Heparinase III with high activity on 6-sulfated heparin and application of heparinase III

Abstract

The invention relates to heparinase III with high activity on 6-sulfated heparin and application of the heparinase III. The amino acid sequence of the heparinase III (BeHepIII) is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention discovers and discloses a novel heparinase III (BeHepIII) for the first time, the heparinase III is derived from Bacteroides eggerthii, has a higher recombinant expression level in escherichia coli, has very high degradation activity on heparin containing a 6-site sulfated structure, and particularly has the enzyme activity which is 93.5 times of that of commercialized heparinase FhHepIII at present, and the heparinase III can be used for degrading heparin containing 6-site sulfated structure. Therefore, the heparinase III (BeHepIII) provided by the invention has a wide application potential in heparin degradation. In addition, by using heparinase III (BeHepIII), directional excision of a reducing end of heparin oligosaccharide synthesized by a chemical enzyme method is realized, and complete chemical enzyme method synthesis of a pentasaccharide structure with anticoagulant factor Xa activity is successfully realized.

Description

Technical Field

[0001] The present invention relates to a heparinase III with high activity against 6-O-sulfated heparin and its applications, belonging to the field of biomedical technology. Background Art

[0002] Heparinases are a class of important polysaccharide-degrading enzymes, mainly used for degrading heparin and heparan sulfate. According to their substrate specificity, heparinases are divided into three families: heparinase I, II, and III. Heparinase I tends to degrade the highly sulfated regions of heparin, but has low activity against heparan sulfate (low-sulfated heparin); while heparinase II has a broad selectivity for heparin and heparan sulfate. In contrast, heparinase III selectively cleaves heparin at the less sulfated structural regions and does not disrupt the antithrombin III binding site. Heparinase III has been widely used as a tool enzyme for determining the sequences of heparan sulfate, heparin, and low molecular weight heparin, as well as for the preparation of low molecular weight heparin.

[0003] In the enzymatic production of low molecular weight heparin (LMWH), heparinase III from Flavobacterium heparinum (FhHepIII) is usually used as a biocatalyst, and this enzyme has been commercialized. However, due to the substrate specificity of heparinase III, the enzyme has low degradation activity against heparin containing 6-O-sulfated structures.

[0004] By simulating the biosynthetic pathway of heparin in vivo, a chemoenzymatic synthesis technology mainly based on multi-enzyme catalysis supplemented by mild chemical synthesis has now been successfully developed. This synthesis technology requires the use of glycosyltransferases to extend a chemically synthesized starting substrate. Previous attempts to remove the chemical groups at the ends of synthetic heparin molecules by chemical means and to maintain the integrity of the sugar chain and antithrombin factor Xa activity as much as possible have not achieved ideal results.

[0005] Therefore, it is highly necessary to develop a novel heparinase III with high activity against 6-O-sulfated heparin, which can be used for preparing oligosaccharide skeletons and synthesizing heparin oligosaccharides. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a heparinase III with high activity against 6-O-sulfated heparin and its applications.

[0007] Term Explanation:

[0008] GlcA-pNP: p-nitrophenyl-β-D-glucuronide;

[0009] UDP-GlcNTFA: uridine diphosphate-N-trifluoroacetylglucosamine;

[0010] UDP-GlcA: Uridine diphosphate-glucuronic acid;

[0011] PAPS: 3'-Phosphoadenosine-5'-phosphosulfate;

[0012] NaKfiA: N-Acetylglucosaminyltransferase;

[0013] PmHS2: Heparin backbone synthase 2;

[0014] NST: N-Sulfotransferase;

[0015] C5-epi: C5-Epimerase;

[0016] 2OST: 2-O-Sulfotransferase;

[0017] 6OST: 6-O-Sulfotransferase;

[0018] 3OST: 3-O-Sulfotransferase;

[0019] FhHepIII: Heparinase III.

[0020] The technical solution of the present invention is as follows:

[0021] In the first aspect of the present invention, there is provided a heparinase III (BeHepIII) highly active against 6-O-sulfated heparin, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0022] Preferably according to the present invention, the heparinase III (BeHepIII) is derived from Bacteroides eggerthii, and this enzyme has a high recombinant expression level in Escherichia coli. More importantly, the enzyme activity of the heparinase III (BeHepIII) of the present invention is 93.5 times that of the currently commercial FhHepIII enzyme derived from Flavobacterium heparinum.

[0023] In the second aspect of the present invention, there is provided a recombinant expression vector, which contains the encoding gene of heparinase III (BeHepIII).

[0024] Preferably according to the present invention, the vector plasmid of the recombinant expression vector is pET28a(+).

[0025] In the third aspect of the present invention, there is provided a recombinant cell, which is obtained by transforming the above recombinant expression vector into a host cell.

[0026] Preferably according to the present invention, the host cell is Escherichia coli; more preferably Escherichia coli BL21(DE3).

[0027] In the fourth aspect of the present invention, there is provided the use of the above-mentioned heparinase III (BeHepIII) in the preparation of low molecular weight heparin.

[0028] Preferably according to the present invention, in the said use, ordinary ungraded heparin is used as a substrate, and the above-mentioned heparinase III (BeHepIII) is added for a degradation reaction to prepare low molecular weight heparin.

[0029] In the fifth aspect of the present invention, there is provided the use of the above-mentioned heparinase III (BeHepIII) in the synthesis of heparin oligosaccharides with a natural structure.

[0030] Preferably according to the present invention, in the said use, the heparinase III (BeHepIII) is used for the directional excision of the reducing end group of the substrate to synthesize heparin oligosaccharides with a natural structure;

[0031] The structural formula of the said substrate is as follows:

[0032]

[0033] In the formula, R1 is: -H, alkyl (such as, but not limited to, -CH3 or -CH2CH3), substituted alkyl, aryl or substituted aryl (such as, but not limited to, p-nitrophenyl); R2 is -Ac or -SO3 - ; n = 1 to 7 and is an integer.

[0034] More preferably, the heparin oligosaccharides with a natural structure are anticoagulant pentasaccharides; specifically, a fully modified heparin anticoagulant hexasaccharide is used as a substrate, and the above-mentioned heparinase III (BeHepIII) is added for a reaction to prepare heparin anticoagulant pentasaccharides with a natural structure.

[0035] During the research process of heparinase by the inventors of the present application, unexpectedly, an amino acid sequence similar to heparinase III of Flavobacterium heparinum was found in Bacteroides eggerthii, which has 56% sequence homology with heparinase III of Flavobacterium heparinum, and it was named BeHepIII. After amplification, sequencing and heterologous expression, it was found that BeHepIII has a high degradation activity for heparin containing a 6-position sulfated structure.

[0036] Beneficial effects:

[0037] 1. The present invention first discovers and discloses a new heparinase III (BeHepIII), which is derived from Bacteroides eggerthii and has a high recombinant expression level in Escherichia coli. It has a high degradation activity towards heparin containing a 6-sulfated structure, especially 93.5 times the enzyme activity of the currently commercialized heparinase FhHepIII, indicating that the heparinase III (BeHepIII) provided by the present invention has great potential for wide application in heparin degradation.

[0038] 2. The heparinase III (BeHepIII) provided by the present invention can be used to prepare oligosaccharide skeletons. The present invention realizes the directional excision of the reducing end of heparin oligosaccharides by chemical enzymatic synthesis using heparinase III (BeHepIII), and successfully realizes the complete chemical enzymatic synthesis of a pentasaccharide structure with antithrombin factor Xa activity. This synthesis method has brought a revolutionary change to the research and development of heparin drugs, expanded the application scope of heparinase, and promoted the research and development of heparin drugs. Therefore, the new heparin-degrading enzyme BeHepIII of the present invention has brought important progress to the research and development of heparin drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Agarose gel electrophoresis pattern of BeHepIII of the present invention

[0040] In the figure: M is Marker; P is the BeHepIII gene.

[0041] Figure 2 SDS-PAGE electrophoresis pattern of BeHepIII of the present invention.

[0042] In the figure: on the left is the protein Marker, and on the right is the purified BeHepIII protein.

[0043] Figure 3 Comparison of heparin cleavage activities between FhHepIII and BeHepIII of the present invention

[0044] In the figure: the abscissa is different heparinase IIIs, and the ordinate is the enzyme activity, with the unit of IU / mg.

[0045] Figure 4 Analysis of the cleavage effect of BeHepIII of the present invention on heparin hexasaccharide by PAMN-HPLC.

[0046] In the figure: the abscissa is the retention time, and the ordinate is the absorption at 310 nm.

[0047] Figure 5 Cleavage of heparin hexasaccharide by BeHepIII of the present invention.

[0048] Figure 6 ESI-MS results of the anticoagulant pentasaccharide synthesized using BeHepIII of the present invention.

[0049] In the figure: the abscissa is m / z and the ordinate is the signal intensity. Detailed implementation manners

[0050] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all well-known methods to those skilled in the art.

[0051] Bacteroides eggerthii in the following embodiments is a conventional strain and can be obtained commercially.

[0052] FhHepIII is the currently commercialized heparinase III derived from Flavobacterium heparinum and can be obtained commercially.

[0053] NaKfiA, PmHS2, NST, C5-epi, 2OST, 6OST and 3OST are all existing enzymes with publicly disclosed amino acid sequences and can be obtained commercially or by artificial synthesis.

[0054] Other raw materials or reagents not described in the present invention can be synthesized according to existing methods or obtained commercially.

[0055] Example 1. Construction of BeHepIII recombinant plasmid and recombinant strain

[0056] 1. Extract the whole genome of Bacteroides eggerthii using the Bacterial DNA Kit (purchased from OMEGA), and then design upstream and downstream primers. Using this whole genome as a template, perform PCR amplification to obtain the gene sequence of heparinase III (BeHepIII).

[0057] The primer sequences for the PCR amplification are as follows:

[0058] F-BeHep: 5′-ATGGGTCGCGGATCCGAAATGGGCAGCAGCCATCAC-3′,

[0059] R-BeHep: 5′-GCCGGATCTCAGTGGTGGTCAGAATTGGAAAAAGCCTTGGTCTTCC-3′.

[0060] Then perform agarose gel electrophoresis on the PCR amplification product, and the result is asFigure 1 as shown

[0061] It can be seen from Figure 1 that the length of the PCR amplification product is about 2000 bp, indicating that the target gene has been successfully amplified from the whole genome of Bacteroides eggerthii.

[0062] 2. To optimize the heterologous expression effect of heparinase III (BeHepIII) derived from Bacteroides eggerthii, the signal peptide of the BeHepIII amino acid sequence was removed, and the part after the signal peptide was retained. After codon optimization in Escherichia coli, the obtained BeHepIII amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. Then, the nucleotide sequence of the encoding gene of BeHepIII was artificially synthesized by Nanjing Genscript Corporation and cloned into the pET28a(+) vector to obtain a recombinant expression vector; then the recombinant expression vector was chemically transformed into BL21(DE3) competent cells to obtain recombinant Escherichia coli.

[0063] Example 2: Expression and purification of BeHepIII recombinant protein

[0064] A single colony of the recombinant Escherichia coli constructed in Example 1 was picked into 20 mL of sterilized LB liquid medium (containing 50 μg / ml kanamycin) for activation culture (37 °C, 225 r / min). The overnight-activated culture broth was inoculated into 1 L of LB liquid medium (containing 50 μg / ml kanamycin) at an inoculation amount of 1% for scale-up culture. It was shaken and cultured at 37 °C and 225 r / min for 4 hours until the OD600 was about 0.8, and IPTG with a final concentration of 0.2 mM was added, and it was induced at 22 °C and 225 r / min for 16 - 18 h. The bacterial cells were collected.

[0065] The collected bacterial cells were resuspended in a equilibration buffer (20 mM Tris-HCl, pH = 8.00; 0.5 M NaCl; 10 mM imidazole), sonicated on ice (working for 3 s, intermittent for 5 s, amplitude 33%, energy 1500 KJ, 4 °C) for 30 min, centrifuged at 12000 rpm for 20 min (4 °C), the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was purified using a nickel column. After loading, the impurities were washed with the equilibration buffer, and finally eluted with an elution buffer (20 mM Tris-HCl, pH = 8.00; 0.5 M NaCl; 250 mM imidazole) to obtain the target protein. The purified protein was stored in 20% glycerol and aliquoted and stored in a -80 °C refrigerator. The purified BeHepIII and mutant recombinant proteins were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were as Figure 2 shown. The protein concentration of BeHepIII was measured using a BCA protein concentration assay kit (Beyotime P0011).

[0066] As Figure 2 can be seen, there was a clearly visible band near the Marker with a molecular weight of 70 kDa. This was consistent with its calculated molecular weight of 75.4 kDa. This indicated that heterologous expression of BeHepIII was achieved by homologous recombination in Escherichia coli.

[0067] Example 3: Determination of BeHepIII Activity

[0068] The reaction system was carried out in a 1 ml volume of Tris-HCl buffer with pH = 7.0 - 7.5 and 50 mmol / L, adding 2 mM calcium chloride, 2 mg / ml unfractionated heparin, and 0.1 mg / mL FhHepIII or BeHepIII. Subsequently, the kinetic changes at 232 nm were detected. The generation of 1 μmol / L of unsaturated double bonds per minute was regarded as 1 IU. Each group was repeated 3 times, and the results were as Figure 3 shown.

[0069] As Figure 3It can be seen that under the same reaction conditions, the degradation activity of commercial FhHepIII towards unfractionated heparin is only 10.23 ± 1.2 IU / mg, while the enzyme activity of the recombinant BeHepIII expressed in the present invention reaches 956.39 ± 10.3 IU / mg, which is 93.5 times that of commercial FhHepII. The reason for the huge difference in enzyme activities between the two is as follows: Since unfractionated heparin has a high degree of 6-O-sulfation modification, and FhHepIII can only degrade the positions without a 6-sulfate group, its activity towards unfractionated heparin is low. However, the heparinase BeHepIII of the present invention has a higher activity towards 6-O-sulfated heparin, which indicates that BeHepIII has great application potential in degrading common unfractionated heparin to prepare low molecular weight heparin.

[0070] Example 4: Chemoenzymatic synthesis of heparin anticoagulant pentasaccharide with natural structure

[0071] 1. The synthesis method of heparin hexasaccharide backbone includes the following steps:

[0072] a. Take 1.0 g of p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP), 1.2 equivalents of UDP-GlcNTFA, and N-acetylglucosaminyltransferase (NaKfiA) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 5 mmol / L MnCl2). The total volume of the reaction solution is 1000 mL, and the concentration of NaKfiA in the reaction solution is 0.2 mg / ml. React the reaction solution overnight at 37°C, and monitor the reaction progress by PAMN-HPLC (detection wavelength: 310 nm). When the substrate conversion rate reaches over 90%, adjust the pH of the reaction solution to 3 with trifluoroacetic acid, and then purify it by C18 column chromatography to obtain heparin disaccharide backbone (GlcNTFA-GlcA-pNP);

[0073] b. Take 518 mg of the heparin disaccharide backbone obtained in step a, 1.2 equivalents of UDP-GlcA, and heparin backbone synthase 2 (PmHS2) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 5 mmol / L MnCl2). The total volume of the reaction solution is 1000 mL, and the concentration of PmHS2 in the reaction solution is 0.2 mg / ml. React the reaction solution overnight at 37°C, and monitor the reaction progress by PAMN-HPLC (detection wavelength: 310 nm). When the substrate conversion rate reaches over 95%, adjust the pH of the reaction solution to 3 with trifluoroacetic acid, and then purify it by C18 column chromatography to obtain heparin trisaccharide backbone (GlcA-GlcNTFA-GlcA-pNP);

[0074] c. Take 500 mg of the heparin trisaccharide backbone obtained in step b, 1.2 equivalents of UDP-GlcNTFA, and N-acetylglucosaminyltransferase (NaKfiA) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 5 mmol / L MnCl2). The total volume of the reaction solution is 500 mL, and the concentration of NaKfiA in the reaction solution is 0.2 mg / ml. React the reaction solution overnight at 37 °C and monitor the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). When the substrate conversion rate reaches over 95%, adjust the pH of the reaction solution to 3 with trifluoroacetic acid, and then purify it by C18 column chromatography to obtain the heparin tetrasaccharide backbone (GlcNTFA-GlcA-GlcNTFA-GlcA-pNP).

[0075] d. Take 300 mg of the heparin tetrasaccharide backbone obtained in step c, 1.2 equivalents of UDP-GlcA, and heparin backbone synthase 2 (PmHS2) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 5 mmol / L MnCl2). The total volume of the reaction solution is 1000 mL, and the concentration of PmHS2 in the reaction solution is 0.2 mg / ml. React the reaction solution overnight at 37 °C and monitor the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). When the substrate conversion rate reaches over 95%, adjust the pH of the reaction solution to 3 with trifluoroacetic acid, and then purify it by C18 column chromatography to obtain the heparin pentasaccharide backbone (GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-pNP).

[0076] e. Take 215 mg of the heparin pentasaccharide backbone obtained in step d, 1.2 equivalents of UDP-GlcA, and N-acetylglucosaminyltransferase (NaKfiA) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 5 mmol / L MnCl2). The total volume of the reaction solution is 1000 mL, and the concentration of NaKfiA in the reaction solution is 0.2 mg / ml. React the reaction solution overnight at 37 °C and monitor the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). When the substrate conversion rate reaches over 95%, adjust the pH of the reaction solution to 3 with trifluoroacetic acid, and then purify it by C18 column chromatography to obtain the heparin hexasaccharide backbone (GlcNTFA-GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-pNP).

[0077] 2. A chemoenzymatic synthesis method of heparin hexasaccharide, comprising the following steps:

[0078] (1) Dissolve the heparin hexasaccharide backbone prepared in Example 1 in an aqueous lithium hydroxide solution with a concentration of 0.1 mol / L, react at 4 °C for 30 min, and detect the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). When the substrate is completely converted, adjust the pH to 7.0, then add the reaction solution to MES buffer (50 mmol / L, pH = 7.5), and continue to add 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and N-sulfotransferase (NST) to construct a 500 mL reaction system. React the reaction system overnight at 25 °C for 16 h. During the reaction, enzymes or PAPS can be added as needed. After the substrate conversion rate > 99%, purify it with a Q-Sepharose strong anion column to obtain N-sulfated heparin hexasaccharide (GlcNS-GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP);

[0079] Among them, in the said reaction system, the concentration of the heparin hexasaccharide backbone is 3 mM, the concentration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is 10 mM, and the concentration of N-sulfotransferase (NST) is 0.2 mg / ml;

[0080] (2) Dissolve about 200 mg of N-sulfated heparin hexasaccharide in MES buffer (50 mmol / L, pH = 7.5), add CaCl2 and C5-isomerase (C5-epi), react in a water bath at 37 °C for 2 h, continue to add 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and 2-O-sulfotransferase (2OST) to construct a 500 mL reaction system. React the reaction system overnight at 25 - 30 °C for 16 h, and detect the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). During the reaction, enzymes or PAPS can be added as needed. After the substrate conversion rate > 99%, purify it with a Q-Sepharose strong anion column to obtain heparin hexasaccharide with IdoA2S (GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP);

[0081] Among them, in the said reaction system, the concentration of N-sulfated heparin hexasaccharide is 3 mM, the concentration of CaCl2 is 5 mM, the concentration of C5-isomerase (C5-epi) is 0.2 mg / ml, the concentration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is 5 mM, and the concentration of 2-O-sulfotransferase (2OST) is 0.2 mg / ml;

[0082] (3) Dissolve all of the prepared heparin hexasaccharide of IdoA2S in MES buffer (50 mmol / L, pH = 7.5), add 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and 6-O-sulfotransferase (6OST) to construct a first reaction system of 300 mL; incubate the first reaction system in a water bath at 37 °C overnight for 12 h; detect the reaction progress by PAMN-HPLC (detection wavelength is 310 nm). When the substrate conversion rate > 99%, continue to add 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and 3-O-sulfotransferase (3OST) to the reacted solution to construct a second reaction system of 400 mL; incubate the second reaction system at 25 °C overnight for 16 h. During the reaction, enzymes or PAPS can be supplemented as needed. When the substrate conversion rate > 99%, purify with a Q-Sepharose strong anion column to obtain fully modified heparin anticoagulant hexasaccharide (GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA-pNP);

[0083] Among them, in the first reaction system, the concentration of heparin hexasaccharide of IdoA2S is 2 mM, the concentration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is 4 mM, and the concentration of 6-O-sulfotransferase (6OST) is 0.3 mg / ml; in the second reaction system, the concentration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is 3 mM, and the concentration of 3-O-sulfotransferase (3OST) is 0.2 mg / ml.

[0084] 3. Dissolve the above fully modified heparin anticoagulant hexasaccharide in Tris-HCl buffer (pH = 7.5, 50 mmol / L), add CaCl2, and then add BeHepIII and FhHepIII respectively to construct a reaction system; incubate the reaction system at 25 °C overnight for 16 h, and detect the reaction progress by PAMN-HPLC (detection wavelength is 310 nm) (as Figure 4 and Figure 5 shown). After the reaction is complete, concentrate the reaction solution using BeHepIII and then purify it with P-2 to prepare the anticoagulant pentasaccharide GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S with a natural structure. The ESI-MS characterization structure of this anticoagulant pentasaccharide is as Figure 6 shown.

[0085] In the reaction system, the concentration of the fully modified heparin anticoagulant hexasaccharide is 1 mM, the concentration of CaCl2 is 5 mM, and the concentrations of BeHepIII and FhHepIII are both 0.3 mg / ml.

[0086] From Figure 4 andFigure 5 It can be seen that the degradation activity of FhHepIII towards the fully modified heparin anticoagulant hexasaccharide is extremely low, while the BeHepIII provided by the present invention has a degradation activity far higher than that of FhHepIII towards the 6-sulfated fully modified heparin anticoagulant hexasaccharide, achieving the excision of the reducing-end ΔUA-pNP and can be used for large-scale and industrial preparation of anticoagulant pentasaccharides with natural structures.

[0087] It can be seen from Figure 6 that the anticoagulant pentasaccharide GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S with a natural structure was successfully prepared in this example.

Claims

1. A heparinase III (BeHepIII) with high activity against 6 - position sulfated heparin, characterized in that, Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. The heparinase III with high activity against 6 - position sulfated heparin as described in claim 1, characterized in that, The heparinase III (BeHepIII) is derived from Bacteroides eggerthii.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene of the heparinase III (BeHepIII) as claimed in claim 1.

4. A recombinant cell, characterized in that, The recombinant cell is obtained by transforming the recombinant expression vector as claimed in claim 3 into a host cell.

5. Use of the heparinase III (BeHepIII) as claimed in claim 1 in the preparation of low molecular weight heparin.

6. The application according to claim 5, characterized in that, The use is to use ordinary ungraded heparin as a substrate, add the heparinase III (BeHepIII) as claimed in claim 1 for a degradation reaction, and prepare low molecular weight heparin.

7. Use of the heparinase III (BeHepIII) as claimed in claim 1 in the synthesis of heparin oligosaccharides with a natural structure.

8. The application according to claim 7, wherein The use is to use the heparinase III (BeHepIII) as claimed in claim 1 for the directional excision of the reducing end group of the substrate to synthesize heparin oligosaccharides with a natural structure; The structural formula of the substrate is as follows: Wherein, R1 is: -H, alkyl, substituted alkyl, aryl or substituted aryl; R2 is -Ac or -SO3 - ; n = 1 to 7 and is an integer.

9. The application according to claim 8, wherein The heparin oligosaccharides with a natural structure are anticoagulant pentasaccharides; specifically, using fully modified heparin anticoagulant hexasaccharides as a substrate, adding the heparinase III (BeHepIII) as claimed in claim 1 for a reaction, and preparing heparin anticoagulant pentasaccharides with a natural structure.

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