KR20020046294A - Two novel acharan sulfate lyases which can specifically degrade acharan sulfate, a preparing method and a use thereof - Google Patents

Abstract

PURPOSE: Novel acharan sulfate lyases specifically degrading acharan sulfate, a preparing method and a use thereof are provided, therefore the acharan sulfate lyase having improved substrate specificity and stability can be produced and it can be useful in producing acharan sulfate oligosaccharides inhibiting the metastasis of cancer. CONSTITUTION: The acharan sulfate lyase capable of degrading glycosaminoglycans(GAG) is isolated from Bacteroides stercoris HJ-15, wherein glycosaminoglycans(GAG) are acharan sulfate, heparin and heparan sulfate; the acharan sulfate lyase has 82,500 Da of molecular weight and optimal pH of 7.0 to 7.2 and optimal temperature of 42 to 45 deg. C; and the activity of enzyme is increased by Mg2+ or Mn2+ and inhibited by Cu2+ or Ni2+. The method for producing the acharan sulfate lyase 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

Two novel acharan sulfate lyases which can specifically degrade acharan sulfate, a preparing method and a use approximately}

The present invention provides a novel acaran sulfate having an acaran sulfate degrading ability derived from bacteroid Bacteroides stercoris HJ-15, which produces a glycosaminoglycan (abbreviated as "GAG") degrading enzyme. The present invention relates to a degrading enzyme, a method for producing the same, and a use thereof, and specifically, a novel acaran sulfate degrading enzyme that is isolated and purified from a bacteroid St. corris HJ-15 strain, which is a human intestinal microorganism, to specifically decompose acaran sulfate. It relates to a manufacturing method and use.

Glycosaminoglycans are structurally diverse and complex through partial changes in the biosynthesis process. GAGs have a basic backbone that is partially sulfided by the repeated uronic acid and glucosamine, and these complex chemical structures allow various kinds of biological activity. 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 cuttlefish (Karamanos et al ., Int. J. Biochem ., 23, 67-72, 1991), and acaran sulfate is found to be abundant in African king snails. Acaran sulfate is structurally similar to heparin and heparan sulfuric acid, and the structure of the disaccharide → 4) -α-D-GlcNAc (1 → 4) -α-L-IdoA2S (1 → is repeated. Sulfuric acid GAG is connected by a 1 → 4 bond in which glucosamine and uronic acid groups are repeated.Heparan sulfuric acid has a single sulfation of disaccharides N-acetyl-D-glucosamine and D-glucuronic acid. Heparin has a monosulfated structure, whereas heparin has a trisulfated structure mainly on the disaccharides N-sulfoyl-D-glucosamine and L-iduronic acid.

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 to 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 clotting.

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 low-density lipoproteinases and triglycerides, which are lipolytic enzymes, into plasma cells. 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 such, 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 drugs, and recently, tonic drinks. Hiyal uronic acid is also widely used as an additive in cosmetics. Heparin is widely used as a glycan drug in the clinic, and heparin is widely used as a anticoagulant for thrombosis, after surgery, kidney dialysis, artificial blood vessels, extracorporeal circulation, and the like. Recently, in addition to anticoagulant action, various biological activities such as complement activity of immune system, inhibition of peripheral angiogenesis, interaction of basic fibroblast growth factor, and antiviral action have been reported.

Acaran sulfate exhibits a variety of biological activities, including the above-mentioned bioactivity, and is characterized by the low side effects of anticoagulants that are structurally modified (eg deacetylation, sulfated, new oligo GAG). It is possible that sulfated oligosaccharides of acaran sulfate provide the structural features necessary for binding to fibroblast growth factors and thus play an important role in cell growth and differentiation. In fact, the compound that was sulfated after deacetylation of -NH ₂ is expected to have a 6-fold increase in cell growth compared to the control group. Acaran sulfate is also ideal as a model compound for reactions using sulfate-based transferases because of its simple structure of repeating disaccharides. In addition, acaran sulfate is very effective for liver function and menopausal disorders, including osteoporosis.

Although GAGs, including acaran sulfate, exhibit various pharmacological effects as described above, their molecular weight is too large, making them difficult to handle and causing side effects. Therefore, it is necessary to prepare low molecular weight GAGs. This has risen greatly. In fact, low molecular weight heparin is being used to overcome the side effects and disadvantages of heparin. This low molecular weight heparin is involved in not only anticoagulant action but also receptor activation of fibroblast growth factor.

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 the method, the fractions of molecular weight of 3,000 Daltons or less are first removed, and then the fragments containing 10-16 sugars or less are removed from the final product, leaving only the heparin fractions of molecular weight of 7,000 Daltons or more. Homogenization of this final mixture produced heparin with the desired antithrombin activity while reduced anticoagulant activity.

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 a single bond. Heparin is a degrading enzyme. Degrading enzymes I, II, III and chondroitin degrading enzymes A, B, and C. Heparin degrading enzyme I acts specifically to decompose heparin, → 4) -α-D-GlcNS (6S or OH) (1 → 4) -α-L-IdoA2S (1 →). There is a clinical trial using heparin degrading enzyme I for the purpose of neutralizing excess heparin in the treatment of heparin (Zimmerman, U.S. Patent Application No. 5,262,325), which the US FDA uses to measure the concentration of heparin. Tejidor et al., Thrombo. Haemost ., 69: 866, 1993. Heparin lyase II is -4) -α-D-GlcNS (6S or OH) (1 → 4) -α-L-IdoA ( 2S or OH) or -β-D-GlcA (→ 1 to decompose heparin and acaran sulfate) Heparin degrading enzyme III is the main bond of heparan sulfate → 4) -α-D-GlcNS (or Ac (1 → 4) -α-L-GlcA (or → IdoA) (specifically acts on 1 → to degrade heparan sulfuric acid (Steffen E., Critic. Rev. in Biochem. And Mol. Biol. , 30 (5): 387-444, 1995).

Most of the above-described heparin degrading enzymes have been isolated from Flavobacterium heparinum, Bacillus sp . (Bellamy et al., US Patent Application No. 5,145,778) and heat-resistant strains other than Flavobacterium heparinum . Heparin degrading enzymes have also been found in the anaerobic strain Bacteroides sp . Bacteroides heparinolyticus ( Bacteroides heparinolyticus) found in the oral cavity was first reported by the genus anaerobic bacteroid (Gesner et al., J. Bacteriol ., 81: 595-604, 1961). Et al. , J. Clin.Microbiol ., 26: 1070-1071, 1988), Bacteroides ovata and Bacteroides thetaiotaomicron in the intestine have been reported (Riley). , TV et al., Microbios Lett., 25: 141-148, 1984; Riley, J. Clin. Pathol., 40: 384-386, 1987). Thus enteric steroid night Omicron Theta Tie Ota, Bacillus Obata (B. ovatus) that live in the human, Bacillus Miss Uni-Four (B. uniformis) and Bacillus hotel's heparin degrading enzymes from the same strain and koriseu (B. stercoris) Has been reported (Riley TV, Microb. Lett., 25: 141-149, 1984), but there have been no reports of purifying acaran sulfate enzyme. Therefore, the study of new acaran sulfate degrading enzyme will be useful for the structural analysis of GAGs such as acaran sulfate and the preparation of low molecular weight acaran sulfate.

The present inventors also recently described bacteroids stericis, a microorganism that synthesizes GAG degrading enzymes in the human intestine. HJ-15 was isolated and identified and patented and registered (Korean Patent Registration No. 10-0257168). The strain decomposes GAG such as acaran sulfate, heparin, heparan sulfate, and chondroitin sulfate.

Therefore, the present inventors are microorganisms producing GAG degrading enzymes, and bacteroid stericis isolated and identified by the present inventors in human intestines. Purification of two new acaran sulfate enzymes specific for acaran sulfate from HJ-15 (KCCM-10096) to reveal their biochemical properties, and the acaran sulfate enzyme was used to analyze the structure of GAGs and low molecular weight aka. The present invention has been completed by revealing that it can be usefully used to prepare lan sulfuric acid.

An object of the present invention is a novel acaran sulfate degrading enzyme that specifically degrades purified acaran sulfate separated from human intestinal microorganisms, which can be usefully used for structural analysis of GAGs and production of low molecular weight acaran sulfate, It is to provide a manufacturing method and use.

Figure 1 shows the elution profile of the cell extract of bacteroids Sterchoris HJ-15 purified by CM- Sephadex C-50 ion exchange chromatography in the present invention,

●: Acaran Sulfase

△: heparin degrading enzyme

-Protein absorbance at 280 nm

Figure 2 shows the elution profile of the acaran sulfate degrading enzyme fraction obtained in Figure 1 purified by SP Sephadex column chromatography,

●: Acaran Sulfase

□: protein absorbance at 280 nm

Figure 3 shows the SDS-PAGE results of the acaran sulfate degrading enzyme isolated and purified from Figure 2 ,

Lane 1: acaran sulfate degrading enzyme 1

Lane 2: acaran sulfate degrading enzyme 2

Lane 3: standard molecular weight

Figure 4 shows the effect of pH on acaran sulfate degrading enzyme isolated and purified in the present invention,

●: Acaran Sulfate Degrading Enzyme 1

■: Acaran Sulfate Enzyme 2

Figure 5 shows the effect of temperature on the acaran sulfate degrading enzyme isolated and purified in the present invention.

●: Acaran Sulfate Degrading Enzyme 1

■: Acaran Sulfate Enzyme 2

In order to achieve the above object, the present invention provides a novel acaran sulfate degrading enzyme that specifically decomposes and refines acaran sulfuric acid separated and purified from human intestinal microorganism bacteroid Stercoris HJ-15, and a method of preparing the same. do.

The present invention also provides the use of the novel acaran sulfate degrading enzyme for the production and structural analysis of GAGs or the production of low molecular weight acaran sulfate.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel acaran sulfate degrading enzyme that specifically decomposes and refines acaran sulfuric acid separated and purified from bacteroids Stericis HJ-15, which is a human intestinal microorganism, and a method for producing the same.

Used in the present invention The bacteroids were identified and identified as follows. After culturing the intestinal flora of humans and rats in anaerobic condition, the cells were obtained from the cell solution, the strains having the activity of decomposing heparin, acaran sulfate, and chondroitin from the cells were identified and identified according to the general strain discrimination method. manual of systemic bacteriology, 1991). As a result, the strain is a bacteroid (BacteroidIt was identified as Bacterial Stercoris HJ-15, and it was deposited on February 19, 1997 by the Korean spawn association of Korea (Accession No. KCCM-10096). GAG degrading enzyme of bacteroids Stericis HJ-15 shows the activity of acaran sulphate as lyase, so it can be used for the production of low molecular weight acaran sulphate. Furthermore, GAG degrading derived from Flavobacterium In addition to exhibiting similar properties to enzymes, it is produced by microorganisms in the intestine.

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

The production method of the novel acaran sulfate degrading enzyme 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) the cell extract is purified by chromatography to obtain enzymatic activity.

In more detail, the step 1, in order to separate the acaran sulfate enzyme from the bacteroid Stercoris HJ-15 strain, the strain first contained trypsin medium containing heparin instead of glucose (glucose) (tryptic soy broth) and incubated in anaerobic conditions ( Can. J. Microbiol., 44, 423-429, 1998). Cells cultured in step 2 are recovered by centrifugation, washed with buffer and suspended. Ultrasonic 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 sulfuric acid is measured for all fractions. Through this process, the present inventors secured three fractions (a, b, c) representing enzyme activity (see FIG. 1 ).

Among them, the fraction a showing the acaran sulfate activity was dialyzed and purified by a hydroxyapatite column and an SP-sepadex C-25 column to specifically decompose acaran sulfate activity. Fractions were obtained (see Table 1 ). The activity of the acaran sulfate lyase of fraction a was divided into two parts in the SP-Sephadex C-25 column, and the first leaked fraction was abbreviated as acharan sulfate lyase I (hereinafter referred to as "ASL1"). And the second leaked fraction was named acharan sulfate lyase II (hereinafter abbreviated as "ASL2").

The present invention also provides the biochemical properties of the acaran sulfate degrading enzyme isolated and purified as described above.

As a result of examining substrate specificity of GAGs of acaran sulfate enzymes ASL1 and ASL2 of the present invention, both acaran sulfate enzymes had not only acaran sulfate but also heparin and heparan sulfate (see Table 3 ). . When the heparin degrading activity of ASL1 and ASL2 of the present invention was regarded as 100%, the activity for acaran sulfate was 549.5 and 339%, respectively, and for heparan sulfuric acid was 128 and 121.7%, respectively.

The acaran degrading enzymes ASL1 and ASL2 seemed identical in appearance on SDS-PAGE, and the molecular weights were all measured at 82,500 Daltons (see FIG. 3 ).

As a result of investigating the optimum pH and temperature during the enzymatic reaction of the acaran sulfate degrading enzyme of the present invention, the enzyme increased the enzyme activity between pH 6.5 and 7.6 and showed the optimum enzyme activity at pH 7.2 (see FIG. 4 ). Optimum enzyme activity was observed at 45 ° C. and activity was drastically decreased at 50 ° C. or higher and completely inhibited at a temperature of 60 ° C. or higher (see FIG. 5 ).

The acaran sulfate degrading enzyme of the present invention was inhibited by most divalent cations except Mn ²⁺ and Mg ²⁺ (see Table 4 ), and the activity was increased when TLCK and PCMS modifying histidine and cysteine groups were added. Although strongly inhibited, the activity was increased by reducing agents such as dl- dithiothreitol (DDT) or 2-mercaptoethanol (ME) (see Table 5 ).

The Km and Vmax values of ASL1 for acaran sulfate were 28.1 μg / ml and 65.0 μmole / min / mg, respectively, and for ASL2, the Km and Vmax values were calculated to be 42.4 μg / ml and 107.6 μmole / min / mg, respectively. It was confirmed that the acaran sulfate degrading enzyme of Acaran sulfate was very excellent substrate specificity and resolution.

In addition, the present invention provides a use of the novel acaran sulfate degrading enzyme for the production and structural analysis of GAGs or low molecular weight acaran sulfate.

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. In particular, acaran sulfate may be an anticoagulant with less side effects when structurally modified (eg, deacetylation, sulfate, new oligo GAG), and sulfated oligosaccharides of acaran sulfate are fibroblast growth factors. It provides the structural features necessary for binding to and plays an important role in cell growth and differentiation. Acaran sulfate is also ideal as a model compound for reactions using sulfate-based transferases because of its simple structure of repeating disaccharides. In addition, acaran sulfate is very effective for liver function and menopausal disorders, including osteoporosis.

Therefore, the novel acaran sulfate degrading enzyme of the present invention is more stable and excellent substrate specificity, so the preparation and structural analysis or basic fibroblast growth factor of acaran sulfate showing the physiological activity as described above (basic fibroblast growth) It can be useful also in the production of acaran sulfate oligosaccharides that can inhibit the metastasis of cancer cells by inhibiting neovascularization in combination with a factor).

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 Acaran Sulfate Enzyme Isolated from Intestinal Bacterial Bacterial Stecoris HJ-15

In order to separate acaran sulfate degrading enzyme, in the present invention, Bacterial Stecoris HJ-15 (Accession No .: KCCM-10096) which produces an enzyme which decomposes GAGs isolated from human intestine by the present inventors Used.

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

The bacteroids Stechoris HJ-15 contains heparin (0.15 g / l), 0.01 w / v% sodium thioglycolate and 0.1 w / v under anaerobic conditions containing 90% nitrogen and 10% carbon dioxide. Incubated at 37 ° C. in 100 L tryptic soy broth containing% ascorbic acid. The cells in the late exponential phase (11-12 hours) were centrifuged at 5000 ° C. for 30 minutes at 4 ° C., and the cell pellet was physiologically containing 50 mM sodium phosphate (pH 7.0). Wash twice with saline. The cell precipitate is suspended in 600 ml of Buffer A (50 mM sodium phosphate buffer, pH 7.0), and 30 ml of the cell suspension is used for 1 second at 80% output using an ultrasonic crusher (ultrasonic processor, Eyela Co.). Crushed for 30 minutes at intervals. Cell debris was removed by centrifugation at 4 ° C. and 18000 rpm for 60 minutes to prepare cell extracts. All examples below were performed at 4 ° C. unless specifically noted.

<1-2> Non-Interacting Column Chromatography

600 ml of the cell extract obtained from Example <1-1> was passed through a 5 × 40 cm QAE cellulose column that was preequilibrated with Buffer A, and eluate of acaran sulfate degrading enzyme in effluent. The column was washed with the same buffer until no activity appeared. The fraction passed through the column was passed through a DEAE cellulose column (5 × 30 cm), again filled with buffer A, and the column was washed with the same buffer until no activity of acaran sulfate lyase appeared. The non-interactive eluate that passed through the column was collected and the total amount of buffer used was 1800 mL.

<1-3> CM-Separdex C-50 Column Chromatography

The eluate collected from Example <1-2> was passed through a CM-Sephadex C-50 column (3 x 30 cm) pre-filled with Buffer A and the column was quenched with 1000 mL of the same buffer. After washing, KCl was dissolved in 2 L of buffer solution and a linear gradient of 0 to 0.6 M was used to elute the enzyme at a flow rate of 105 mL / h. The activities of heparin and acaran sulfate enzymes were examined in all eluted fractions, and three fractions (fraction a, fraction b and fraction c) representing the activities of the enzymes were collected ( Fig. 1 ). Among them, fractions b and c were considered to be similar to heparin degrading enzyme II / III and heparin degrading enzyme I derived from Flavobacterium heparinum because of their high activity against heparan sulfate and heparin, respectively. ( Table 1 ). Therefore, the present inventors further performed the following purification process after dialysis with buffer A fraction A, which shows a particularly strong enzyme activity against acaran sulfate, among the fractions.

Substrate Specificity of GAG Degradation Fraction in Bacterial Stecoris HJ-15 enzyme activation (%) Acaran sulfate Heparin Heparan sulfate Fraction a fraction b fraction c 48500 100100100 12126830

<1-4> hydroxyapatite ultrogel column chromatography

330 mL of fraction a obtained from <1-3> was passed through a hydroxyapatite Ultrogel column (2.5 × 10 cm) filled with buffer A. After washing the column with 500 ml of the same buffer, sodium phosphate buffer (pH 7.0) was mixed with 800 ml of buffer A, and the mixture was eluted at a flow rate of 120 ml / h by giving a linear concentration gradient from 50 to 400 mM. Fractions showing enzymatic activity were collected and dialyzed with 2 L of Buffer A.

<1-5> SP- Sephadex C-25 Chromatography

165 ml of the enzyme active fraction obtained from Example <1-4> was passed through 3 × 30 cm SP- Sephadex C-25 filled with buffer A and the unadsorbed protein was washed with 800 ml of the same buffer. After dissolving KCl in buffer A and giving a linear concentration gradient at a concentration of 0 to 0.5 M, the solution was eluted at a flow rate of 75 ml / h. Two fractions (ASL1, fractions 91-99; ASL2, fractions 118-120) showing acaran sulfate degrading enzyme activity were separated therefrom. The protein concentration, enzyme activity, substrate specificity, and the like of the purified and purified acaran sulfate degrading enzyme are thus shown in Table 2 below.

Purification of Acaran Sulfase process Total active (U ^a ) Total protein (mg) Inactive (U / mg Protein) Yield Fold Primary extract 198.1 7636.8 0.026 100 One CM-Sephadex C-50 Column Chromatography 137.5 39.2 3.51 69.4 135 Hydroxyapatite Ultrogel Column Chromatography 135.6 14.1 9.62 68.5 370 SP-Sephadex C-25 Column Chromatography 30.8 ^b 23.0 ^c 0.61 ^b 0.30 ^c 50.5 ^b 76.7 ^c 15.5 ^b 11.6 ^c 1942 ^b 2950 ^c

a; 1 unit (U) is the activity of an enzyme that forms 1 μmole disaccharide per minute

b; Acaran Sulfate I

c; Acaran Sulfate II

As shown in Table 2 above, the inactivation of the first outflowed fraction (ASL1) was 50.5 U / mg protein at 15.5% yield and the inactivation of the second outflowed fraction (ASL2) was 76.7 U / mg at 11.6% yield. It was a protein. The molecular weights of ASL1 and ASL2 of the present invention were measured to be 82.500 Daltons on SDS-electrophoresis ( FIG. 3 ).

Example 2 Measurement of Novel Acaran Sulfate Degrading Enzyme Activity against GAG

The activity of GAG degrading enzymes including acaran sulfate degrading enzyme was measured according to the method of the prior art (Linhardt, RJ, Current Protcols in Molecular Biology., 17.13.17-17.13.32, 1994). Adjust the UV spectrophotometer (Jasco Co. V-530) to 40 ° C, add 100 µg of substrate, 650 µL of 50 mM sodium phosphate buffer (pH 7.2) to 1 ml quartz cuvette, and equilibrate the temperature. Maintained. At this time, heparin, heparan sulfuric acid, chondroitin sulfate A, B, C, acaran sulfate, O-desulfurized acaran sulfate, N-sulfurized acaran sulfate was used as a substrate, the concentration of the substrate is shown in Table 3 below It was. 50 μl of the enzyme solution was added thereto, and the change in absorbance at 232 nm was measured for 3 minutes at 1 second intervals. The activity of the enzyme was calculated as the change in absorbance per minute, where the extinction coefficient of the product was 3800 M ^−1, and the enzyme activity of 1 U was 1 μmole per minute of non-reducing terminal of saturated uronic acid as 1 molecule of water was lost. Defined as conversion to unsaturated uronic acid.

Substrate Specificity of Acaran Sulfase temperament Substrate concentration (mg / ml) Relative Activity (%) ^a ASL1 (HepII-1) ASL2 (HepII-1) Flavobacterium-derived heparin degrading enzyme ^b I II III Heparin sulfuric acid (pork) Heparan sulfuric acid (small) Acaran sulfate O-desulfurized acaran sulfate N-sulfurized acaran sulfate Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C 111 0.1 0.10.1111 100128nd 549.5 0nd000 100121.7nd 339 0nd000 10030nd^c0 ndnd000 100172nd 100^dndnd000 0100nd 0 ndnd000

a; Relative activity when the activity for heparin (heparan sulfate in the case of Flavobacterium-derived heparin degrading enzyme III) is 100%

b; Data from Physiological reviews , 71, 481-539, 1991

c; Do not measure

d; Unpublished material

As a result, the two acaran sulfate degrading enzymes ASL1 and ASL2 isolated and purified from Example <1-5> had not only acaran sulfate but also heparin and heparan sulfate degradability ( Table 3 ). When the heparin degrading activity of ASL1 and ASL2 of the present invention was regarded as 100%, the activity for acaran sulfate was 549.5 and 339%, respectively, and for heparan sulfuric acid was 128 and 121.7%, respectively. However, there was no overall activity for de-O-sulfated acharan sulfate and chondroitin sulfate.

Example 3 Biochemical Characterization of Novel Acaran Sulfate Degrading Enzymes

<3-1> Molecular weight measurement

In order to determine the molecular weights of the two acaran sulfate degrading enzymes of the present invention isolated and purified from Example <1-5>, SDS-PAGE was carried out by the method described by Laemmli (Laemmli UK. Nature 227, 680). -685, 1970). Gels were stained with Coomassie brilliant blue R-250 solution and, if necessary, further silver salts.

As a result of SDS-PAGE, the molecular weights of both acaran sulfate degrading enzymes of the present invention were measured to be 82,500 Daltons ( FIG. 3 ).

<3-2> pH and Temperature Measurement of Optimum Reaction for Acaran Sulfate Degrading Enzyme Activity

The optimum pH of the novel acaran sulfate degrading enzyme activity of the present invention was determined using 50 mM sodium phosphate buffer solution at various pHs ranging from pH 6.0 to 8.5, and enzymes at various temperatures from 25 ° C. to 60 ° C. under standard conditions. The optimum temperature of enzyme activity was determined by measuring the activity of. The cuvette containing 50 μl of substrate and 50 mM sodium phosphate buffer (pH 7.2) was pre-heated for 1 minute at each temperature, and then the reaction was initiated by adding 10 μl of each of the acaran sulfate degrading enzyme of the present invention. . Enzyme reaction was measured for 3 minutes in the same manner as in Example 2.

As a result, the acaran sulfate degrading enzyme of the present invention increased the enzyme activity between pH 6.5 and 7.6 and showed the optimum enzyme activity at pH 7.2 ( FIG. 4 ). The optimum reaction temperature was 40 to 50 ° C. and showed the optimum enzyme activity at 45 ° C., and activity was rapidly reduced at 50 ° C. or higher and completely inhibited at a temperature above 60 ° C. ( FIG. 5 ).

<3-3> Effect of Divalent Metal Ion and Amino Acid Modification on Acaran Sulfate Degrading Enzyme Activity

In order to investigate the effect of divalent cations on the enzymatic activity of the novel acaran sulfate degrading enzyme of the present invention, various divalent cations were added to the enzyme at a final concentration of 50 to 100 μM and enzymes were prepared in the same manner as in Example 2. Activity was measured and the results are shown in Table 4 below. In addition, in order to investigate the effect of the amino acid modifying agent (enzyme activity) of the acaran sulfate degrading enzyme of the present invention, after adding various amino acid modification reagents shown in Table 5 to the enzyme Example 2 and The enzyme activity was measured in the same manner, and the results are shown in Table 5 below.

Effect of Divalent Metal Ion on Acaran Sulfate Dehydrogenase Activity Metal ions Residual activity (%) Acaran Sulfate I Acaran Sulfate II Mg ^{2 + a} Ni ^{2 + a} Ca ²⁺ Zn ²⁺ Pb ²⁺ Co ²⁺ Mn ²⁺ Cu ²⁺ 100113 8 91 37 74 14 121 0 100126 6 99 54 8 53 138 0

a; Final concentration 1 mM

As shown in Table 4 , the activity of the enzyme slightly increased when Mg ²⁺ or Mn ²⁺ was added, whereas the addition of Cu ²⁺ and Ni ²⁺ was strongly inhibited. In particular, ASL2 was strongly inhibited by Pb ²⁺ .

Effect of Amino Acid Modification Reagents on Acaran Sulfase reagent Residual activity (%) Acaran Sulfate I Acaran Sulfate II Control groupSFSFParaoxoneTPCKIAACARBDDTTME ^a PCMS ^a TLCK ^a 1007689968510073174173 0 0 1009586991009989167184 0 0

a; Final concentration 0.05 mM

As shown in Table 5 , both enzymes were strongly inhibited by the addition of TLCK and PCMS modifying histidine and cysteine groups, but activity was increased by reducing agents such as DDT and ME.

<3-4> Kinetic Constant Determination of Acaran Antioxidase

In order to determine the kinetics constants of the two acaran sulfate degrading enzymes of the present invention, the substrate concentration was set to 200, 400, 600, 1,000, 2,000, and 3,000 μg. It was determined by measuring the initial rate. Michaelis-Menten constants were determined by calculating the reaction rates at different substrate concentrations under optimal reaction conditions.

As a result, the Km and Vmax values of ASL1 for acaran sulfate were 28.1 μg / ml and 65.0 μmole / min / mg, respectively, and for ASL2, the Km and Vmax values were 42.4 μg / ml and 107.6 μmole / min / mg, respectively. The Km and Vmax values of ASL1 for heparin and heparan sulfuric acid were calculated to be 8.8 μg / ml, 11.6 μmole / min / mg and 7.5 μg / ml and 14.3 μmole / min / mg, respectively, and 20.6 μg / ml for ASL2, respectively. Calculated as ㎖, 22.9 μmole / min / mg and 16.4 μg / ㎖, 31.3 μmole / min / mg it was confirmed that the substrate specificity and resolution of the acaran sulfate degrading enzyme of the present invention to acaran sulfate is very excellent.

As described above, the novel acaran sulfate degrading enzyme isolated from the bacteroid Stercoris HJ-15 of the present invention has excellent substrate specificity and shows stable enzyme activity, thus effectively decomposing not only acaran sulfate but also heparin and heparan sulfate. Acaran sulfate, heparin, heparan sulfate can be used to prepare oligosaccharides, such as a basic fibroblast growth factor (basic fibroblast growth factor) to produce an acaran sulfate oligosaccharide that inhibits neovascularization by inhibiting neovascularization It can be usefully used.

Claims (11)

Bacteroides stercoris A new acaran sulfate degrading enzyme, a GAG degrading enzyme isolated from HJ-15. The novel acaran sulfate degrading enzyme according to claim 1, wherein the GAGs are acaran sulfate, heparin and heparan sulfate. The novel acaran sulfate enzyme according to claim 1, wherein the acaran sulfate is specifically decomposed and the molecular weight is 82,500 Daltons in SDS-PAGE. The new acaran sulfate enzyme according to claim 3, wherein the optimum pH of the acaran sulfate enzyme is 7.0 to 7.2, and the optimum reaction temperature is 42 ° C to 45 ° C. 4. The novel acaran sulfate of claim 3 wherein the enzyme activity is increased by a divalent cation of Mg ²⁺ or Mn ²⁺ and the enzyme activity is inhibited by a divalent cation of Cu ²⁺ or Ni ²⁺ . Degrading enzymes. 1) culturing the bacteroids stericis in a suitable medium; 2) recovering the cultured cells and preparing a cell extract; 3) purifying the cell extract by chromatography to obtain an enzymatic activity fraction; 4) obtaining only a fraction that specifically degrades acaran sulfate in the purified enzyme active fraction; And 5) A method for preparing a novel acaran sulfate enzyme according to claim 1, characterized in that the step 4 is purified by chromatography. The method of claim 6, wherein the chromatography of steps 3 and 5 is performed by QAE-cellulose column, DEAE-cellulose column, CM-Sephadex C-50 column, hydroxyapatite ultrogel A) and at least one column selected from the group consisting of SP-Sephadex C-25 column. 7. A process according to claim 6, characterized in that from step 5 two elution fractions showing degradative activity for acaran sulfuric acid are separated. 7. A process according to claim 6 wherein the first fraction of the two eluting fractions is 91-99 and the second fraction is 118-120. The novel acaran sulfate degrading enzyme of claim 1 used for preparing oligosaccharides of acaran sulfate, heparin and heparan sulfate. 11. The novel acaran sulfate degrading enzyme of claim 1, which is used for the preparation of acaran sulfate oligosaccharide that binds to basic fibroblast growth factor, inhibits neovascularization and inhibits cancer cell metastasis.