JPH03108486A - Plasmid to code heparinase, heparinase producing strain having same plasmid and production of heparinase - Google Patents

Abstract

NEW MATERIAL:A recombinant plasmid comprising heparinase gene DNA and a vector plasmid by providing a host bacterium belonging to the genus Escherichia coli with ability of producing heparinase. USE:For producing heparinase. PREPARATION:A heparinase gene DNA fragment is cut out from chromosome DNA with a restriction enzyme, a vector plasmid is cleft with a proper restriction enzyme not interfering in genetic information of plasmid essential for plasmid duplication and a recombinant plasmid is formed from chromosome fragrant and DNA ligase.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a novel plasmid encoding heparinase,
The present invention relates to a new heparinase producing strain containing this plasmid and a new method for producing heparinase.

(Prior Art) Heparinase is derived from heparin, i.e., -4)-2-deoxy-2-sulfamino-α as its main repeating unit.
-D-glucobylanose-6-sulfate-(1,4
) -α-L-idopyranosyluronic acid-2-sulfuric acid-
(1-) is an enzyme that cleaves certain glycosidic bonds of sulfated gelucosaminoglycans. The product of this enzymatic activity is a shortened fragment of the heparin chain. Heparinase, also known as heparitin (heparan sulfate In other words, it cleaves a sulfated polymer with a chemical structure similar to heparin, producing short-chain fragments of heparitin.

The substrate heparin is widely used as an anticoagulant, and thus heparinase has diverse applications in the study of heparin structure, the study of blood clotting mechanisms, and the enzyme of heparin in body tissues and fluids.

Heparinase is also used to prepare small heparin fragments that have potential therapeutic value as antithrombin or antitumor agents.

A relatively small number of microorganisms are known to produce enzymes that can degrade heparin. Heparinase (heparin lyase, EC4, 2, 2, 7) is produced by Flavobacterium hevarinum, Flavobacterium sp.
It has been found in cultures of Bacteroides sp., Bacteroides heparinolyticus, Peptostreptococcus and Niebacterium. These heparinases are described in the Journal of Biochemistry [
Vol. 233, p. 853 (1956) 1, Exverientia [Vol. 41, p. 1541 (1985) 1
, Matsumoto Dental University Bulletin (Japan) [Vol. 8, p. 15 (198
2nd year) 1, Journal of Applied Acid Environmental Microbiology [Volume 46
No. 1252 (1983) 1, Journal of Clinical Microbiology [Volume 26, No. 10
70 pages (1988) 1, and Journal of the South African Veterinary Association [
It is disclosed in Vol. 53, p. 214 (1982) 1. In addition, two of the inventors have recently discovered the only known Bacillus species capable of producing heparinase. This bacterium, named Bacillus sp.
No. 8) was given. This strain was obtained by patent application No. 63-2978.
No. 07 (November 25, 1988), in a new method for producing heparinase.

A "plasmid" is a non-chromosomal genetic element composed of circular molecules of deoxyliponucleic acid found within microbial cells. By using conventional gene cloning techniques, fragments of DNA carrying the genetic information for the production of useful proteins or peptides can be introduced into suitable plasmids, called "vector" plasmids. When the resulting recombinant plasmid is introduced into a suitable host organism, the transformed host organism often produces useful plasmid-encoded proteins or peptides. Typically, E. coli is used as the host organism. A plasmid carrying a heparinase gene is new, a heparinase-producing strain of E. coli carrying such a plasmid is also new, and a method for producing heparinase using an E. coli strain carrying a plasmid encoding heparinase is also new.

[Problem to be solved by the invention]

Traditional methods for producing heparinase using Flavobacterium hevarinum, Bacteroides hevarinolyticus or Bacillus sp. 811100 strain required the addition of heparin to the growth medium to induce heparinase synthesis. The need to add heparin as an inducer has added considerable cost to traditional heparinase production methods. A method of producing heparinase in a ligneous manner, ie, using a plasmid-carrying E. coli strain that does not require the addition of heparin as an inducer, is advantageous in producing the enzyme at a lower cost.

With the intention of creating a strain that essentially produces heparinase, we developed Bacillus sp.

Construct a new recombinant plasmid consisting of heparinase gene DNA from the chromosome of B11100 strain (FBRM P-10408) and vector plasmid pBR329,
Next, this new recombinant plasmid was introduced into E. coli II B 101 strain, which is a host bacterium. The host bacteria transformed by the recombinant plasmid produced heparinase without the need for the addition of heparin as an inducer, and as a result, the inventors completed this invention.

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

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

Another object of the invention is to provide a new method for heparinase production using plasmid-carrying strains.

[Means to solve 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 produced by excising the heparinase gene DNA fragment from the chromosomal DNA using a restriction enzyme, then cutting the vector plasmid with an appropriate restriction enzyme that does not interfere with the plasmid genetic information essential for plasmid replication, and then cutting this chromosomal fragment and the vector cut with the restriction enzyme. It can be produced by a method consisting of treating a plasmid with DNA ligase to form a recombinant plasmid and isolating the recombinant plasmid.

The novel strain according to the present invention belongs to the genus Escherichia that contains a novel plasmid and produces heparinase, for example, Escherichia coli strain H8101 (pPDF
16) (FERM P-11011) i? be.

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

The present invention will be explained in detail below.

(2) New plasmid and its construction According to the present invention, it is possible to construct a new plasmid that provides the host with the ability to produce heparinase. 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. A typical example is pBR322゜pBR329, Cot
El, pMB9. pscloL, pHc79 are listed.

The DNA fragment that can be used in the present invention includes a chromosomal gene encoding heparinase, and includes Bacillus sp.
It is produced by cutting DNA from BII100 strain (FERM P-10408) with restriction enzymes. Bacillus sp. strain 88100 isolated from soil is the first
Indicates the taxonomic properties shown in the table.

Table 1 Morphological Properties Durham Staining Cell Size Spore Amount Swelling Spore Location Paraspore Crystal Motility Growth Characteristics Aerobic Growth Anaerobic Growth Growth Temperature Maximum Minimum Optimal Lysozyme (0,001%) Positive 0.6-0 .8μta 5% 7% 10% Growth at pH 6,8 (Nutritional Medium) Growth at pH 5,7 (Sabouraud Medium) Requirements for NaCl and KCI Requirements for Growth Factors Biochemical Properties Catalase VP Test Sea P Medium pH Acid production From D-ribose to D-xylose to mustard - from Rhamnose to D-galactose to D-fructose Negative Positive Negative Negative Negative Positive Positive No Negative Positive Negative 6.5-7.0 Positive Positive Positive Positive Positive D-arabinose Mustard - From arabinose to raffinose to D-mannose to maltose to sucrose to lactose to D-glucose to D-trehalose to glycerol to D-mannitol to sorbitol to m-erythritol to inositol to adonitol to fermentation Gas production from carbohydrates Indole production Dihydroxyacetone Formation Crystalline Dextrin Formation Reduction of Nitrate Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive Negative Negative Negative Assimilation of Citrate Positive Assimilation of Propionate Negative Decomposition of Tyrosine
Hydrolysis of negative starch Hydrolysis of negative casein Degradation of negative heparin Degradation of positive heparan sulfate
Degradation of positive chondroitin-4-sulfate
Degradation of positive dermatan sulfate Degradation of positive chondroitin-6-sulfate Degradation of positive hyaluronic acid Degradation of positive polygalacturonic acid
GC content in negative DNA 57
Conventional methods are used to construct a plasmid carrying the mole % heparinase gene of the present invention. for example,
Bacillus sp, BH100 strain (FERM P-104
08) The derived chromosomal DNA is cut with an appropriate restriction enzyme to generate fragments. The chromosomal DNA fragments are then fractionated depending on their size, for example, by sucrose density gradient ultracentrifugation or agarose gel electrophoresis. can.

This fragment can also be used without such size fractionation. The vector plasmid can be linearized by cutting with an appropriate restriction enzyme and then treated with an appropriate phosphatase to dephosphorylate it. Furthermore, linear vector plasmids can also be used without dephosphorylation. After being treated in this manner, the vector plasmid is mixed with the chromosomal DNA fragment, and this mixture is suitably treated with gauze to produce recombinants. This recombinant DNA is then introduced into a suitable host bacterium, such as E. coli strain HB101. The transformed host bacterium that produces heparinase is
For example, it is detected by using enzyme-linked immunoassays with antisera specific for purified Bacillus heparinase, or by testing host cell heparinase activity, or both. Plasmid DN containing genetic information for heparinase production
A is isolated from heparinase-producing host bacteria by conventional methods.

The new plasmid pPDH6 is constructed as follows. Chromosomal DNA isolated from Bacillus sp. strain 88100 is partially digested with the restriction enzyme Sau 3 A and size fractionated by sucrose density gradient ultracentrifugation. A fragment of approximately 5-7 kilobase pairs is recovered. this DNA
is digested with BamHr restriction enzyme, recombined into dephosphorylated vector plasmid ρBR329, and introduced into E. coli strain 18101. Transformed host bacteria that produce heparinase can be tested using an enzyme-linked immunoassay with purified Bacillus heparinase-specific antisera, or the heparinase activity of the host bacteria can be tested, either or both. Detected by Plasmid pPDR6 is isolated from heparinase-producing host bacteria by conventional methods. As shown in Figure 1, the restriction enzyme map of the novel plasmid pPD)16 shows that plasmid pPD)16 is a circular molecule of DNA consisting of about 9.7 kilobase pairs, and Bacillus sp.
It shows that it is composed of vector plasmid pBR329 into which a chromosomal DNA fragment derived from strain 1 was inserted.

2. Production of heparinase-producing strain A novel heparinase-producing strain of the genus Escherichia was obtained according to the present invention. A recombinant plasmid constructed by recombining chromosomal DNA derived from Bacillus sp. strain 81100 and a vector plasmid is introduced into a host bacterium of the Escherichia genus by a conventional transformation method. The host bacteria that can be used in the present invention may be any strain of the genus Escherichia.

Typical host bacteria used are E. coli and E. coli.
! This is the large intestine i1) 18101 strain, which is a type H strain (hybrid 5 train) of B strain A. A transformed strain produced by introducing a heparinase-encoding plasmid into an Escherichia strain is microbiologically identical to the host, except for the plasmid-encoded characteristics, such as resistance to antibiotics and the ability to produce heparinase. have characteristics. For example, plasmid pPDH6 was transferred to E. coli 1810
Transformed E. coli I produced by introducing into one strain
I B 101 strain (pPDH6) has the same microbiological characteristics as the old E. coli H81 strain [genotype: F'',
hsds20 (rB-+ mB-), rect 1
3+ ara-14, ProA2.1acYi gal
2. rpsL20(Sm), xyl-5, mtl
-1,5upE44 λ) and is further characterized by the properties encoded by plasmid pPDH6: ampicillin resistance, chloramphenicol resistance and heparinase production.

As a culture medium for the heparinase-producing strain of the present invention, a conventional medium containing a carbon source, a nitrogen source, and inorganic salts is used. Any utilizable carbon source 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, tryptone, meat extract, cornstarch liquor amino acid solution, or readily available inorganic nitrogen sources such as ammonium sulfate and ammonium chloride. This can be exemplified as Furthermore, in addition to the carbon source and nitrogen source mentioned above, it is also possible to add various salts such as magnesium chloride, sodium phosphate, potassium phosphate, potassium chloride, calcium chloride, and the like. It is also possible to add vitamins and other growth factors such as folic acid, calcium pantothenate, biotin, riboflavin, and thiamine. Furthermore, if necessary, it is also possible to add a gelling agent such as powdered agar or gelatin. The medium is prepared by adding acid or alkali as appropriate.
t5. Adjust the temperature within the range of o to 8.0 and sterilize by autoclaving.

As a specific example of a suitable medium, tryptone per 11
0g 1 yeast extract 5g 1 glucose 1g and NaC
An example is a liquid medium containing 110 g and adjusted to pH 7.0.

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

3. Method for obtaining heparinase The heparinase producing strain of the present invention can be grown aerobically at 35 to 37 degrees in any suitable medium as mentioned above.
Cultured at C for 12-70 hours.

Separation and purification of heparinase is performed, for example, as follows. Cells are harvested from the culture medium by centrifugation, filtration, or other conventional methods and disrupted by sonication, French press pressure, 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 conventional methods well known in the field of protein purification, such as conventional ion exchange and gel filtration methods, loftography methods, chromatofocusing, preparative isoelectric focusing methods, The heparinase is fractionated by using one or both of electrophoresis and the like, thereby obtaining heparinase.

Suitable methods for obtaining heparinase are illustrated 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 solution is centrifuged at 8000×g for 30 minutes to collect cells. Furthermore, the collected cells are subjected to ultrasonic pulverization to form a crude heparinase solution, which is used as a sample for the next step. The sample is applied to a sulfated cellulofine force column equilibrated in pH 7, 410 mM IIEPES buffer, and the adsorbed enzyme is eluted using the same buffer with a concentration gradient of 10 to 0.3 M NaCl. The collected active fraction was transferred to Amicon P
Concentrate by ultrafiltration using a MIO membrane. The sample is further fractionated by gel filtration chromatography using a 5HODEX WS-2003 column equilibrated with the above buffer containing 100 mM NaCl. The active fraction thus obtained contained only a single heparinase, which was detected as one protein band when analyzed by SO3-polyacrylamide gel electrophoresis.

The activity measurement method using ultraviolet absorption used for quantitative analysis of heparinase activity is as follows.

20 μl of appropriately diluted enzyme solution and p) 17.4.50
50μ of a 5μ/ml heparin solution dissolved in mM HEPES buffer! The containing reaction mixture is kept at 40°C for 4 hours.

After that, the reaction was carried out at pH 2,0,20mM KCI-MCI
The solution was stopped by adding 2.0-2% of this reaction mixture.
Measure the absorbance at 32 nm. One international type of activity was based on the molar extinction coefficient of double bonds, 5100c+r'M-', and was defined as the amount of enzyme required to generate 1M mol of double bonds in 1 minute at 40°C.

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

) Action: Heparinase catalyzes the elimination reaction of heparin and heparasulfate, producing short-chain glycosaminoglycan fragments. Since double bonds are simultaneously generated at this time, these exhibit ultraviolet absorption at 232 nm.

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

iii) Optimal temperature and thermal stability: 5. OmM Ca
The optimum reaction temperature in the presence of Cl'' was 45°C (30 minutes reaction), and more than 80% of the activity remained after heating at 45°C for 60 minutes.

Calcium ions are required for stabilization of this enzyme.

Note that this enzyme is completely inactivated by heating at 60°C for 15 minutes.

iv) Optimal pH: The optimal pH for the reaction is pH 1.2 to 7.8.
is within the range of

■) Influence of inorganic ions: Enzyme activity is “””+ Mg”
In the presence of chloride of and Ba'', it increases by about 50%.

0.5mM co”°+ Zn”, Fe”, Hg”
, Co'' and 1.0 mM Ni'' the activity decreases in the presence of chloride.

Mn2Z Li'' and 8g1 chloride have no substantial effect on activity at concentrations up to 5.0 mM.

Example 1 The construction of a novel plasmid containing a heparinase gene and the production of a heparinase producing strain are shown below.

(1) Production of chromosomal DNA containing heparinase gene Heparinase-producing Bacillus sp, 88100 strain (F
Chromosomal DNA was isolated from ERM P-10408) using the phenol extraction method. This DNA sample was digested with the restriction enzyme Sau 38 at 37°C for 5.10°20
and partially cut for 30 minutes to produce fragments.

The fragments were size fractionated by sucrose density gradient ultracentrifugation using a 5-20% sucrose gradient. After centrifugation at 23,000 rpm for 17 hours at 4°C, fractions were collected, and the size of the DNA fragments in each fraction was confirmed by agarose gel electrophoresis.

(2) Insertion of heparinase gene chromosomal DNA into vector plasmid Plasmid pBR used as vector plasmid
329 (International Biotechnology rn
c, + USA; carries ampicillin resistance, chloramphenicol resistance, and tetrazycline resistance genes.

) was treated with the restriction enzyme BamHI for 1 hour at 37°C and then with bacterial alkaline phosphatase for 1 hour at 37°C. These enzymes were inactivated and removed by phenol extraction and ethanol precipitation of the plasmids. Approximately 5 to 7 kilobase pairs (2
The fragment of gg) was mixed with Ban+HI-cut, dephosphorylated vector plasmid pBR329 (0.5 μg) and this mixture was treated with bacteriophage T4 DNA ligase for 16 hours at 16°C.

A recombinant plasmid containing the inserted chromosomal DNA was thus produced. This reaction requires a sample of 65
It was stopped by heating at °C for 5 minutes. Recombinant plasmids were then ethanol precipitated and recovered.

(3) Transformation of a plasmid carrying a heparinase gene into a host bacterium E. coli strain H8101 was prepared using 10 ml of LB medium (10 g of tryptone and 5 g of yeast extract per liter).

The cells were cultured at 37°C in a medium (containing glucose Ig and 110 g of NaC, pH 7.0) with shaking until the late logarithmic growth phase. Cells were collected by centrifugation and suspended in 1 m CaC1z at a final concentration of 30 mM with the addition of 0.1 ml of ice-cold CaCl□ to obtain competent cells. Combine 50 μl of this cell suspension and 5 μl of the recombinant DNA solution obtained in step (2), and place on ice for 25 min.
Hold for minutes. The mixture contains plasmid DNA in host cells.
To introduce A, heat at 42°C for 1-2 minutes. This cell suspension was inoculated into fresh LB medium, and the culture was
The cells were incubated at 7°C for 60 minutes with shaking. Cells were then collected by centrifugation, diluted appropriately in LB medium, and plated on solid growth medium containing chloramphenicol to obtain transformants.

(4) Isolation of heparinase producing strain carrying plasmid pPDI+6 The transformed host bacterium is Bacillus sp.

Heparinase production was tested by an enzyme-linked immunoassay using an eel polyclonal antiserum specific for purified heparinase from strain 811100 (FERM P-10408).

According to the directions for use, use the picoBLUE Immu-noDETEcTI
Colony assays were performed on nitrocellulose filters using ON KIT (S, tratagene)].

Among the 1200 colonies showing insertional inactivation of the tetracycline gene in the plasmid vector, 7 recombinant clones showing strong immune signals were obtained. One example of these transformations is with plasmid pPD)I6,
E. coli 11BI that produces enzymatically active heparinase
The OI strain (pPDH6) was isolated as a single strain. This strain has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
The deposit number FERM P-11011 was given.

Example 2 The production and purification of plasmid pPDl16 is illustrated below.

E. coli HBIOI (pPDHI6) (FERM P-
No. 11011) and 500 mN of B medium (10.0 g of tryptone and 5.0 g of yeast extract per liter).
Guru]-su1. Contains Og and NaC110, Og,
The cells were cultured at 37° C. with shaking until the late logarithmic growth phase. This bacterial cell is 4°CC15000
Collected by centrifugation for 20 minutes at 50mM glucose, 25mM Trif! , tlcI (pH 8,0
), 10mM EDTA, 5mg/ml lysozyme? It was suspended in 10 ml of liquid.

The suspension was kept at room temperature for 5 minutes and then 20ml of 0.2N NaOH containing 1% sodium dodecyl sulfate was added with slow stirring. Samples were kept on ice for 10 minutes and then treated with 15 tttl of 5M potassium acetate pH 4,
8 was added. After stirring gently, place the sample on ice for 1 hour.
It was held for 0 minutes. The sample was heated at 2000×g at 4°C for 20
The mixture was centrifuged for 1 minute, and the precipitate containing cell debris and chromosomal DNA was removed. 0.6 volumes of isopropanol was added to the supernatant fraction, and the samples were incubated at room temperature for 15 minutes. Plasmid DNA was recovered by centrifugation at 12,000 x g for 30 minutes. The precipitate was washed with ethanol, dried in a vacuum desiccator, and washed with 10111M Tris 11C+, 1
Dissolved in mM EDTA pH 8.0 solution. R.N.
A was removed by treatment with deoxyribonuclease-free ribonuclease at a final concentration of 10 μg/ml at room temperature. After this treatment, the sample was 1.0 d
of I M NaCl and centrifuged at 40,000 x g for 6 hours. Precipitate with 10mM) Lis HCI.

It was extracted with an equal volume of buffer-saturated phenol, dissolved in a 1 mM EDTA pH 8.0 solution and collected.

Plasmid DNA was prepared using 10mM) Squirrel HCI,
Blo-Ge1 A150 (1 cu + X loc
It was further purified by chromatography using a column of m) and eluted with the same buffer.

Fractions of 0.5 rtrfl were collected. Both aliquots containing plasmid were collected and plasmid DNA was collected by ethanol precipitation. The purity of the plasmid was confirmed by agarose gel electrophoresis. Approximately 35 μg of purified plasmid pPDH6 DNA was recovered.

Example 3 Production of heparinase is exemplified as follows.

E. coli HBIOI (pPDH6) (FERM P-1
1011) at 37°C with shaking, and cultured in LB medium [tryptone 10 per liter] until the late logarithmic growth phase.
．． Og, yeast extract 5.0g, glue: 7-su 1
．． Contains Og and NaC110, Og, pH 7.0
) Cultured in 3f. Cells at 4°C, 5000Xg
Collect by centrifugation for 20 minutes and add 10mM of 50In1.
It was suspended in HEPES buffer, pH 7.4, and frozen at 20'C. After thawing at room temperature, the cell suspension was
Using a Model UR100P ultrasonic generator, the mixture was sonicated for 10 minutes at 20 second intervals while cooling on ice to obtain a crude heparinase solution.

This crude heparinase solution had a heparinase activity of 0.61.0 per liter.

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme map of plasmid pPDH6. Diagram

Claims (1)

[Scope of Claims] 1. A host bacterium belonging to the genus Escherichia that has the ability to produce heparinase, and has heparinase gene DNA.
A recombinant plasmid consisting of a vector plasmid and a vector plasmid. 2. DNA of Bacillus sp. BH100 stock (FERM
The plasmid according to claim 1, wherein the plasmid is DNA derived from P-10408). 3. The plasmid according to claim 1, wherein the plasmid is pPDH6. 4. A heparinase-producing strain belonging to the genus Escherichia that carries a recombinant plasmid consisting of heparinase gene DNA and a vector plasmid; 5. A heparinase-producing strain belonging to the genus Bacillus having a heparinase gene BH100 strain (FERMP-1040
8) The heparinase producing strain according to claim 4, which retains a recombinant plasmid containing DNA derived from the above. 6. The heparinase producing strain according to claim 4, wherein the heparinase producing strain retains plasmid pPDH6. 7. The heparinase producing strain is E. coli HB101 strain (pPD
The heparinase producing strain according to claim 4, which is FERMP-11011). 8. Cultivating in a medium a heparinase producing strain belonging to the genus Escherichia that holds a recombinant plasmid composed of heparinase gene DNA and a vector plasmid,
A method for producing heparinase, which comprises collecting heparinase from a culture. 9. Bacillus sp. in which the heparinase-producing strain has the heparinase gene. HB100 strain (FERMP-1040
8) The production method according to claim 8, wherein the recombinant plasmid containing the derived DNA is retained. 10. The heparinase producing strain is E. coli HB101 (pPD
H6) (FERMP-11011), the manufacturing method according to claim 8.