JP2001057884A - New heparitinase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme capable of cleaving a glucosaminide bond of heparin and heparan sulfate and forming a double bond between carbon atoms of cleavage site, useful as a reagent for analytical study, a degrading agent for preparation of low molecular heparin useful as an antithrombotic agent, etc. SOLUTION: This new heparitinase T-II is a lyase acting on a glucosaminide bond of heparin and heparan sulfate and having an action of forming a double bond between carbon atoms at the 4-position and the 5-position of glucuronic acid and iduronic acid of cleavage site, hardly acts, on heparin, mainly acting on heparan sulfated material, produces an unsaturated disaccharide as a decomposition product which is a non-sulfate substance and a small amount of uronic acid-glucosamine-N-sulfate, has an optimum pH of 5.5-6.5, a stable pH range of 5.0-9.5, an optimal temperature of 55 deg.C, a stable temperature range of <=50 deg.C, increases enzyme activity by Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ and inhibits enzyme activity by Zn2+. The enzyme is obtained from a culture solution or an extracted solution of microbial cell prepared by culturing Bacillus circulans Hpt 298 strain (FERM BP-3765) belonging to the genus Bacillus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme heparitinase which decomposes heparan sulfate and heparin, and a method for producing the same.

Heparitinase is a complex polysaccharide of heparan sulfate (hereinafter abbreviated as HS) or heparin (hereinafter abbreviated as Hep), which has a basic structure of repeating disaccharide units of N-acetyl-D-glucosamine and uronic acid. It is an enzyme that cleaves glucosaminide bonds, and is useful as a function of HS or Hep in vivo or as a reagent for analysis and research of these substances in biological components. In addition, as a low molecular weight agent for preparing low molecular weight heparin, which is being developed as an antithrombotic agent in recent years, or as a material (Hep removing agent) for reducing the side effect of Hep, which is a problem when treating with an extracorporeal circulation device ) Is attracting attention, and is expected to have various uses for diagnostic and therapeutic purposes.

[0003]

BACKGROUND OF THE INVENTION Enzymes which can be used in these applications include the presence or absence of a sulfate group bound to a sugar chain,
It is necessary to have a variety of heparitinases with different substrate specificities that recognize differences in the sugar chain structure such as the binding position, and it is desirable that the enzyme source can be stably supplied in large quantities. From this point of view, the present inventors have sought an enzyme satisfying the above-mentioned uses from microbial sources, searched for the enzyme-producing bacterium, and have already found three types of heparitinase from the genus Flavobacterium (Japanese Patent Application Laid-Open No. 2-57183).

[0004] Other reports detailing the purification and properties of heparitinase of microbial origin include other enzymes collected from bacteria belonging to the genus Flavobacterium or Bacillus. For example, the summary of the 10th International Glycoconjugate Symposium (Page 330, 1989) and JP-A-2-142470. Although these known enzymes are useful for the above purpose, new heparitinases having different substrate specificities are required to achieve the above object.

[0005] For the above reason, the present inventors further searched for a novel heparitinase-producing bacterium in nature and found that Bacillus circulans (Ba) isolated from soil in Saitama Prefecture.
cillus circulans) HpT298 strain was found to be capable of producing novel heparitinase. These heparitinases were fractionated and purified, and four new heparitinases having different physicochemical properties and specificities were isolated.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and is intended to solve the above problems.
A novel enzyme that decomposes heparitinase T-I, heparitinase T-II, heparitinase T-III and heparitinase T-IV, and relates to the production of the enzyme. The present invention relates to a method for efficiently producing the present enzyme from a bacterium belonging to the genus Bacillus. Hereinafter, the present invention will be described in more detail.

[0007] The microorganism used for the production of the present enzyme may be any strain as long as it is a bacterium belonging to the genus Bacillus capable of producing the present enzyme. The Bacillus circulans HpT298 strain used in the examples of the present invention is the present inventors. Are new strains isolated from soil in Saitama prefecture by searching for Hep-assimilating bacteria, and their bacteriological properties are as follows.

(A) Morphological properties Gram stain: negative Cell shape: bacilli (0.4-0.5 μm × 2.0-
3.6μm) Spore shape: Oval Spore swelling: Positive Paraspore crystal: Negative Motility: Positive

(B) Growth characteristics Aerobic growth: Positive Anaerobic growth: Negative Growth temperature 15 ° C: Positive 20 ° C: Positive 30 ° C: Positive 40 ° C: Positive 50 ° C: Positive 51 ° C: Positive 52 ° C: Negative lysozyme Growth in the presence of (0.001%): Positive Growth in the presence of NaCl 2%: Positive 5%: Negative 7%: Negative Growth at pH 6.8 (nutrient medium): Positive pH 5.7 (Sabouraud dextrose medium) ): Positive Growth pH: 5.0-9.0, especially 6.5-7.5 is optimal Requirement of NaCl and KCl: Negative

(C) Physiological properties and other properties Catalase: Positive VP test: Negative pH in VP medium: 6.0 or less Acid production From D-glucose: Positive From D-xylose: Positive D- From mannose: Positive From L-arabinose: Positive From D-mannitol: Positive From sorbitol: Negative Gas production from D-glucose: Negative Indole production: Negative Casein hydrolysis: Negative Starch hydrolysis: Positive Tyrosine degradation: Negative Phenylalanine deamination: Negative Nitrate reduction: Positive Citric acid assimilation: Positive Propionic acid assimilation: Negative Heparin assimilation: Positive Heparan sulfate assimilation: Positive Chondroitin sulfate assimilation: Positive assimilation of keratosulfate: Negative Guanine + cytosine (G + C) content: 53.0 Mol% Main isoprenoid quinone: menaquinone-7 (MK
-7) Diaminopimelic acid (DA) in cell wall peptidoglycan
P): meso-DAP

The taxonomic position of the HpT298 strain having the above-mentioned mycological properties is examined with reference to the Barjay's Manual of Systematic Bacteriology, 1st edition, Volume 2, (1986). Then, this bacterium forms a spore with a motile aerobic Gram-negative bacillus, and the main isoprenoid quinone is menaquinone-7, and the cell wall peptidoglycan contains meso-diaminopimelic acid. Was done. Compared with other known species of the genus Bacillus, the strain was found to be Bacillus circulan
s). However, known Bacillus circulans strains such as IFO 1363
2. IFO 13635 and IFO 13636 are new strains that do not produce the enzyme of the present invention and are distinguished from known strains in this regard.

Bacillus sp. (Bacillus sp.) BH100 strain is known as a bacterium-producing enzyme which degrades HS and Hep belonging to the genus Bacillus (Japanese Patent Application Laid-Open No. 2-142470). , PH in VP medium, hydrolytic ability of starch, and G + C content in DNA.
Eight strains differ from the BH100 strain. The HpT2
98 strains have been deposited with the National Institute of Microbial Industry and Technology under the International Microorganism Accession No. BP-3765.

[0013] The novel heparitinase T-II of the present invention comprises:
The Bacillus circulans HpT298 strain or the present enzyme-producing bacterium belonging to the genus Bacillus is cultured in a nutrient medium usually used for culturing microorganisms, preferably a medium supplemented with Hep, HS, or a substance containing these in order to enhance the enzyme-producing ability. As a result, the enzyme is produced and accumulated in the medium or the cells, and thus the purified enzyme can be obtained by a known method.

More specifically, the present enzyme-producing bacterium belonging to the genus Bacillus is cultured in an appropriate nutrient medium, for example, a medium containing an appropriate carbon source, nitrogen source, inorganic salts and Hep or HS, or a substance containing these. Is cultured to produce and accumulate the enzyme in the medium or in the cells. As the carbon source, any substance that can be assimilated can be used. For example, D-glucose, D-xylose, D-mannose, L-arabinose, D-mannitol, starch and its hydrolyzate, molasses, citric acid Salts, various peptones and the like. As the nitrogen source, organic or inorganic nitrogen compounds such as yeast extract, malt extract, various peptones, various meat extracts, soybean powder, defatted soybean powder, corn stapler, amino acid solution, ammonium salt, and the like, can be used. As the inorganic salt, various salts such as phosphates, magnesium, potassium, sodium, and calcium are used. If necessary, various inorganic and organic substances necessary for the growth of bacteria or enzyme production, for example, antifoaming agents such as silicone oil, sesame oil, various surfactants, and vitamins can be added to the medium.

In the present invention, the present enzyme can be produced in a large amount by adding Hep, HS or a substance containing them as an inducer of the present enzyme. These inducers may be added from the beginning of the culture or during the culture.
The addition amount is typically 0.2% to 2% as Hep or HS.
Good results are obtained with the addition.

The form of culture may be liquid culture or solid culture, but liquid culture is usually preferred, and industrially, it is advantageous to carry out deep aeration stirring culture. Culture conditions in the present invention, appropriately select the most advantageous conditions for the production of the enzyme,
Adjust and do. The culturing temperature can be appropriately changed within a range of 15 to 51 ° C, but particularly preferred is 40 to 45 ° C.
° C. The culturing time varies depending on the culturing conditions, but is 1-2.
The cultivation may be terminated on the order of days, when the enzyme reaches the maximum accumulation amount. The pH of the medium may be around neutral at the time of preparation of the medium, and usually does not need to be adjusted.

The above four enzymes can be obtained from both the supernatant of the culture solution and the cell extract thus obtained. For the culture supernatant, add ammonium sulfate,
After dialysis of the precipitate precipitated at 0.6 saturation, the enzyme is fractionated and purified using hydroxyapatite, an ion exchange resin, a gel filtration agent and an adsorbent. For the intracellular enzymes, the cells are suspended in an appropriate buffer, the cells are disrupted by ultrasonication or mechanical disruption to extract the enzyme, and the centrifuged supernatant is added to the culture supernatant. Purification can be performed by the same method as used. However, the present invention is not at all limited by these purification techniques.

The titer of the present enzyme is that it is a lyase that acts on the hexosaminide bond, and the double bond formed between carbons 4 and 5 of the uronic acid at the stump of the cleavage site causes ultraviolet absorption. It is determined by taking advantage of having and measuring the increase.

The substrates of the enzyme include heparitinase TI,
As for heparitinase T-II and heparitinase T-III, HS derived from bovine kidney was used for heparitinase T-IV.
Is used for Hep derived from porcine intestinal mucosa.

That is, 10 μl of the enzyme solution and 100 mM Tris / acetate buffer (p
H7.0) 25 μl, 25 μl of 20 mM calcium chloride and 15 μl of water are added and reacted at 45 ° C. for 10 minutes. To this solution, 500 μl of a 0.06 N hydrochloric acid solution was added to stop the reaction, and the ultraviolet absorption A at 232 nm was measured. As a control solution for measuring the ultraviolet absorption A ₀ at zero time in the same solution.

The enzyme titer is expressed in terms of a titer that gives a decomposition amount of 1 μmol per minute under the above reaction conditions as one unit.
It is calculated from the following equation. A−A ₀ /5.5 (molar correction using molecular extinction coefficient) ×
600/10 (correction of enzyme dilution) x 1/10 (correction per minute) = U / ml (unit per 1 ml of enzyme used)

The physicochemical properties of the novel heparitinase T-II of the present invention and other heparitinases are shown. (1) a lyase which acts on Gurikosaminido binding of either enzymes heparin or heparan sulfate for operation to form a double bond between the 4-position and 5-position carbons of glucuronic acid or iduronic acid of the cut.

(2) Substrate specificity (FIGS. 1-2) Heparitinase T-1 and heparitinase T-II are composed of He
p has little effect and acts mainly on HS, and the unsaturated disaccharide produced as a decomposition product is a non-sulfuric oxide (hereinafter referred to as “△ DiHS
-OS ") and a small amount of uronic acid-glucosamine-
N-sulfuric acid (hereinafter referred to as "@ DiHS-NS").
Heparitinase T-III hardly acts on Hep, acts mainly on HS, and unsaturated disaccharides generated as degradation products are ΔDiHS-OS and ΔDiHS-NS. Heparitinase T-IV acts on Hep and HS, and the unsaturated disaccharide generated as a decomposition product is ΔDiHS-NS, uronic acid-glucosamine-N, 6-disulfate (hereinafter “ΔDiHS-di
N, 6S "), uronic acid-2-sulfate-glucosamine-N-sulfuric acid (hereinafter referred to as" △ DiHS-diU, NS ") and uronic acid-2-sulfate-glucosamine-N, 6-
Disulfuric acid (hereinafter referred to as “@ DiHS-triS”).

(3) Optimum pH (FIG. 3) The optimum pH of the enzyme and the like is determined by a reaction at 45 ° C. for 10 minutes using 50 mM acetate buffer, Tris / acetate buffer and Tris / hydrochloric acid buffer. As a result, both heparitinase T-I and heparitinase T-II have a pH of 5.5 to 6.5, heparitinase T-III has a pH of 7.0 to 8.0, and heparitinase T-IV has a pH of 7.5 to 8.0. It is.

(4) Stable pH range (FIG. 4) The stable pH range of the present enzyme and the like was adjusted by using 100 mM acetate buffer, Tris / acetate buffer, Tris / HCl buffer, and glycine / sodium hydroxide buffer solution. When heparinase TI was treated at pH 4.5 to 9.5 for 30 minutes.
5. Heparitinase T-II is stable at pH 5.0 to 9.5, heparitinase T-III is stable at pH 5.0 to 9.5, and heparitinase T-IV is stable at pH 5.0 to 10.0.

(5) Optimal temperature of action (FIG. 5) The optimal temperature of the present enzyme and the like was adjusted to 50 mM Tris / acetate buffer pH.
When 7.0 was used and the reaction was performed for 10 minutes, heparitinase T-I and heparitinase T-II were 5
5 ° C, 50 ° C for heparitinase T-III and 40 ° C for heparitinase T-IV.

(6) Stable temperature range (FIG. 6) The stable temperature range of the present enzyme and the like was adjusted to 50 mM Tris / acetate buffer.
When treated at each temperature for 60 minutes using pH 7.0 and examined, heparitinase T-I and heparitinase T-II were 50 ° C or less, and heparitinase T-III was 45 ° C.
Hereinafter, heparitinase T-IV is each stable at 40 ° C. or lower.

(7) Conditions for deactivation by pH, temperature, etc. (FIGS. 4 and 6) The enzyme and the like were buffered in 100 mM acetate buffer, Tris / acetate buffer, Tris / hydrochloride buffer and glycine / sodium hydroxide buffer. The solution was subjected to a treatment at 37 ° C. for 30 minutes to examine that heparitinase T-I, heparitinase T-II and heparitinase T-III had a pH of 4.5 or less and a pH of 10.0 or more, and heparitinase T-IV had a pH of 4 or more. .
It rapidly deactivates at pH 5 or less and pH 10.5 or more, respectively. The enzyme and the like were treated with 50 mM Tris / acetate buffer (pH 7.0) for 60 minutes at each temperature and examined. As a result, heparitinase T-I and heparitinase T-II were 5 mM each.
At 5 ° C. or higher, heparitinase T-III rapidly deactivates at 50 ° C. or higher, and heparitinase T-IV rapidly deactivates at 45 ° C. or higher.

(8) Influence of Inorganic Ions (Table 1) The activity of the present enzyme is activated or inhibited by various ions. Heparitinase TI is Ca ²⁺ , Co ²⁺ , Mg ²⁺ ,
It is activated by Mn ²⁺ and inhibited by Zn ²⁺ . Heparitinase T-II are ^{Ba 2+, Ca 2+, Co 2+} , Mg 2+, activated with Mn ^2+, it is inhibited by Zn ^2+. Heparitinase T-II
I is inhibited by Zn ²⁺ . Heparitinase T-IV is Ba
^2+, Ca ^2+, activated with Mg ^2+, Co ^2+, is inhibited by Zn ^2+.

[0030]

[Table 1]

The enzymatic chemical properties of these four heparitinases were compared with those of known enzymes.
I, heparitinase T-II and heparitinase T-III
Act on heparan sulfate with little effect on heparin, and because the degradation products are different from those of known enzymes of the genus Flavobacterium, and that heparitinase T-IV acts on heparin and heparan sulfate, but flavobacteria The above four types of heparitinases have different properties from known enzymes because of the fact that their degradation products are different from those of the known enzymes of the genus Rume and that the optimum temperature and temperature stability are different from those of the enzymes of the genus Bacillus. It was identified as an enzyme.

[0032]

The present invention will be described in more detail with reference to the following Examples, which do not limit the scope of the present invention. Example Peptone A (manufactured by Far Eastern Pharmaceutical Co., Ltd.) 0.75%, Yeast extract (manufactured by Far Eastern Pharmaceutical Company) 0.5%, heparin sodium (manufactured by Syntex) 0.5%, K ₂ HPO ₄ 0.1%, MgSO _4.
7H ₂ O 0.02%, NaCl 0.1%, defoamer Adecanol LG109 (manufactured by Asahi Denka) 0.005% (pH 7.0)
20,000 ml of the production medium having the composition of 0)
Charge in a jar fermenter with a volume of 20 ml at 120 ° C.
After steam sterilization for one minute, pre-culture at 45 ° C for one day on a Heart Infusion agar medium (manufactured by Eiken Chemical Co., Ltd.). The medium was inoculated, and 600 ml (3%) of a culture of Bacillus circulans HpT298 strain, which had been shake-cultured at 45 ° C for 20 hours, was aseptically inoculated.
Culture was performed with aeration (1 v. Vm) and stirring (300 rpm) for a time. After completion of the culture, the culture solution was treated by continuous centrifugation to collect the cells, and half of the cells (wet amount: 110 g) was 300 ml.
Was suspended in 0.1 M phosphate buffer (pH 6.8) and crushed using an ultrasonic crusher. After crushing, insolubles were removed by centrifugation, and ammonium sulfate was added to the obtained supernatant to make it 0.6 saturated. The precipitate was collected, dialyzed overnight against 20 mM Tris / acetate buffer pH 7.0, loaded onto a DEAE-Sephacel column (4.2 × 25 cm) and eluted with the same buffer. The eluate was loaded on a hydroxyapatite column (3.2 × 24 cm) and eluted by linearly increasing the salt concentration from 0 to 0.5 M in the same buffer. When the activity was measured using Hep and HS as substrates, heparitinase T-I was eluted in the passing solution, heparitinase T-II was eluted at around 0.2 M, and heparitinase T-III and heparitinase T-IV were eluted at around 0.4 M. . Each fraction was desalted using an ultrafiltration membrane, and then replaced with a 50 mM Tris / acetate buffer. Among them, each of the fractions of heparitinase T-I and heparitinase T-II was loaded on a Sephacryl S-300 column (3.8 × 100 cm) and contained 5% of 0.2 M salt.
Gel filtration was performed with a 0 mM Tris / acetate buffer, and the respective active fractions were collected and concentrated and desalted using an ultrafiltration membrane to obtain an enzyme solution. The fraction from which heparitinase T-III and heparitinase T-IV were simultaneously eluted was loaded on a sulfated Cellulofine column (3.2 × 20 cm) and subjected to 5
Elution was performed by linearly increasing the salt concentration from 0 to 0.3 M in 0 mM Tris / acetate buffer. Heparitinase T-III was eluted at about 0.1 M and heparitinase T-IV was eluted at about 0.15 M. Each fraction was concentrated and desalted using an ultrafiltration membrane to obtain an enzyme solution.

Yield of each enzyme Heparitinase TI: 12 U Heparitinase T-II: 6 U Heparitinase T-III: 50 U Heparitinase T-IV: 6 U

[0034]

According to the present invention, a novel heparitinase T-II capable of decomposing heparin and heparan sulfate can be provided.

[Brief description of the drawings]

FIG. 1 shows the resolution of each enzyme for heparin (derived from porcine intestinal mucosa) and heparan sulfate (derived from bovine kidney).

FIG. 2 shows the results of examining the disaccharide components of the enzyme hydrolyzate.

FIG. 3 shows the optimum pH.

FIG. 4 shows a stable pH range.

FIG. 5 shows the optimal temperature of action.

FIG. 6 shows a stable temperature range.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroshi Maruyama 3-1253 Tateno, Higashiyamato-shi, Tokyo Inside the Tokyo Research Laboratory (72) Inventor Keiichi Yoshida 3-1253 Tachino, Higashiyamato-shi, Tokyo Gaku Kogyo Co., Ltd.

Claims (2)

[Claims] 1. Heparitina having the following physicochemical properties:
-Sease T-II. (A) Action Acts on glycosaminide binding of heparin and heparan sulfate.
Lyase, glucuronic acid or
A double bond is formed between carbons at the 4- and 5-positions of the acid. (B) Substrate specificity It hardly acts on heparin, and mainly acts on heparan sulfate.
Unsaturated disaccharide produced as a decomposition product
A small amount of uronic acid-glucosamine-N-sulfuric acid. (C) Optimum pH 5.5-6.5 (D) Stable pH range 5.0-9.5 (E) Optimum temperature 55 ° C (F) Stable temperature range 50 ° C or less (G) Inhibition and activation Enzyme activity is Ba²⁺, Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺so
Activated, Zn ²⁺Inhibited by. 2. Heparitinase T- belonging to the genus Bacillus.
2. The method for producing heparitinase T-II according to claim 1, wherein a bacterium having II-producing ability is cultured, and heparitinase T-II is collected from the culture solution or the cell extract.