Construction method of heparin 2-O-sulfotransferase high-activity mutant

Abstract

The invention discloses a construction method of a heparin 2-O-sulfotransferase high-activity mutant, and belongs to the technical field of biology. According to the heparin 2-O-sulfotransferase and the application of the heparin 2-O-sulfotransferase, a dissolution promoting label is fused at the N end of the heparin 2-O-sulfotransferase, the coding sequence of the dissolution promoting label is optimized, and active expression of the heparin 2-O-sulfotransferase is achieved; according to the invention, the heparin 2-O-sulfotransferase is mutated, so that a mutant with high stability and catalytic activity is obtained; furthermore, fermentation conditions of the heparin 2-O-sulfotransferase are optimized, so that the expression quantity and the enzyme activity are improved, and the application potential of the heparin 2-O-sulfotransferase in industrial production is enhanced.

Description

Technical Field

[0001] The invention relates to a method for constructing a high-activity mutant of heparin 2-O-sulfotransferase, and belongs to the field of biotechnology. Background Art

[0002] Heparin is a highly sulfonated glycosaminoglycan composed of repeating disaccharide units of uronic acid and glucosamine. As the most commonly used anticoagulant, heparin plays a key role in the prevention and treatment of thrombosis and is an indispensable part of modern medical practice. It is naturally synthesized in the endoplasmic reticulum and Golgi apparatus of mast cells. Its biosynthesis process begins with the synthesis of intracellular heparin precursor (heparosan), and after a series of enzymatic modifications, it produces fully functional heparin.

[0003] Currently, the supply of heparin in the market still relies on extraction from animal tissues. However, challenges such as the long growth cycle of animal donors and the high risk of cross-species pathogen transmission limit the ability to meet the growing global demand for heparin. Therefore, bioengineering strategies have emerged as an emerging method for heparin synthesis and have become an effective alternative to traditional extraction methods.

[0004] In the complex process of heparin modification, heparin 2-O-sulfotransferase is the key enzyme in the second step of sulfonation reaction. It uses the sulfonic acid donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to transfer the sulfonic acid group to the C2 position of the hexuronic acid residue to form 2-O-sulfonated IdoA2S or GlcA2S. This rate-limiting step is crucial for the subsequent sulfonation reaction. Heparin modified by heparin 2-O-sulfotransferase has specific biological activity. If 2-O-sulfonation is insufficient or absent in heparin, its binding affinity with antithrombin will be significantly reduced, impairing the conformational activation of antithrombin and weakening its inhibitory effect on thrombin and factor Xa.

[0005] Although heparin 2-O-sulfotransferase can be expressed now, there are still many challenges in terms of expression efficiency and catalytic performance of existing heparin 2-O-sulfotransferase to meet the needs of industrial production. Summary of the invention

[0006] The present invention provides a heparin 2-O-sulfotransferase mutant, which has one or more mutations at the following sites based on the parent shown in SEQ ID NO.1: A85G, A98K, Y102F, H103F, G137A, S144A, Y145F, I160V, I167L, F179Y, R190P, A210D, F221L, L237M, K241I, N243R, F249L, L295P, N314F, F316L, Q323W.

[0007] In one embodiment, the heparin 2-O-sulfotransferase mutant is based on the parent shown in SEQ ID NO.1, with the tyrosine at position 145 mutated to phenylalanine, and the obtained mutant is named Y145F.

[0008] In one embodiment, the heparin 2-O-sulfotransferase mutant is based on the parent shown in SEQ ID NO.1, with the 98th alanine mutated to lysine and the 145th tyrosine mutated to phenylalanine. The obtained mutant is named A98K / Y145F.

[0009] The present invention also provides a heparin 2-O-sulfotransferase mutant fusion protein, which is a fusion protein with a lytic tag connected to the N-terminus of the heparin 2-O-sulfotransferase mutant; the lytic tag includes but is not limited to SUMO, MBP, TrxA, SET2, NusA, and SUMO mutant.

[0010] In one embodiment, the amino acid sequences of the lytic tags SUMO, MBP, TrxA, SET2, and NusA are shown in SEQ ID NOs. 3-7.

[0011] In one embodiment, the SUMO mutant replaces the SUMO gene whose nucleotide sequence is shown in SEQ ID NO.13.

[0012] In one embodiment, the heparin 2-O-sulfotransferase mutant and the lytic tag are connected via a connecting peptide.

[0013] In one embodiment, the connecting peptide includes but is not limited to PAPAP, G4S, (Gly)6 and EAAAK.

[0014] In one embodiment, the nucleotide sequences encoding the connecting peptides PAPAP, G4S, (Gly)6, and EAAAK are shown as SEQ ID NOs. 8 to 11, respectively.

[0015] The present invention also provides a gene encoding the heparin 2-O-sulfotransferase mutant or the fusion protein.

[0016] The present invention also provides a recombinant microorganism expressing the heparin 2-O-sulfotransferase mutant or the fusion protein.

[0017] The present invention also provides a recombinant Escherichia coli expressing the heparin 2-O-sulfotransferase mutant or the fusion protein.

[0018] In one embodiment, the recombinant Escherichia coli uses E. coli OrigamiB (DE3) or E. coli BL21 (DE3) as a host.

[0019] In one embodiment, the recombinant Escherichia coli also contains a molecular chaperone plasmid pGro7.

[0020] The present invention also provides a method for expressing the heparin 2-O-sulfotransferase mutant, which comprises culturing the recombinant Escherichia coli in a culture medium for a period of time and collecting the enzyme solution.

[0021] In one embodiment, the culture is carried out at 25-37°C to OD 600 The pH value was 0.6-0.8, induced with arabinose and IPTG, cultured for 6-24 hours, the bacteria were collected, crushed and centrifuged to obtain the crude enzyme solution.

[0022] In one embodiment, Ni-NTA affinity chromatography is used to purify the target protein in the crude enzyme solution.

[0023] The invention also provides a method for hydrolyzing heparin by using the heparin 2-O-sulfotransferase.

[0024] In one embodiment, the method is a reaction in the following catalytic system: the buffer is 10-50 mM PBS, the pH is 6.0-8.0, and the final concentration is 0.1-0.5 g / L completely desulfurized N-sulfonated (CDSNS) heparin, 1.0-3.0 g / L p-nitrobenzenesulfonic acid (pNPS), 1.0-3.0 g / L PAPS, 1.0-3.0 g / L acylsulfotransferase IV (AST IV) and 20-50 μg / mL of pure heparin 2-O-sulfotransferase enzyme or 0.5 mL of crude enzyme extract.

[0025] In one embodiment, the reaction temperature is 35-45° C., and the catalytic time is 2-12 hours.

[0026] The present invention also provides the application of the heparin 2-O-sulfotransferase mutant, or the fusion protein, or the recombinant Escherichia coli, or the method in the field of food, medicine or health care products for preparing products containing heparin or heparan sulfate.

[0027] Beneficial effects:

[0028] 1. The present invention achieves the active expression of heparin 2-O-sulfotransferase and improves its expression level by fusing the SUMO lytic tag to the N-terminus of heparin 2-O-sulfotransferase via a connecting peptide PAPAP and optimizing the codons of the first ten amino acids of SUMO.

[0029] 2. The present invention uses relevant software to identify hot spots of heparin 2-O-sulfotransferase, adopts an evolution-based regression consensus strategy, and combines multiple homologous sequences for comprehensive analysis, carefully selecting 21 potential key amino acid sites. These sites not only cover amino acids that are critical to the stability of the distal region, but also include amino acids located near the substrate pocket binding pocket. On this basis, single-point mutations are performed on the selected sites, and mutants with significantly improved enzyme activity are screened for combined mutations, and finally a mutant with high stability and catalytic activity is obtained.

[0030] 3. The present invention further improves the expression level and enzyme activity of heparin 2-O-sulfotransferase by optimizing the fermentation conditions, thereby enhancing its application potential in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of heparin 2-O-sulfotransferase catalysis.

[0032] Figure 2 This is a gel image of heparin 2-O-sulfotransferase fused with different solubilizing tags; the lanes from left to right are the intracellular supernatant (left) and precipitate (right) of pET32a empty load (control), Ga2OST, SUMO-Ga2OST, MBP-Ga2OST, TrxA-Ga2OST, SET2-Ga2OST, and NusA-Ga2OST; the red arrows point to Ga2OST with different fusion tags, and the blue arrows point to GroEL.

[0033] Figure 3 It is a gel image of pure enzyme of some heparin 2-O-sulfotransferase mutants; wherein, the lanes from left to right are: Marker, wild type (WT), A98K, Y145F, A98K / Y145F.

[0034] Figure 4 This is a gel image of batch feeding sampling of heparin 2-O-sulfotransferase mutants; wherein, the lanes from left to right are the marker and the protein gel images after cell wall breaking after sampling in the upper tank at different times after induction. DETAILED DESCRIPTION

[0035] 1. E. coli OrigamiB (DE3) and E. coli Top 10 are commercial strains; pET32a (+) and pGro7 are commercial plasmids.

[0036] 2. Plasmid construction reagents and sequencing verification were purchased and completed at Shanghai Biotech Co., Ltd.

[0037] 3. Various analytical reagents were purchased from Sinopharm Group.

[0038] 4. Culture medium:

[0039] LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder.

[0040] TB medium, 2.31g / L KH2PO4, 12.54g / L K2HPO4, 12g / L tryptone, 24g / L yeast powder, 5g / L glycerol.

[0041] 5. Enzyme activity detection method:

[0042] Heparin 2-O-sulfotransferase activity detection method: 0.1g / L completely desulfurized N-sulfonated (CDSNS) heparin, 1.5g / L p-nitrobenzenesulfonic acid (pNPS), 1.0g / L 3′-phosphoadenosine-5′-phosphosulfate (PAPS), 2.0g / L AST IV and 20μg / mL pure heparin 2-O-sulfotransferase enzyme or 0.5mL crude enzyme extract were added to the reaction system. The reaction temperature of the catalytic system was 40℃ and the catalytic time was 2-12h. The enzyme activity was determined by measuring the absorbance of nitrophenol (pNP) formed during the reaction at 400nm using an enzyme-linked immunosorbent assay. Heparin 2-O-sulfotransferase activity (U) is defined as the amount of enzyme required to synthesize 1μM pNP per hour at 40℃.

[0043] Heparin 2-O-sulfotransferase reaction product (ΔIdoA2S-GlcNS) detection method: Agilent 1600 HPLC system, Spherisorb strong anion exchange column (4.0×250 mm, 5.0 μm), mobile phase: 1.8 mM NaH2PO4 and 1 M NaClO4 solution, flow rate: 1 mL min -1 , injection volume: 20μL, detection time 35min, detector: UV232nm.

[0044] Example 1: Expression and purification of heparin 2-O-sulfotransferase in Escherichia coli

[0045] Heparin 2-O-sulfotransferase from Gallus gallus (shown in SEQ ID NO.1) was selected for codon optimization according to the codon preference rule of E. coli, and the optimized nucleotide sequence (shown in SEQ ID NO.2) was ligated between the BamH I and Hind III restriction enzyme sites of plasmid pET32a(+) to obtain the recombinant plasmid pET32a-Ga2OST.

[0046] The lytic tag genes SUMO, MBP, TrxA, SET2, and NusA (as shown in SEQ ID NO.3-7) were amplified using specific primer pairs, and integrated into the pET32a-Ga2OST plasmid linearized by Not I / Xho I restriction endonucleases by Gibson assembly to achieve the N-terminal fusion of the lytic tag and heparin 2-O-sulfotransferase. Then, the same homologous recombination technology was used to insert connecting peptides PAPAP, G4S, (Gly)6, and EAAAK (nucleotide sequences are shown in SEQ ID NO.8-11, respectively) between the lytic tag and the heparin 2-O-sulfotransferase. Recombinant plasmids such as pET32a-SUMO-PAPAP-Ga2OST were constructed.

[0047] E. coli OrigamiB (DE3) was selected as the host to express the above recombinant plasmid, and the molecular chaperone plasmid pGro7 was introduced to promote the correct folding of the protein. The recombinant plasmid was transferred into E. coli OrigamiB (DE3), and after verification, it was streaked on a plate containing ampicillin (100 μg / L) and chloramphenicol (35 μg / L). A single colony was picked and inoculated in LB seed medium. After culturing to seed liquid, the seed liquid was transferred to 50 mL TB fermentation medium at a volume fraction of 1 mL / 50 mL. After continuing to culture for 2 hours, 1 g / L arabinose and 0.4 mM IPTG were added and induced at 25 ° C for 12 hours. After the end, the bacteria were collected and crushed by high-pressure homogenization for purification and analysis.

[0048] The collected bacteria were crushed by high-pressure homogenization and centrifuged at high speed to remove cell debris. The supernatant was filtered with a 0.22μm water membrane, and the target protein was purified by Ni-NTA affinity chromatography. After the column was equilibrated with liquid A, the crude enzyme solution was loaded, and the chromatography column was equilibrated with liquid A. After that, the chromatography column was rinsed with liquid B of different concentrations and the rinse solution was collected. The purified components were verified by SDS-PAGE, and the purest components were desalted with a PD-10 desalting column. Low salt buffer (20mM PBS, 150mMNaCl; pH 7.5) was used for desalting, and the purified and desalted protein was collected. Among them, liquid A: 20mM PBS buffer pH 7.5, 500mMNaCl; liquid B: 20mM PBS buffer pH 7.5, 500mM NaCl, 500mM imidazole.

[0049] Example 2: Comparison of the expression of heparin 2-O-sulfotransferase by different lytic tags and connecting peptides

[0050] The fermentation broth of the recombinant bacteria constructed in Example 1 was centrifuged, the cells were collected, washed and resuspended, and the cell OD was adjusted. 600The concentration of 1g / L CDSNS heparin, 1.5g / L pNPS, 1.0g / L PAPS, 2.0g / L AST IV and 0.5mL crude enzyme extract were added to the crude enzyme solution (calculated according to the final concentration). The reaction temperature of the catalytic system was 40°C and the catalytic time was 4h. The enzyme activity was determined by detecting the absorbance of pNP formed during the reaction at 400nm using an enzyme-linked immunosorbent assay. The results showed that the enzyme activity of the fusion of SUMO tag and PAPAP connecting peptide at the N-terminus of heparin 2-O-sulfotransferase was higher. The protein gel images of different solubilizing tags and heparin 2-O-sulfotransferase are shown in Figure 2 : Lanes from left to right are the cell supernatants (left) and precipitates (right) of pET32a empty load (control), Ga2OST, SUMO-Ga2OST, MBP-Ga2OST, TrxA-Ga2OST, SET2-Ga2OST, and NusA-Ga2OST; red arrows point to Ga2OST with different fusion tags. Blue arrows point to GroEL (without connecting peptide).

[0051] Table 1 Comparison of different solubility-promoting tags and linker peptides on the expression of heparin 2-O-sulfotransferase

[0052] name Enzyme activity (U / mL) SUMO-Ga2OST 340.80±13.06 MBP-Ga2OST 324.80±7.47 TrxA-Ga2OST 183.73±13.15 SET2-Ga2OST 14.11±1.36 NusA-Ga2OST 309.6±13.11 SUMO-PAPAP-Ga2OST 387.40±19.87 <![CDATA[SUMO-G4S-Ga2OST]]> 269.65±13.55 <![CDATA[SUMO-(Gly)6-Ga2OST]]> 192.28±8.83 SUMO-EAAAK-Ga2OST 361.81±14.13

[0053] Example 3: Improving the expression level of heparin 2-O-sulfotransferase by optimizing the N-terminal synonymous codon

[0054] In order to generate the synonymous mutant of N-terminal coding sequence (NCS), superfolded green fluorescent protein (sfGFP, shown in SEQ ID NO.14) was first fused to the C-terminus of the construct in a manner similar to pET-32a-Ga2OST. Subsequently, a specific forward primer was designed for the first 10 amino acids (excluding the start codon) of the N-terminus of SUMO encoded by the sequence shown in SEQ ID NO.12 for denaturation amplification, and a standard reverse primer was combined to introduce synonymous mutations in the first 10 amino acid residues of SUMO. After digesting the parent DNA with Dpn I, the PCR product was cyclized using a DNA ligation kit Ver.2.1 and then introduced into E. coli OrigamiB (DE3). This process ultimately generated a strain expressing the synonymous mutant of NCS.

[0055] Fluorescence-activated cell sorting (FACS) technology was used to screen mutants with significantly enhanced fluorescence from 5,000 cells. Subsequently, these mutants were inoculated into 96-well plates, cultured with LB medium, and their biomass and fluorescence intensity were measured respectively. We selected the 16 strains with the strongest fluorescence and further screened them using shake flasks. Afterwards, the enzyme activity of the strain without the reporter gene sfGFP was measured. By comparison, the strain with the highest enzyme activity was determined, and the optimal nucleotide sequence corresponding to the first 10 amino acids at the N-terminus of its SUMO is shown in SEQ ID NO:13.

[0056] Example 4: Obtaining a single-point mutant of heparin 2-O-sulfotransferase

[0057] Based on the pET-32a-SUMO-PAPAP-Ga2OST plasmid containing the SUMO mutation shown in SEQ ID NO:13 constructed in Example 3, a single point mutation was introduced into Ga2OST using specific primers (Table 2) through site-directed mutagenesis technology. The entire plasmid was PCR amplified using the specified primers to introduce the desired mutation. After amplification, the PCR product was digested with Dpn I restriction endonuclease and purified by a column method. The obtained linearized fragments were assembled by the Gibson assembly method, then transformed into E. coli Top 10 cells, and the cells were plated on a medium containing ampicillin. Positive clones were selected for further culture, and the plasmids were extracted to confirm their purity, and then transformed into E. coli Origami B (DE3) for expression. According to the same method as in Example 1, the crude enzyme activity in the broken cells was determined, and the results are shown in Table 3. The enzyme activity of the single point mutant Y145F was increased by 86%. The specific enzyme activity was determined after the protein was purified according to the method of Example 1, and the results are shown in Table 3. Figure 3 As shown, the specific enzyme activities of mutant A98K and mutant Y145F were 573.95 U / mg and 665.00 U / mg, respectively.

[0058] Table 2 Site-directed mutagenesis primers

[0059]

[0060]

[0061] Table 3 Heparin 2-O-sulfotransferase single point mutant information

[0062]

[0063]

[0064] Example 5: Obtaining multi-point superposition mutants of heparin 2-O-sulfotransferase

[0065] Based on the single mutation constructed in Example 4, the same strategy was used to further introduce mutations to construct mutants as shown in Table 3. The PCR amplification product was digested with Dpn I restriction endonuclease and purified by a column method. The obtained linearized fragments were assembled by the Gibson assembly method, and then transformed into E. coli Top 10 cells, and the cells were plated on a medium containing ampicillin. Positive clones were selected for further culture, and the plasmids were extracted to confirm their purity, and then transformed into E. coli OrigamiB (DE3) for expression. The mutants were obtained by the method in Example 1. According to the same method as Example 1, the crude enzyme activity in the broken cells was determined, and the protein was purified and the specific enzyme activity was determined. The results are shown in FIG. Figure 3 As shown in Table 4, the two-point superposition mutant A98K / Y145F had the greatest increase in enzyme activity, with a specific enzyme activity of 827.39 U / mg.

[0066] Table 4 Heparin 2-O-sulfotransferase superimposed mutant information

[0067]

[0068]

[0069] Example 6: Optimizing the fermentation conditions of heparin 2-O-sulfotransferase to increase its yield

[0070] The fermentation culture conditions for expressing the heparin 2-O-sulfotransferase mutant A98K / Y145F constructed in Example 5 were optimized. The fermentation conditions included: the recombinant strain was cultured in TB medium at 37°C until OD 600 The final concentration of arabinose was 1 g / L, and the culture temperature was adjusted to 25°C. IPTG was added half an hour later to continue induction culture.

[0071] The post-induction culture temperature, culture time and inducer concentration in the fermentation process were optimized, including:

[0072] (1) The fermentation was carried out according to the above method, except that the culture temperature after induction was adjusted to 20-30°C. The results showed that the crude enzyme activity could be increased to 649.39 U / mL by induction at 25°C.

[0073] (2) Fermentation was performed according to the above method, except that the induction was performed at 25°C and the induction culture time was adjusted to 12 to 48 h. The results showed that the crude enzyme activity after 12 h of culture was increased to 758.31 U / mL.

[0074] (3) Fermentation was carried out according to the above method, except that the concentration of the inducer arabinose was adjusted to 0-1 g / L and the concentration of IPTG was adjusted to 0.2-0.8 mM, respectively. Induction was carried out at 25°C for 12 h. The results showed that when the concentrations of arabinose and IPTG were 1 g / L and 0.4 mM, respectively, the crude enzyme activity was increased to 837.75 U / mL.

[0075] Example 7: Heparin 2-O-sulfotransferase fermentation in a 5-L fermenter

[0076] Subsequently, in order to verify the applicability of the fermentation parameters optimized in Example 6 in the larger-scale cultivation of heparin 2-O-sulfotransferase mutants, a fed-batch culture strategy was used in a 5-L fermenter for further verification. The specific steps are as follows:

[0077] (1) Preparation of seed solution: a single colony was selected and inoculated into LB seed medium, and cultured at 37°C, 220 rpm for 8-10 h to prepare the first-level seed solution; the first-level seed solution was transferred into 50 mL TB fermentation medium at a volume fraction of 1 mL / 50 mL, and cultured at 37°C, 220 rpm for 4-6 h to prepare the second-level seed solution.

[0078] (2) Fermentation condition control: The strain was inoculated with 10% of the inoculum into a fermenter containing 2L of TB medium, and glycerol was added at an initial concentration of 20 g / L. The culture was first cultured at 37°C for 2-3 hours until the OD 600 About 8-10; then, the temperature was lowered to 25°C for induction, and arabinose and IPTG were added to a final concentration of 1g / L and 0.4mM and cultured for 30h. During the culture process, the ventilation volume was always maintained at 2vvm. When the dissolved oxygen DO dropped below 50%, glycerol was fed and stopped until DO reached 80%. When DO dropped below 50%, the above feeding operation was continued.

[0079] At the end of fermentation, heparin 2-O-sulfotransferase in the fermentation broth was detected. The results showed that the production reached 37.23 mg protein / g DCW 22 h after fermentation induction, and the enzyme activity reached 5.72×10 6 U / L.

[0080] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A heparin 2-O-sulfotransferase mutant, characterized in that Based on the parent shown in SEQ ID NO.1, it has mutations in one or more of the following sites: A85G, A98K, Y102F, H103F, G137A, S144A, Y145F, I160V, I167L, F179Y, R190P, A210D, F221L, L237M, K241I, N243R, F249L, L295P, N314F, F316L, Q323W.

2. The heparin 2-O-sulfotransferase mutant according to claim 1, characterized in that Based on the parent shown in SEQ ID NO.1, the alanine at position 98 is mutated to lysine, and / or the tyrosine at position 145 is mutated to phenylalanine.

3. A fusion protein, characterized in that The N-terminus of the heparin 2-O-sulfotransferase mutant of claim 1 or 2 is connected to a lytic tag; the lytic tag includes but is not limited to SUMO, MBP, TrxA, SET2, NusA, and SUMO mutant.

4. The fusion protein according to claim 3, characterized in that The heparin 2-O-sulfotransferase mutant and the lytic tag are connected via a connecting peptide.

5. A gene encoding the heparin 2-O-sulfotransferase mutant according to claim 1 or 2, or the fusion protein according to claim 3 or 4.

6. A recombinant microorganism expressing the heparin 2-O-sulfotransferase mutant according to claim 1 or 2 or the fusion protein according to claim 3 or 4.

7. A recombinant Escherichia coli, characterized in that Using E. coli OrigamiB (DE3) or E. coli BL21 (DE3) as a host, the heparin 2-O-sulfotransferase mutant of claim 1 or 2 or the fusion protein of claim 3 or 4 is expressed.

8. The recombinant Escherichia coli according to claim 7, characterized in that The recombinant Escherichia coli also contains a molecular chaperone plasmid pGro7.

9. A method for preparing the heparin 2-O-sulfotransferase mutant according to claim 1 or 2, characterized in that: The recombinant Escherichia coli according to claim 7 or 8 is cultured in a culture medium for a period of time, and the enzyme solution is collected; arabinose and / or IPTG are used for induction during the culture process.

10. Use of the heparin 2-O-sulfotransferase mutant according to claim 1 or 2, or the fusion protein according to claim 3 or 4, or the gene according to claim 5, or the recombinant microorganism according to claim 6, or the recombinant Escherichia coli according to claim 7 or 8, or the method according to claim 9 in the preparation of products containing heparin or heparan sulfate in the field of food, medicine or health products.

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