JP2833793B2 - Plasmid encoding heparinase, heparinase-producing strain carrying this plasmid, and method for producing heparinase - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel plasmid encoding heparinase, a novel heparinase-producing strain containing this plasmid, and a novel method for producing heparinase.

[Conventional technology]

Heparinase is heparin, ie, -4) -2-deoxy-2-sulfamino- as the major repeating unit.
α-D-glucopyranose-6-sulfate- (1,
4) -α-L-Idopyranosyluronic acid-2-sulfuric acid-
(An enzyme that cleaves certain glycosidic bonds of sulfated glucosaminoglycans having 1-. The product of this enzymatic activity is a shortened fragment of the chain of heparin. Heparinase or heparitin (heparan sulfate) That is, it cleaves a sulfated polymer having a chemical structure similar to heparin to produce a fragment of a shorter chain of heparitin.

Heparin, a substrate, is widely used as an anticoagulant, so heparinase is used for structural studies of heparin,
There are a variety of uses in studying the mechanism of blood coagulation and in enzymes of heparin in body tissues and fluids.

Heparinase is also used in the preparation of low molecular weight heparin fragments of potential therapeutic value as antithrombin or antitumor agents.

It is known that relatively few types of microorganisms produce enzymes that can degrade heparin. Production of heparinase (heparin lyase, EC 4.2.2.7) has been found in the culture of Flavobacterium heparinum, Flavobacterium sp., Bacteroides sp. . These heparinases are described in Journal of Biochemistry [233, 853 (1956)], Experience [41
Vol. 1541 (1985)], Journal of Matsumoto Dental University (Japan)
[Vol.8, p.15 (1982)], Journal of Applied Amide Environmental Microbiology [Vol.46, p.1252 (1983)], Journal of Clinical Microbiology [ Volume 26
1070 (1988)], and the Journal of South African Veterinary Association [53
Vol. 214 (1982)]. in addition,
Two of the present inventors have recently found the only known Bacillus that can produce heparinase. Bacillus
This bacterium, named sp. BH100 strain, was deposited with the Research Institute of Microbial Industry and Technology, and given the deposit number FERM P-10408 (Microbial Lab No. 10408). This strain is a
-297807 (November 25, 1988) used in a novel method for producing heparinase.

"Plasmids" are non-chromosomal genetic elements made up of circular molecules of deoxyribonucleic acid found in microbial cells. By using conventional gene cloning techniques, fragments of DNA carrying genetic information for the production of useful proteins or peptides can be introduced into a suitable plasmid called a "vector" plasmid. When the resulting recombinant plasmid is introduced into a suitable host organism, the transformed host organism often produces useful plasmid-encoded proteins or peptides.
Usually, E. coli is used as the host organism. A plasmid having a heparinase gene is novel, a heparinase-producing strain of Escherichia coli carrying such a plasmid is also novel, and a method for producing heparinase using an Escherichia coli strain carrying a plasmid encoding heparinase is also novel.

[Problems to be solved by the invention]

Flavobacterium, Heparinum, Bacteroides
Conventional methods for producing heparinase using heparinolitis or Bacillus sp. Strain BH100 required the addition of heparin to the growth medium to induce heparinase synthesis. The need to add heparin as an inducer has led to a considerable increase in the cost of conventional heparinase production methods. Methods that essentially produce heparinase, that is, use a plasmid-carrying E. coli strain that does not require the addition of heparin as an inducer, are advantageous for performing enzyme production at lower cost.

With the intention of creating a strain that essentially produces heparinase, the inventors have determined that the strain Bacillus sp.
A new recombinant plasmid consisting of the heparinase gene DNA from the chromosome of P-10408) and the vector plasmid pBR329 was constructed, and this new recombinant plasmid was then introduced into the host bacterium Escherichia coli HB101. Host bacteria transformed with the recombinant plasmid produce heparinase without the need for the addition of heparin as an inducer, and as a result, the present inventors have completed the present invention.

An object of the present invention is to provide a novel plasmid having a heparinase gene.

It is another object of the present invention to provide a novel strain carrying a plasmid having a heparinase gene.

It is another object of the present invention to provide a novel method for heparinase production using a plasmid-bearing strain.

[Means for solving the problem]

The novel plasmid according to the present invention provides a host bacterium with the ability to produce heparinase, and is composed of a vector plasmid and heparinase gene DNA.

This plasmid is used to cut out the heparinase gene DNA fragment from the chromosomal DNA with a restriction enzyme, cut the vector plasmid with an appropriate restriction enzyme that does not interfere with the plasmid genetic information essential for plasmid replication, and cut the vector with this chromosome fragment and the restriction enzyme. The plasmid can be produced by a method comprising treating it with DNA ligase to form a recombinant plasmid, and isolating the recombinant plasmid.

The novel strain according to the present invention contains a novel plasmid and belongs to the genus Escherichia which produces heparinase. For example, Escherichia coli HB101 strain (pPDH6) (FE
RM P-11011).

The present invention also provides a novel method for producing heparinase, which comprises retaining a plasmid having a heparinase gene and culturing a heparinase-producing Escherichia coli strain to obtain heparinase.

 Hereinafter, the present invention will be described in detail.

1. Novel Plasmid and Constitution Thereof According to the present invention, a novel plasmid that provides a host with the ability to produce heparinase can be constructed. This new plasmid is composed of a vector plasmid and a fragment of heparinase gene DNA.

Vector plasmids that can be used in the present invention include any plasmid that can replicate in E. coli.
Typical examples are pBR322, pBR329, Col E1, pMB9, pSC10
1, pHC79 is listed.

Fragments of DNA that can be used in the present invention include a chromosomal gene encoding heparinase, and Bacillus sp.
It is produced by cutting DNA from strain 00 (FERM P-10408) with restriction enzymes. Bacillus s isolated from soil
The p.BH100 strain exhibits the taxonomic properties shown in Table 1.

For constructing the plasmid having the heparinase gene of the present invention, a usual method is used. For example, chromosomal DNA from Bacillus sp. Strain BH100 (FERM P-10408) is cut with an appropriate restriction enzyme to generate a fragment. Next, the chromosomal DNA fragment can be fractionated according to the size, for example, by sucrose density gradient ultracentrifugation or agarose gel electrophoresis. This fragment can also be used without fractionation by such size. Vector plasmids are cut into linear forms with appropriate restriction enzymes, and can be treated with appropriate phosphatases and dephosphorylated. The linear vector plasmid can be used without dephosphorylation. After being treated in this manner, the vector plasmid is mixed with the chromosomal DNA fragment, and the mixture is treated with an appropriate ligase to produce a recombinant. This recombinant DNA is then introduced into a suitable host bacterium, for example, E. coli strain HB101. The transformed host bacterium producing heparinase can be used, for example, to use an enzyme-linked immunoassay with an antiserum specific for purified bacillus heparinase, or to test the heparinase activity of the host cell. It is detected by using both. Plasmid with genetic information for heparinase production
DNA is isolated from heparinase-producing host bacteria by conventional methods.

The novel plasmid pPDH6 is constructed as follows. The chromosomal DNA isolated from the Bacillus sp. BH100 strain is partially cut with a restriction enzyme Sau3A, and then subjected to size fractionation by sucrose density gradient ultracentrifugation. Recover fragments of about 5-7 kilobase pairs. This DNA is digested with BamHI with a restriction enzyme, recombined into a dephosphorylated vector plasmid pBR329, and introduced into E. coli strain HB101. The transformed host bacterium producing heparinase should use an enzyme-linked immunoassay with an antiserum specific for purified Bacillus heparinase, or use one or both to test the heparinase activity of the host bacterium. Is detected by Plasmid pPDH6 is isolated from heparinase-producing host bacteria by conventional methods. As shown in FIG. 1, the restriction map of the novel plasmid pPDH6 shows that the plasmid pPDH6 has a DNA of about 9.7 kilobase pairs.
And a chromosome derived from Bacillus sp.
This shows that the DNA fragment is composed of the inserted vector plasmid pBR329.

2. Production of heparinase-producing strain According to the present invention, a novel heparinase-producing strain of the genus Escherichia was obtained. Chromosome DN from Bacillus sp.BH100 strain
The recombinant plasmid constructed by recombination between A and the vector plasmid is introduced into a host bacterium of the genus Escherichia by a conventional transformation method. The host bacterium that can be used in the present invention may be any strain of the genus Escherichia.

A typical host bacterium used is E. coli HB101, which is a hybrid strain of E. coli K-12 and E. coli B strains. Transformants produced by introducing a heparinase-encoding plasmid into a strain of the genus Escherichia will have the same microbiological characteristics as the host, except for plasmid-encoded properties such as antibiotic resistance and the ability to produce heparinase. Has characteristics. For example, the plasmid pPDH
The transformed E. coli HB101 strain (pPDH6) produced by introducing the E. coli 6 into the E. coli HB101 strain has the same microbiological characteristics as the E. coli HB101 strain [genotype: F ⁻ , hsdS20 (rB ⁻ , mB
^- ), Rect 13, ara-14, ProA2, lacY1, galK2, rpsL20 (S
m), xy1-5, mtl-1, supE44 λ], and further characterized by the properties encoded by plasmid pPDH6, namely ampicillin resistance, chloramphenicol resistance and heparinase production.

As the culture medium for the heparinase-producing strain of the present invention,
An ordinary medium containing a carbon source, a nitrogen source and inorganic salts is used. Any carbon source that can be used can be used as the carbon source. For example, D-glucose, sucrose, lactose, tryptone or peptone can be exemplified as typical examples. A wide variety of conventional nitrogen sources can be used, such as yeast extract, peptone,
Examples include tripton, meat extract, corn steep liquor, amino acid solutions, and the like, or inorganic nitrogen sources such as ammonium sulfate and ammonium chloride, which are easily available. Further, in addition to the above-mentioned carbon source and nitrogen source, various salts such as magnesium chloride, sodium phosphate, potassium phosphate, potassium chloride, and calcium chloride can be added. It is also possible to add vitamins and other growth factors such as folic acid, calcium pantothenate, biotin, riboflavin, thiamine and the like. further,
If necessary, a gelling agent such as agar powder or gelatin can be added. The medium is adjusted to pH 5.0 to 8.0 by appropriately adding an acid or alkali, and sterilized by an autoclave.

As an example of a suitable medium, tryptone per one
A liquid medium containing 10 g, yeast extract 5 g, glucose 1 g and NaCl 10 g and adjusted to pH 7.0 can be exemplified.

The heparinase-producing strain of the present invention is cultured at 20 to 37 ° C, and preferably at 35 to 37 ° C under aerobic conditions.

3.Method of obtaining heparinase The heparinase-producing strain of the present invention may be aerobically at 35 to 37 ° C in any suitable medium as described above.
Incubate for 12-70 hours.

Separation and purification of heparinase are performed, for example, as follows. The cells are recovered from the culture by centrifugation, filtration or other conventional methods, and are disrupted by sonication, pressure by a French press or other conventional methods. At this stage, the nucleic acid may be removed by treatment with deoxyribonuclease and ribonuclease, precipitation with protamine sulfate, and the like. The crude heparinase-active fraction can be obtained by a conventional method well-known in the field of protein purification, such as ordinary ion exchange and chromatography methods such as gel filtration, or chromatofocusing, preparative isoelectric focusing, electrophoresis. Fractionation is performed by using one or both of the methods, thereby obtaining heparinase.

A preferred method for obtaining heparinase is exemplified below.

The strain of the present invention is cultured aerobically at 35-37 ° C for 24-36 hours in a suitable medium, as described above. The resulting culture is centrifuged at 8000 × g for 30 minutes to recover the cells.
Further, the collected cells are made into a crude heparinase solution by ultrasonic pulverization, and used as a sample for the next step. Sample is pH 7.
4 Apply to a sulfated Cellulofine column equilibrated in 10 mM HEPES buffer, and adsorb the enzyme
Elution is performed with a concentration gradient of Cl0 to 0.3M. The collected active fraction is concentrated by ultrafiltration using an Amicon PM10 membrane. The sample is further fractionated by gel filtration chromatography using a SHODEX WS-2003 column equilibrated with the above buffer containing 100 mM NaCl. The active fraction thus obtained is detected as one protein band by SDS-polyacrylamide gel electrophoresis analysis,
Contains only a single heparinase.

The activity measurement method by ultraviolet absorption used for the quantitative analysis of heparinase activity is as follows. Dissolved in appropriately diluted 20μ enzyme solution and pH 7.4, 50mM HEPES buffer
The reaction mixture containing 50μ of a 5 mg / ml heparin solution is kept at 40 ° C for 4 hours. Thereafter, the reaction was performed at pH 2.0, 20 mM KCl-HCl
The solution was stopped by adding 2.0 ml of the solution, and the
The absorbance at is measured. One international unit of activity was based on the molar extinction coefficient of the double bond of 5100 cm ⁻¹ M ⁻¹ , and was the amount of enzyme required to produce 1 μmol of the double bond in one minute at 40 ° C.

4. Characteristics of heparinase The enzymatic characteristics of heparinase produced by this method are as follows.

i) Action: Heparinase catalyzes the elimination reaction of heparin and heparasulfate to produce short-chain glycosaminoglycan fragments. At this time, to generate a double bond at the same time,
These exhibit ultraviolet absorption at 232 nm.

ii) Substrate specificity: This enzyme cleaves heparin and heparan sulfate, but not chondroitin-6-sulfate, chondroitin-4-sulfate, dermatan sulfate, hyaluronic acid, dextran sulfate and polygalacturonic acid.

iii) Optimum temperature and thermal stability: The optimum reaction temperature in the presence of 5.0 mM CaCl ² was 45 ° C. (reaction for 30 minutes), and after heating at 45 ° C. for 60 minutes, 80% or more of the activity remained. To stabilize this enzyme,
Calcium ions are required. In addition, this enzyme, 60 ℃
Complete inactivation by heating for 15 minutes.

iv) Optimum pH: The optimum pH for the reaction is in the range of pH 7.2 to 7.8.

v) Influence of inorganic ions: Enzyme activities are Ca ²⁺ , Mg ²⁺ , and Ba
^It increases about 50% in the presence of ²⁺ chloride. 0.5mM CO ²⁺ ,
The activity decreases in the presence of Zn ²⁺ , Fe ⁺² , Hg ²⁺ , CO ²⁺ and 1.0 mM Ni ²⁺ chloride. Chloride of Mn ²⁺ , Li ²⁺ and Ag ²⁺ is 5.0mM
At concentrations up to, there is no substantial effect on activity.

Example 1 The construction of a novel plasmid having a heparinase gene and the production of a heparinase-producing strain are described below.

(1) Production of chromosomal DNA having heparinase gene Heparinase-producing Bacillus sp. Strain BH100 (FERM P-10
408), chromosomal DNA was isolated by the phenol extraction method. A sample of this DNA was obtained using the restriction enzyme Sau3A at 37 ° C.
The fragments were cut for 5, 10, 20, and 30 minutes to produce fragments.
The fragments were size fractionated by sucrose density gradient ultracentrifugation using a 5-20% sucrose gradient. 23000r for 17 hours at 4 ° C
After centrifugation at pm, the fractions were collected and the size of the DNA fragment in each fraction was confirmed by agarose gel electrophoresis.

(2) Insertion of chromosomal DNA of heparinase gene into vector plasmid Plasmid pBR3 used as vector plasmid
29 (International Biotechnology Inc., U
SA; retains ampicillin resistance, chloramphenicol resistance and tetracycline resistance genes. ) Was treated with the restriction enzyme BamHI at 37 ° C. for 1 hour and then with bacterial alkaline phosphatase at 37 ° C. for 1 hour. These enzymes were inactivated and removed by phenol extraction and ethanol precipitation of the plasmid. The fragment of about 5 to 7 kilobase pairs (2 μg) obtained in the step (1) is BamHI-
The cut, dephosphorylated vector plasmid pBR329 (0.
5 μg), and the mixture is bacteriophage T
The cells were treated with 4 DNA ligase at 16 ° C. for 16 hours. Recombinant plasmids containing the inserted chromosomal DNA were thus produced. The reaction was stopped by heating the sample at 65 ° C. for 5 minutes. The recombinant plasmid was then precipitated with ethanol and recovered.

(3) Transformation of plasmid carrying heparinase gene into host bacteria Escherichia coli HB101 strain was cultured in 10 ml of LB medium (per liter,
Tryptone 10g, yeast extract 5g, glucose 1g, and Na
The cells were cultured at 37 ° C. in 10 g of Cl at pH 7.0 with shaking until late logarithmic phase. Cells are harvested by centrifugation and 0.1 ml of ice-cold CaC to obtain competent cells.
l _2, and the mixture was suspended in CaCl ₂ 1 ml of final concentration 30 mM. This cell suspension (50 μ) and the recombinant DNA solution (5 μ) obtained in step (2) were combined and kept on ice for 25 minutes. The mixture was heated at 42 ° C. for 1-2 minutes to introduce the plasmid DNA into the host cells. This cell suspension was inoculated into fresh LB medium, and the culture was cultured at 37 ° C. with shaking for 60 minutes. Next, cells were harvested by centrifugation, appropriately diluted in LB medium, and plated on solid growth medium containing chloramphenicol to obtain transformants.

(4) Isolation of Heparinase-Producing Strain Carrying Plasmid pPDH6 The transformed host bacterium was an enzyme using a purified heparinase-specific rabbit polyclonal antiserum derived from Bacillus sp. Strain BH100 (FERM P-10408). Heparinase production was tested by a binding immunoassay.

According to the usage, pico blue immunodetection kit [picoBLUE Immu-noDETECTION KIT (Startagen
e)] on a nitrocellulose filter using
A colony assay was performed. Among 1200 colonies showing the insertion and inactivation of the tetracycline gene of the plasmid vector, 7 recombinant clones showing a strong immune signal were obtained. One example of these transformations, the E. coli strain HB101 (pPDH6) harboring plasmid pPDH6 and producing enzymatically active heparinase, was isolated as a single strain. This strain was deposited with the Institute of Microbial Industry and Technology, and given the deposit number FERM P-11011.

Example 2 The production and purification of plasmid pPDH6 is exemplified below.

500 ml of Escherichia coli HB101 (pPDH6) (FERM P-11011)
LB medium (containing 10.0 g of tryptone, 5.0 g of yeast extract, 1.0 g of glucose and 10.0 g of NaCl per liter, pH
Was shaken at 37 ° C in 7.0) until the late logarithmic growth phase. The cells were recovered by centrifugation at 4 ° C., 5000 × g for 20 minutes, and then 50 mM glucose, 25 mM Tris HCl were added.
(PH 8.0), 10 mM EDTA, suspended in 10 ml of a solution containing 5 mg / ml lysozyme.

The suspension was kept at room temperature for 5 minutes, then 20 ml of 0.2 N NaOH containing 1% sodium dodecyl sulfate was added with slow stirring. Samples are kept on ice for 10 minutes, then
ml of 5M potassium acetate pH 4.8 was added. After gentle stirring, the samples were kept on ice for 10 minutes. Sample is 4 ℃, 2
Centrifuged at 000 xg for 20 minutes, cell debris and chromosomal DNA
The precipitate containing was removed. To the supernatant fraction, 0.6 volumes of isopropanol were added and the samples were left at room temperature for 15 minutes. Plasmid DNA was recovered by centrifugation at 12000 × g for 30 minutes. The precipitate was washed with ethanol, dried in a vacuum desiccator, and dissolved in a 10 mM Tris HCl, 1 mM EDTA pH 8.0 solution. RNA was removed by treatment with ribonuclease free deoxyribonuclease at a final concentration of 10 μg / ml at room temperature. After processing in this way, the sample is 1.0 ml
And then centrifuged at 40,000 × g for 6 hours. The precipitate was dissolved in 10 mM Tris-HCl, 1 mM EDTA pH8.0 solution and collected, and extracted with the same volume of buffer-saturated phenol.

Plasmid DNA is 10 mM Tris HCl, 1 mM EDTA (pH 8.0)
Was further purified by chromatography using a Bio-Gel A-150 (1 cm × 10 cm) column equilibrated with the same buffer and eluted with the same buffer. 0.5 ml fractions were collected.
The fractions containing the plasmid were collected and
DNA was collected by ethanol precipitation. Plasmid purity was confirmed by agarose gel electrophoresis. About 35 μg of the purified plasmid pPDH6 DNA was recovered.

Example 3 Production of heparinase is exemplified as follows.

Escherichia coli HB101 (pPDH6) (FERM P-11011) was
LB culture medium (containing 10.0 g of tryptone, 5.0 g of yeast extract, 1.0 g of glucose and 10.0 g of NaCl, pH 7.0, per liter) until the late logarithmic growth phase 3
Cultured in The cells were collected by centrifugation at 5000 xg for 20 minutes at 4 ° C, suspended in 50 ml of 10 mM HEPES buffer, pH 7.4, and frozen at -20 ° C. After thawing at room temperature, the cell suspension was sonicated for 10 minutes at intervals of 20 seconds using a TOMY Model UR100P ultrasonic generator to obtain a crude heparinase solution.

This crude heparinase solution had 0.6 IU of heparinase activity per one.

[Brief description of the drawings]

 FIG. 1 is a restriction map of plasmid pPDH6.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:19) (72) Inventor Peter Dobrvorski East Germany Leipzig 7050, Pelmoserstrasse 15, East Germany Science Academy Biotechnology Research Institute (56) References Experientia, Vol. 41 (1985) p. 1541-1543 Journal of Clinical Microbiology, Vol. 25 [5] (1988) p. 1070-1071 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/60 C12N 1/21 C12N 9/88 BIOSSYS (DIALOG) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A recombinant plasmid comprising a DNA fragment of about 5 to 7 kilobase pairs containing a DNA encoding heparinase derived from Bacillus sp. Strain BH100 and having the following restriction map, and a vector plasmid. . 2. The recombinant plasmid according to claim 1, wherein the recombinant plasmid is pPDH6 shown in the restriction map shown in FIG. 3. A recombinant plasmid comprising a DNA fragment of about 5 to 7 kilobase pairs containing a DNA encoding heparinase derived from Bacillus sp. And a heparinase-producing strain belonging to the genus Escherichia. 4. The heparinase-producing strain according to claim 3, wherein the recombinant plasmid is pPDH6 shown in the restriction map shown in FIG. 5. The method according to claim 5, wherein the heparinase-producing strain is transformed E. coli HB10.
The heparinase-producing strain according to claim 3, wherein the strain is one strain (pPDH6). 6. A recombinant plasmid comprising a DNA fragment of about 5 to 7 kilobase pairs having the following restriction enzyme map and containing a DNA encoding heparinase derived from Bacillus sp. Strain BH100, and a vector plasmid. A method for producing heparinase, comprising culturing a heparinase-producing strain belonging to the genus Escherichia, which retains the above, in a medium, and collecting heparinase from the culture. 7. The transformed heparinase-producing Escherichia coli HB10
7. The method according to claim 6, wherein the strain is one strain (pPDH6).