Sphingobacterium sphingobacterium, purified product heparinase III thereof, purification method and application

Abstract

The invention discloses a strain of sphingobacterium, a purified product heparinase III of the sphingobacterium as well as a purification method and application of the sphingobacterium and the purified product heparinase III, and aims to provide a strain of sphingobacterium capable of degrading heparan sulfate, which is screened and cultured from soil, and the relatively pure heparinase III is obtained through a series of purification steps. The preservation number of the sphingobacterium multiforme A1 is GDMCC No: 65091, and the sphingobacterium multiforme A1 is preserved in the Guangdong Province Microbial Culture Collection Center on September 2, 2024, the preservation number of the sphingobacterium multiforme A1 is CGMCC NO: 65091, and the sphingobacterium multiforme A1 is CGMCC NO: 65091. Belongs to the technical field of biology.

Description

Technical Field

[0001] The invention discloses a sphingobacterium, a heparinase III purified product of the sphingobacterium, a method for purifying and preparing the heparinase III by using the sphingobacterium, and an application of the heparinase III, belonging to the field of biotechnology. Background Art

[0002] Heparinase is mainly isolated from some bacteria that use heparin as a carbon source, and was originally derived from Flavobacterium heparinum or Pedobacter heparinus. In addition, heparinase has also been found in many microorganisms, such as Corynebacterium sp., Sphingobacterium sp., Bacillus subtilis, Bacillus circulans, Prevotella heparinolytica, and Bacteroides stercoris HJ-15. However, Flavobacterium heparinum is still the only source of commercial heparinase.

[0003] Heparanase III is a highly active sulfated glycoprotein that mainly acts on heparan sulfate, has a molecular weight of 66 KDa, and an isoelectric point of about 9. Due to its relatively single source (Flavobacterium heparinum), its use is severely limited. Summary of the invention

[0004] In order to solve the above problems, the present invention screened and cultured a strain of Sphingobacillus that can degrade heparan sulfate from soil, and obtained relatively pure heparinase III through a series of purification steps.

[0005] Yet another object of the present invention is a method for the purification / production of the above-mentioned heparinase III.

[0006] A final object of the present invention is the use of the above-mentioned heparinase III.

[0007] To this end, the first technical solution provided by the present invention is as follows:

[0008] A strain of SpHingobacterium multivorum A1, with a deposit number of GDMCC No: 65091, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024.

[0009] The second technical solution provided by the present invention is a heparinase III, which is the heparinase III (SMHep-III) produced by the Sphingobacterium multivorum A1 described in the first technical solution.

[0010] The third technical solution provided by the present invention is the method for producing heparinase III described in the second technical solution, which comprises the following steps in sequence: 1) culturing the sphingobacterium sphingosine under conditions suitable for the sphingobacterium sphingosine to produce the heparinase III described in the first technical solution; and

[0011] 2) Isolating and purifying the heparinase III.

[0012] Furthermore, the culturing is carried out in TSB medium and / or seed medium.

[0013] Furthermore, the production method of heparinase III comprises the following steps in sequence:

[0014] 1. Screening of Heparinase III Producing Bacteria

[0015] 1) Enrichment culture

[0016] Add 1g soil and 30mL sterile water to the test tube and mix well. Pipette 5mL supernatant into 30mL TSB medium and culture at 37℃, 200rpm for 24h. Then take 5mL culture solution and add it into 30mL fresh enrichment medium and culture at 150rpm, 37℃ for 24h to obtain bacterial solution.

[0017] 2) Initial screening

[0018] Use an inoculation loop to streak the bacterial solution prepared in step 1) on the primary screening plate culture medium. After 48-72 hours, use the photocopy method to transfer the cultured colonies to a new primary screening plate culture medium. Take the corresponding colonies for enrichment culture for 1-7 days, and detect whether the heparan sulfate concentration in the culture medium decreases. When the heparan sulfate concentration in the culture medium decreases by 20%, proceed to the next step;

[0019] 3) Fermentation

[0020] In step 2), the colony was inoculated into the seed culture medium, incubated at 37°C and 180 rpm for 24 h, and then inoculated into the fermentation medium at a volume ratio of 5%, incubated at 20-30°C and 180 rpm for 24 h, and the fermentation broth and crude enzyme extract were collected to detect whether there was heparan sulfate enzyme activity;

[0021] 4) Storage in glycerol tubes

[0022] Pick the colony corresponding to the culture medium in which the heparan sulfate concentration in the fermentation broth decreases from 100 μg / ml to 80 μg / ml for streak culture, determine the effective single colony and use an inoculation loop to streak for monoclonal culture; inoculate the single colony after monoclonal culture into the seed culture medium, cultivate it in a constant temperature shaker at 37°C at 180 rpm for 24 hours, take 500 μL of the bacterial solution and add it to a sterilized 1.5 mL centrifuge tube, then add 500 μL of sterilized 50% glycerol, mix well and store at -40°C and -80°C respectively.

[0023] 2. Fermentation of Heparinase III Producing Bacteria

[0024] Take out 10-50ul of the seed liquid stored in the glycerol tube and inoculate it into 50ml of the first-level seed culture medium, and culture it at 23-30 degrees Celsius for 16-32 hours; then transfer 5-10ml from the seed culture liquid and inoculate it into the second-level seed culture medium, and culture it under the same conditions for 16-32 hours; then inoculate the second-level seed culture liquid into the fermentation medium at an inoculation rate of 5%, culture it at 23-30 degrees Celsius for 1 day, and collect the bacteria by centrifugation.

[0025] 3. Isolation and purification of heparinase III

[0026] 1) Preparation of crude enzyme solution

[0027] The bacterial cells obtained in step 2 are suspended in 10-50 mM Tris-HCl buffer at pH 7.0, and the bacterial cells are broken by ultrasound or high pressure until the solution is clear. The supernatant and the precipitate are separated by centrifugation at 4°C and 10,000 rpm using a high-speed refrigerated centrifuge, and the supernatant is collected to obtain a crude enzyme solution, and the crude enzyme activity of the supernatant is determined; the crude enzyme solution is precipitated with ammonium sulfate and dialyzed to obtain a crude heparinase III enzyme solution to be used for column separation.

[0028] 2) SP-sepHarose column purification

[0029] The crude enzyme solution in step 1) is loaded onto a SP-sepHarose column equilibrated with 10-50 mM Tris-HCl buffer at pH 7.0. The column is washed with the same buffer and then gradient eluted with an eluent containing 0-1 M NaCl in the same buffer to collect the active portion, which is heparinase III.

[0030] Furthermore, in the above-mentioned method for purifying heparinase III, the SP-sepHarose column uses SP-sepHarose gel filler purchased from Nanovita Technologies.

[0031] Furthermore, in the above-mentioned method for purifying heparinase III, in the screening of heparinase III producing bacteria, the TSB medium described in step 1) contains 17 g / L tryptone, 3 g / L soy peptone, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose; the primary screening plate medium described in step 2) contains 1 g / L heparan sulfate, 5 g / L yeast powder, 10 g / L tryptone, 10 g / L NaCL, 20 g / L agar powder, pH 7.0; the seed culture medium described in step 3) contains 5 g / L beef extract, 10 g / L tryptone, 5 g / L yeast powder, 5 g / L NaCL, pH 7.0; the fermentation medium contains 2 g / L tryptone, 8 g / L heparan sulfate, and 10 g / L NaCL. 5g / L, (NH4)2SO41g / L, KH2PO42.5g / L, MgSO4·7H2O 0.5g / L, pH7.0;

[0032] Furthermore, in the above-mentioned method for purifying heparinase III, the ultrasound is at 422W, on for 5s and off for 5s, for 10min / 20ml.

[0033] Furthermore, the above-mentioned method for purifying heparinase III, the specific purification steps are as follows:

[0034] 1) Wash the column with sterile water and then add the filler;

[0035] 2) Equilibrate with 10 times column volume of Tris-HCl buffer (50 mM pH 7), making sure the filler is kept moist at all times;

[0036] 3) Add the treated crude enzyme to the column, and fill the column bed with buffer until all the samples enter the column bed;

[0037] 4) Connect the eluent, set the eluent gradient to 0-1M NaCl Tris-HCl buffer (50mM pH7), and take the collection tube with absorption peak to measure enzyme activity;

[0038] 5) The collected liquid was concentrated using a 30kDa ultrafiltration centrifuge tube.

[0039] The last technical solution provided by the present invention is that the above-mentioned heparinase III is used to analyze the structure of heparan sulfate and its analogs; or to detect the quality of heparan sulfate; or to prepare low molecular weight heparan sulfate.

[0040] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0041] In order to solve the problem of enzyme supply, the present invention screened and cultured a strain of bacteria capable of degrading heparan sulfate from soil, and obtained relatively pure heparinase III through a series of purification steps.

[0042] The heparinase III described in the present invention is a novel heparinase III that has not been reported before. Its physical and chemical properties are different from those of the currently known heparinase III, and it has strong selectivity for the cleavage site when enzymolyzing heparan sulfate, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a curve chart showing the effect of the pH of the reaction system on the enzyme activity of SMHep-III.

[0044] Figure 2 This is a curve chart showing the effect of reaction system temperature on SMHep-III enzyme activity.

[0045] Figure 3 This is a bar graph showing the effects of metal ions and EDTA on the enzyme activity of SMHep-III.

[0046] Figure 4 It is the thermal stability curve of SMHep-III.

[0047] Figure 5 It is the pH stability curve of SMHep-III.

[0048] Figure 6 It is a standard disaccharide analysis HPLC spectrum.

[0049] Figure 7 It is the HPLC spectrum of the structure of heparan sulfate degraded by Flavobacterium enzyme III.

[0050] Figure 8 This is the HPLC spectrum analyzed by enzymatic hydrolysis of heparan sulfate structure by heparinase III provided in Example 1. DETAILED DESCRIPTION

[0051] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0052] Source of strain: A strain was isolated from the soil of Xiansheng Village, Xinpi Town, Xingning City, Meizhou City, Guangdong Province, China. Heparinase III can be produced when using a culture medium containing heparan sulfate. The strain was cultured on a slant culture medium and stored at -70°C. Identified by Shanghai Shenggong Company, the strain is a sphingobacterium SpHingobacterium multivorum, and was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 2, 2024, with a collection number of GDMCC No: 65091, and the collection address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, and the classification name is: Sphingobacterium multivorum.

[0053] Example 1

[0054] This embodiment provides a method for preparing SMHep-III, which comprises the following steps in sequence:

[0055] 1) Screening of heparinase III-producing bacteria

[0056] 1.1) Enrichment culture

[0057] Add 1 g of soil and 30 mL of sterile water to the test tube and mix well. Pipette 5 mL of supernatant into 30 mL of TSB medium and culture at 37°C, 200 rpm for 24 h. Then take 5 mL of culture solution and add it to 30 mL of fresh enrichment medium and culture at 150 rpm, 37°C for 24 h.

[0058] The TSB culture medium described in this step contains 17 g / L tryptone, 3 g / L soy peptone, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose.

[0059] 1.2) Initial screening

[0060] Use an inoculation loop to streak the bacterial solution in 1.1) on the primary screening plate culture medium. After 60 hours, when the strain grows to a certain size, use the copy method to transfer the cultured colonies to a new primary screening plate culture medium. Take the corresponding colonies for enrichment culture for 3 days and detect whether the heparan sulfate concentration in the culture medium decreases. When the heparan sulfate concentration in the culture medium decreases by 20%, further verify it by detecting whether the crude enzyme extract has enzyme activity.

[0061] The primary screening plate culture medium in this step contains 1 / L heparan sulfate, 5g / L yeast powder, 10g / L tryptone, 10g / L NaCL, 20g / L agar powder, pH 7.0;

[0062] 1.3) Fermentation

[0063] The colonies on the photocopy master plate were inoculated into the seed culture medium, and cultured at 37°C and 180 rpm for 24 h. Then, 5% of the inoculum was inoculated into the fermentation culture medium, and incubated at 20-30°C and 180 rpm for 24 h. The fermentation broth and crude enzyme extract were collected to detect whether there was heparan sulfate enzyme activity of 0.13 U / ml.

[0064] The seed culture medium in this step contains 5 g / L beef extract, 10 g / L tryptone, 5 g / L yeast powder, 5 g / L NaCL, and a pH of 7.0; the fermentation culture medium contains 2 g / L tryptone, 8 g / L heparan sulfate, 5 g / L NaCL, 1 g / L (NH4)2SO4, 2.5 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O, and a pH of 7.0.

[0065] 1.4) Glycerol tube storage

[0066] Pick the colony corresponding to the culture medium in which the heparan sulfate concentration in the fermentation broth decreases from 100 μg / ml to 80 μg / ml for streak culture, determine the effective single colony and use an inoculation loop to streak for monoclonal culture; inoculate the single colony after monoclonal culture into the seed culture medium, cultivate it in a constant temperature shaker at 37°C at 180 rpm for 24 hours, take 500 μL of the bacterial solution and add it to a sterilized 1.5 mL centrifuge tube, then add 500 μL of sterilized 50% volume fraction glycerol aqueous solution, mix well and store at -40°C and -80°C respectively.

[0067] The seed culture medium in this step contains 5 g / L beef extract, 10 g / L tryptone, 5 g / L yeast powder, 5 g / L NaCL, and pH 7.0;

[0068] 2) Fermentation of heparinase III (SMHEP-III) producing bacteria:

[0069] Take out 25ul of the seed liquid stored in the glycerol tube and inoculate it into 50ml of the first-level seed culture medium, and culture it at 23-30 degrees Celsius for 24 hours; then transfer 7.5ml from the seed culture liquid and inoculate it into the second-level seed culture medium, and culture it under the same conditions for 24 hours; then inoculate the second-level seed culture liquid into the fermentation medium at an inoculation rate of 5%, culture it at 28 degrees Celsius for 1 day, and collect the bacteria by centrifugation.

[0070] 3) Isolation and purification of heparinase III (SMHEP-III):

[0071] 3.1) Preparation of crude enzyme

[0072] The bacterial cells obtained in step 2) are suspended in 25 mM Tris-HCl buffer at pH 7.0, and the bacterial cells are broken by ultrasound (the ultrasound is at 422 W, on for 5 s and off for 5 s, 10 min / 20 ml) until the solution is clear. The supernatant and the precipitate are separated by centrifugation at 4°C and 10000 rpm using a high-speed refrigerated centrifuge. The supernatant is collected and precipitated with ammonium sulfate and dialyzed to obtain a crude enzyme for determining the crude enzyme activity and further purification.

[0073] 3.2) SP-sepHarose column purification

[0074] The crude enzyme solution was loaded onto the SP-sepHarose column using the SP-sepHarose gel filler purchased from Nanovita Technologies. The specific purification steps are as follows:

[0075] 1) Wash the column with sterile water and then add the filler;

[0076] 2) Equilibrate with 10 times column volume of Tris-HCl buffer (10-50 mM, containing 10 mM CaCl, pH 7), making sure the filler is kept moist at all times;

[0077] 3) Add the treated crude enzyme to the column, and fill the column bed with buffer until all the samples enter the column bed;

[0078] 4) Connect the eluent, set the eluent gradient to 0-1M NaCl Tris-HCl buffer (10-50mM, containing 10mM CaCl, pH7), and take the collection tube with the absorption peak to measure the enzyme activity;

[0079] 5) The collected liquid was concentrated using a 30kDa ultrafiltration centrifuge tube and compared with the whole genome. It was preliminarily determined to be SMHep-III, and then further cloned and confirmed to be SMHep-III.

[0080] Example 2

[0081] The other steps and process parameters provided in this embodiment are the same as those in Example 1, except that the parameters of the fermentation of the heparinase III producing bacteria in step 2) are different, as follows:

[0082] Take out 10ul of the seed liquid stored in the glycerol tube and inoculate it into 50ml of the first-level seed culture medium, and culture it at 23-30 degrees Celsius for 16 hours; then transfer 5ml from the seed culture liquid and inoculate it into the second-level seed culture medium, and culture it under the same conditions for 16 hours; then inoculate the second-level seed culture liquid into the fermentation medium at an inoculation rate of 5%, culture it at 23 degrees Celsius for 1 day, and collect the bacteria by centrifugation.

[0083] Example 3

[0084] The other steps and process parameters provided in this embodiment are the same as those in Example 1, except that the parameters of the fermentation of the heparinase III producing bacteria in step 2) are different, as follows:

[0085] Take out 50ul of the seed liquid stored in the glycerol tube and inoculate it into 50ml of the first-level seed culture medium, and culture it at 23-30 degrees Celsius for 32 hours; then take out 10ml of the seed culture liquid and inoculate it into the second-level seed culture medium, and culture it under the same conditions for 32 hours.

[0086] hours; then the secondary seed culture solution was inoculated into the fermentation medium at a rate of 5%, cultured at 30 degrees Celsius for 1 day, and the bacteria were collected by centrifugation.

[0087] Example 4

[0088] The other steps and process parameters provided in this embodiment are the same as those in Example 1, except that the fermentation method and parameters for preparing the crude enzyme in 3.1) are different, as follows:

[0089] The bacterial cells obtained in step 2) were suspended in 10 mM Tris-HCl buffer at pH 7.0, and the bacterial cells were broken by ultrasound (the ultrasound was at 422 W, on for 5 s and off for 5 s, 10 min / 20 ml) until the solution was clear. The supernatant and precipitate were separated by centrifugation at 4 ° C and 10000 rpm using a high-speed refrigerated centrifuge. The supernatant was collected and precipitated with ammonium sulfate and dialyzed to obtain a crude enzyme for determining the crude enzyme activity and further purification.

[0090] Example 5

[0091] The other steps and process parameters provided in this embodiment are the same as those in Example 1, except that the fermentation method and parameters for preparing the crude enzyme in 3.1) are different, as follows:

[0092] The bacterial cells obtained in step 2) are suspended in a 150 mM Tris-HCl buffer solution with a pH of 7.0, and the bacterial cells are broken by high pressure (the high pressure is to use a high-pressure homogenizer to break the cells for 3 to 5 cycles at 4 degrees Celsius and 1600 to 1800 bar) until the solution is clear, and the supernatant and precipitate are separated by centrifugation at 4°C and 10,000 rpm using a high-speed refrigerated centrifuge, and the supernatant is collected and precipitated with ammonium sulfate and dialyzed to obtain a crude enzyme for determining the crude enzyme activity and further purification.

[0093] In order to verify the SMHep-III provided by the present invention, the property research experiment of SMHep-III is given below.

[0094] 1) Effect of reaction system pH on SMHep-III enzyme activity

[0095] Prepare a Tris-hcl (50mM) solution with a pH of 6-11, place it at room temperature, and add HS within 30 minutes before measuring enzyme activity to make the concentration reach 1%. Take 2.5ml of the buffer solution with HS added and mix it with 20ul of enzyme solution for 15 minutes to detect the change in the absorbance value at 232nm. For enzyme activity measurement, the negative control should be tested. Repeat all data three times and calculate the standard deviation; the results are referred to Figure 1 It can be seen that under the condition of pH 7, the enzyme activity value of heparinase III (0.575 IU / ml) is the largest, so this pH is the optimal pH of heparinase III; and in the pH range of 7-8, the enzyme activity is higher; when the pH is greater than 8, the enzyme activity decreases rapidly.

[0096] 2) Effect of reaction system temperature on SMHep-III enzyme activity

[0097] The optimal reaction pH = 7 buffer and substrate solution (1 mg / mL HS, 50 mM Tris-HCl) were incubated at 55 ° C, 50 ° C, 45 ° C, 40 ° C, 35 ° C, 30 ° C, and 25 ° C for 10 min, 2.5 ml of the optimal pH = 7 buffer (1 mg / mL HS, 50 mM Tris-HCl) and 20 μL of enzyme solution were mixed evenly for the determination of the enzyme activity of SMHep-III, and the incubation temperature corresponding to the maximum enzyme activity was selected as the optimal reaction temperature. Figure 2 It can be seen that at a temperature of 35°C, the enzyme activity value of heparinase III (0.6 IU / ml) is the largest, so this temperature is the optimal temperature for heparinase III; in the range of 25-45°C, the enzyme activity is relatively high; above 45°C, the enzyme activity decreases rapidly.

[0098] 3) Effects of metal ions and EDTA on the activity of SMHep-III enzyme

[0099] Add 10 mM Na + Mg 2+ , Ca 2+ , Ba 2+ , Mn 2+ and EDTA to the optimal pH = 7 buffer (1mg / mL HS, 50mMTris-HCl), and incubate at the optimal reaction temperature of 35°C for 10min. Add each component according to the enzyme reaction system (add 20μl of enzyme to 2.5ml of 1mg / mL heparan sulfate 50mMTris-HCl buffer) and use a UV spectrophotometer to measure the change in absorbance at 232nm, and calculate the SMHep-III specific activity to study the effects of metal ions and EDTA on the activity of SMHep-III. Figure 3 It can be seen that Mn 2+and EDTA inhibited the activity of heparinase III, with EDTA completely inactivating the enzyme; while Na + , Ca 2+ Mg 2+ , Ba 2+ They all play an enhancing role in the activity of heparinase III, among which Ba 2+ The effect (0.88IU / ml) is the strongest.

[0100] 3.4 Thermal stability of SMHep-III

[0101] The enzyme solution was incubated in a water bath with buffer solution at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 5°C, and 60°C for 1 h, and each component was added according to the enzyme reaction system (20 μl of enzyme was added to 2.5 ml of 1 mg / mL heparan sulfate 50 mM Tris-HCl buffer) and the change in absorbance at 232 nm was measured using an ultraviolet spectrophotometer, and the specific activity of SMHep-III was calculated to study its thermal stability. Figure 4 It can be seen that SMHep-III maintains a high activity in the range of 25-35°C. Once the temperature exceeds 40°C, the enzyme activity decreases rapidly. Therefore, heparinase III is sensitive to temperature.

[0102] 3.5 pH stability of SMHep-III

[0103] Store 180 μl of buffer (1 mg / mL heparan sulfate, 50 mM Tris-HCl) with pH values ​​of 6, 7, 8, 9, 10, 11, and 12 and 20 μl of enzyme solution at 4°C for 14 h, take 20 μl and add it to 2.5 ml of buffer containing substrate (1 mg / mL heparan sulfate, 50 mM Tris-HCl), add each component according to the enzyme reaction system (add 20 μl of enzyme to 2.5 ml of 1 mg / mL heparan sulfate 50 mM Tris-HCl buffer) and use a UV spectrophotometer to measure the change in absorbance at 232 nm, and calculate the specific activity of SMHep-III to study the pH stability of SMHep-III. Figure 5 It can be seen that SMHep-III can maintain a high activity in the pH range of 6-8, and the optimal storage pH is 8.

[0104] Application Example 1

[0105] The heparinase III provided in Example 1 is used to analyze the structure of heparan sulfate and its analogs. The heparan sulfate is accurately weighed, dissolved in water and quantitatively diluted to a 20 mg / mL solution; 20uL of the above solution is accurately measured and mixed with 70uL of calcium acetate solution (pH7.0) and 100uL of SMHep-III enzyme solution; enzymolysis is carried out in a water bath at 25 degrees Celsius for 48 hours to obtain a test solution. The test solution is loaded onto HPLC for disaccharide component analysis to obtain a disaccharide spectrum, see Figure 6-8 The disaccharide profile obtained by this enzyme has completely different disaccharide components from the disaccharide profile obtained by Flavobacterium enzyme III. This new heparinase III can provide a more comprehensive perspective for the structural analysis of heparan sulfate and its analogs.

Claims

1. A strain of SpHingobacterium multivorum A1, characterized in that: The deposit number is GDMCCNo:65091, and it was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024.

2. A heparinase III, characterized in that The heparinase III is produced from the Sphingobacterium multivorum A1 according to claim 1.

3. A method for producing heparinase III, characterized in that: The method comprises the following steps in sequence: 1) culturing the Sphingobacterium sphingosine under conditions suitable for the Sphingobacterium sphingosine of claim 1 to produce the heparinase III; and 2) Isolating and purifying the heparinase III.

4. The method according to claim 3, characterized in that The culturing is carried out in TSB medium and / or seed medium.

5. The method according to claim 4, characterized in that The TSB culture medium contains 17 g / L of tryptone, 3 g / L of soy peptone, 5 g / L of sodium chloride, 2.5 g / L of dipotassium hydrogen phosphate, and 2.5 g / L of glucose; and / or the seed culture medium contains 5 g / L of beef extract, 10 g / L of tryptone, 5 g / L of yeast powder, 5 g / L of NaCL, and the pH value is 7.0; the fermentation culture medium contains 2 g / L of tryptone, 8 g / L of heparan sulfate, 5 g / L of NaCL, 1 g / L of (NH4)2SO4, 2.5 g / L of KH2PO4, and 0.5 g / L of MgSO4·7H2O, and the pH value is 7.

0.

6. The method according to claim 3, characterized in that The separation and purification steps include i) disrupting the bacterial cells and centrifuging to separate the supernatant and the precipitate; ii) precipitating the supernatant with ammonium sulfate and dialyzing; and iii) chromatographic purification, such as purification using an SP-sepHarose column.

7. Use of the heparinase III according to claim 2 for analyzing the structure of heparan sulfate and its analogs.

8. Use of the heparinase III according to claim 2 for detecting the quality of heparan sulfate; or for preparing low molecular weight heparan sulfate.

9. Use of the heparinase III according to claim 2 for preparing heparan sulfate for degrading in vivo and in vitro.

10. A composition or kit for analyzing the structure of heparan sulfate and its analogs, for detecting the quality of heparan sulfate and / or for preparing low molecular weight heparan sulfate, characterized in that: Contains the heparinase III as described in claim 2.

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