-IdoA2S (1->), which is a heparan sulfate decomposition of the present invention. It has been shown that heparinase has broad substrate specificity for iduronic acid or glucuronic acid including binding and for highly sulfated domains. Izu is N-sulfo acaran sulfate It also showed weak substrate specificity for desulfurized acaran sulfate, which indicates that the heparinase of the present invention is-> 4) -α-D-GlcNS (1-> 4) -α-L-IdoA2S (1-> and -> 4) -α-L-IdoA (1-> also act on binding. Flavobacterium heparinase II used as a comparative group also showed activity against all these substrates, among which-> 4) Acaran sulfate containing α-D-GlcNAc (1-> 4) -α-L-IdoA2S (1-> bond) is a hepatic agent of the present invention, despite being an excellent substrate for Flavobacterium heparinase II. Ran sulfate disintegrated heparanases showed no substrate specificity for acaran sulfate.

Example 3 Effect of NaCl Concentration on Heparin Degraded Heparinase

In order to investigate the effect of NaCl concentration on the heparin decomposed heparinase of the present invention, various concentrations of NaCl from 0.1 to 1.0 M were added to the enzyme reaction solution, and the enzyme activity was measured in the same manner as in Example <1-2>. Measured.

As a result, the enzyme activity gradually increased at NaCl concentrations from 100 mM to 500 mM, indicating the optimum enzyme activity at 500 mM, and the enzyme activity gradually decreased at higher concentrations ( FIG. 9 ). It can be seen from this that NaCl affects the stabilization of heparinase.

Example 4 Effect of Amino Acid Conversion Reagent on the Activity of Heparan Sulfate Decomposed Heparinase

In order to investigate the effect of amino acid modifying agents on the heparinase of the present invention that specifically degrades heparan sulfate, phenylmethylsulfonyl fluoride (PMSF), diethyl para, shown in Table 7 -nitrophenyl phosphate (diethyl p -nitrophenyl phosphate), TPCK , dl- ditti haute ray Tall (dl-dithiothreitol), Io FIG acetate (iodoacetic acid), carbodiimide (carbodiimide), butane diol (butanediol), p-chloro Mercury phenyl sulfonic acid (p-chloromercuryphenyl sulfonic acid), TLCK , and 2-mercaptoethanol Merlin (2-mercaptoethanol) was measured the enzymatic activity in the same manner followed by the addition of such as the above example <1-2> and the results table 7 is shown.

Effect of Amino Acid Conversion Reagent on Heparan Sulfate-Decomposed Heparinase Amino Acid Conversion Reagent Concentration (mM) Relative activity ^a (%) Control PMSF diethyl para-nitrofanyl phosphate TPCKdl-dithiotratol iodoacetic acid carbodiimbutanediol p-chloromercurifanyl sulfoneTLCK2-mercaptoethanol 0.10.10.10.10.10.10.10.10.050.050.05 100959894209969594071193

a: 0.03 unit of enzyme activity was considered 100%.

In Table 7 , the serine protease inhibitors such as PMSF and the cysteine protease inhibitors such as iodoacetic acid are used for the enzymatic activity of heparan sulfate decomposed heparinase of the present invention. Little effect. On the other hand, other cysteine-derived reagent p -chloromercuryfanylsulfonate significantly inhibited the activity of the enzyme, while dithiotratol and mercaptoethanol increased the enzyme activity. In addition, trypsin inhibitor TPCK did not affect the enzyme activity, while another inhibitor TLCK slightly inhibited the enzyme activity.

<4-2> Amino Acid Composition Analysis

In order to analyze the amino acid composition of the heparan sulfate decomposed heparinase of the present invention, the enzyme was first decomposed with 6 N hydrochloric acid at 155 ^° C. for 1 hour to form a derivative using phenylisothiocyanate. The amino acid derivative thus formed was analyzed by an amino acid analyzer (Amino acid Analyzer, Applied Biosystem model 420/130 Derivatizer) and the results are shown in Table 8 below.

Amino Acid Composition Analysis amino acid pmol Mol% CysAsx ^a Glx ^a SerGlyHisArgThrAlaProTyrValMetIleLeuPheTrpLys ND ^b 368.56439.19127.62162.00110.28173.72196.72278.25214.34209.24294.53113.59158.99378.59321.02ND ^b 423.15 0.009.2811.063.214.082.784.384.967.015.405.277.422.864.009.548.090.0010.66 gun 3,969.79 100.00

Asx ^a ; Asparagine and Aspartic Acid

Glx ^a ; Glutamine and Glutamic Acid

b; Not determined

As shown in Table 8 , the amino acid composition analysis of heparan sulfate-dissolved heparinase showed that the enzyme contains a large amount of lysin, which is consistent with the pI value of 8.7, which results in flavobacterium hepa. It was slightly lower compared with the pI value of Linease 8.9-10.1 ( J. Biol. Chem ., 267: 24347-24355, 1992).

<4-3> Internal Amino Acid Sequence Analysis

In order to determine the internal amino acid sequence of heparan sulfate decomposed heparinase of the present invention, the enzyme was digested with trypsin to analyze the internal amino acid sequence of the peptide, and the results are shown in Table 9 below.

Internal Amino Acid Sequencing enzyme Internal amino acid sequence Sequence Listing Homology (%) Heparan Sulfate Heparinase Flavobacterium Heparinase II Flavobacterium Heparinase III ---- MADEALQHTFFA ---- 329 ---- YKDEYLNYEFLK ---- 98 ---- MADKALVHQFQP ---- 123 4873

The N-terminus of the heparan sulfate decomposed heparinase of the present invention was blocked and could not be sequenced, which is consistent with that of the N terminus of Flavobacterium heparinase.

As a result of analyzing the internal amino acid of the heparan sulfate decomposed heparinase of the present invention, it was composed of the amino acid of SEQ ID NO: 1 . It shows 48% and 73% homology with the amino acid sequence of Flavobacterium II as depicted in SEQ ID NO: 2 and the amino acid sequence of Flavobacterium III as depicted in SEQ ID NO: 3 , respectively, for Flavobacterium III. Had a higher similarity.

<4-3> Kinetic constant determination

Of the present invention In order to determine the kinetic constants of heparan sulfate decomposed heparinase, enzyme activity was measured in the same manner as in Example <1-2> with a substrate concentration of 200, 400, 600, 1,000, 2,000 and 3,000 µg. After that, the Micheles-Menten constants were determined using a Hanes plot.Table 10and10Shown in

Determination of Heparan Sulfate Kinetic Constants enzyme Vmax (μM) Km (μM) Heparin Heparan Sulfate Heparin Heparan Sulfate Heparan Sulfate Heparinase Flavobacterium Heparinase I ^a Flavobacterium Heparinase II Flavobacterium Heparinase III 38.2 58.4219- ^b 16.7 28.6- 141 90.5 15.317.8 -57.4 11.2- 29.4

a: J. Biol. Chem. , 267: 24347-24355, 1992.

b: not determined.

remindTable 10As shown in the figure, Km and Vmax for heparin of the heparan sulfate decomposed heparinase of the present invention were 9.05 × 10, respectively.^-5M, 38.2 μmol / min / mg, and Km and Vmax for heparan sulfuric acid were 1.53 × 10, respectively.^-5M, measured at 58.4 μmol / min / mg. This is a high value in comparison with the Km and Vmax values of Flavobacterium II, indicating that the heparan sulfate-dissolved heparinase of the present invention has superior substrate specificity for heparan sulfate compared to the conventional heparinase III. .

Heparan sulphate heparinase has a Vmax greater than Flavobacterium heparinase II and smaller than Flavobacterium heparinase III. However, Flavobacterium heparinase III did not degrade heparin at all, whereas heparin sulfate heparin sulfate isolated in this study did not degrade heparin. This feature is thought to be a new enzyme completely different from the existing heparanases.

As described above, the present invention provides two novel heparinases, which are isolated and purified from human enteric microorganisms bacteroids Stechoris HJ-15, and specifically decompose heparin and heparan sulfuric acid, respectively, and their It is about a use. The novel heparinase of the present invention has excellent substrate specificity and shows stable enzymatic activity, and thus can be usefully used to reveal oligosaccharide production techniques such as heparin, heparan sulfuric acid, acaran sulfate, and structural features thereof, and to prepare low molecular weight heparin. And at high doses of heparin can be used to neutralize it.

Claims (15)

A novel heparinase, a GAG degrading enzyme isolated and purified from Bacteroides stercoris HJ-15 (Accession No .: KCCM-10096). The novel heparinase according to claim 1, wherein the GAGs are heparin, heparan sulfuric acid, and acaran sulfuric acid. The novel heparinase of claim 1, wherein the heparin is specifically degraded and has a molecular weight of 48 kDa. According to claim 3, Optimum NaCl concentration in the enzyme reaction of heparinase is 200-900 mM, optimum pH 6.0-7.5, and optimum reaction temperature 35-55 ° C. According to claim 3, Heparinase is characterized in that the enzyme activity is promoted by a divalent cation selected from the group consisting of EDTA, Co ²⁺ , Cu ²⁺ , Ni ²⁺ , Ba ²⁺ and Zn ²⁺ New heparanaise. The novel heparinase of claim 1, wherein the heparan sulfuric acid is specifically decomposed and has a molecular weight of 70 kDa. The novel heparinase of claim 6, wherein the optimum pH of the enzyme reaction of heparinase is 6.0-7.25 and the optimum reaction temperature is 40-45 ℃. The novel heparinase of claim 6, wherein the heparinase is promoted by a divalent cation selected from the group consisting of Mg ²⁺ , Cu ²⁺ and Co ²⁺ . The novel heparinase of claim 6, wherein the heparinase is promoted by a reducing agent selected from the group consisting of dithiothreitol and 2-mercaptoethanol. . The novel heparinase of claim 6, wherein the heparinase contains a large amount of lysine in the amino acid composition and comprises an internal amino acid sequence represented by SEQ ID NO: 1 . 1) culturing the bacteroids stericis in a suitable medium; 2) recovering the cultured cells and preparing a cell extract; And 3) Purifying the cell extract by chromatography to obtain an enzyme active fraction Method of producing a novel heparinase of claim 1, characterized in that consisting of steps. The process of claim 11 wherein the chromatography of step 3 is QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, CM-Sephadex C-25 and high A method for producing one or more of the columns selected from the group consisting of Hi-Trap SP. The novel heparinase of claim 1 used in the preparation and structural analysis of GAG oligosaccharides. The novel heparinase of claim 1 used for the production of low molecular weight heparin. The novel heparinase of claim 1, which is used for heparin neutralization during heparin overdose.