A method for constructing a heparin 2-o-sulfotransferase high-activity mutant

Abstract

The application discloses a construction method of a heparin 2-O-sulfotransferase high-activity mutant, and belongs to the technical field of biology.1、The application fuses a solubility-promoting label to the N terminal of heparin 2-O-sulfotransferase, optimizes the coding sequence of the solubility-promoting label, and realizes the active expression of heparin 2-O-sulfotransferase. The application also mutates heparin 2-O-sulfotransferase, and obtains a mutant with high stability and catalytic activity. Further, the fermentation conditions of heparin 2-O-sulfotransferase are optimized, the expression amount and enzyme activity are improved, and the application potential in industrial production is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a heparin 2-O-sulfotransferase high-activity mutant, belonging to the field of biotechnology. BACKGROUND

[0002] Heparin is a highly sulfated glycosaminoglycan composed of repeating disaccharide units of uronic acid and glucosamine. As the most commonly used anticoagulant, heparin plays a key role in preventing and treating thrombosis, and is an indispensable part of modern medical practice. It is naturally synthesized in the endoplasmic reticulum and Golgi body of mast cells, and its biosynthesis process begins with the synthesis of intracellular heparosan, which is modified by a series of enzymes to form fully functional heparin.

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

[0004] In the complex process of heparin modification, heparin 2-O-sulfotransferase is a key enzyme for the second sulfation reaction. It uses the sulfonic acid donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to transfer a sulfonic acid group to the C2 position of the hexuronic acid residue, forming 2-O-sulfated IdoA2S or GlcA2S. This rate-limiting step is crucial for subsequent sulfation reactions. Heparin modified by heparin 2-O-sulfotransferase has specific biological activity, and insufficient or missing 2-O-sulfation in heparin can significantly reduce its binding affinity to antithrombin, impair the conformational activation of antithrombin, and weaken its inhibition of thrombin and factor Xa.

[0005] Although heparin 2-O-sulfotransferase can be expressed at present, 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

[0006] The present application provides a heparin 2-O-sulfotransferase mutant, which has one or more mutations in the following positions 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 an embodiment, the heparin 2-O-sulfotransferase mutant is a mutant in which the tyrosine at position 145 is mutated to phenylalanine based on the parent shown in SEQ ID NO. 1, and the mutant is named Y145F.

[0008] In an embodiment, the heparin 2-O-sulfotransferase mutant is a mutant in which the alanine at position 98 is mutated to lysine and the tyrosine at position 145 is mutated to phenylalanine based on the parent shown in SEQ ID NO. 1, and the mutant is named A98K / Y145F.

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

[0010] In an embodiment, the amino acid sequences of the solubility-promoting tags SUMO, MBP, TrxA, SET2, and NusA are shown in SEQ ID NO. 3-7.

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

[0012] In an embodiment, the heparin 2-O-sulfotransferase mutant and the solubility-promoting tag are connected by a connecting peptide.

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

[0014] In an embodiment, the nucleotide sequences encoding the connecting peptides PAPAP, G4S, (Gly)6, and EAAAK are shown in SEQ ID NO. 8-11, respectively.

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

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

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

[0018] In an embodiment, the recombinant E. coli is E. coli OrigamiB(DE3) or E. coli BL21(DE3).

[0019] In an embodiment, the recombinant E. coli further comprises a chaperone plasmid pGro7.

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

[0021] In an embodiment, the culturing is performed at 25-37℃ to OD 600 0.6-0.8, inducing with arabinose and IPTG, culturing for 6-24h, collecting the bacterial cells, breaking and centrifuging to obtain the crude enzyme solution.

[0022] In an embodiment, the target protein in the crude enzyme solution is purified by Ni-NTA affinity chromatography.

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

[0024] In an embodiment, the method is performed in a catalytic system comprising 10-50mM PBS as the buffer, pH 6.0-8.0, 0.1-0.5g / L completely desulfated N-sulfonated (CDSNS) heparin, 1.0-3.0g / L p-nitrophenylsulfate (pNPS), 1.0-3.0g / L PAPS, 1.0-3.0g / L acylsulfate transferase IV (AST IV), and 20-50μg / mL heparin 2-O-sulfotransferase pure enzyme or 0.5mL crude enzyme extract.

[0025] In an embodiment, the reaction temperature is 35-45℃, and the catalytic time is 2-12h.

[0026] The present application also provides the use of the heparin 2-O-sulfotransferase mutant, the fusion protein, the recombinant E. coli, or the method in the medical field for preparing a product containing heparin or heparan sulfate.

[0027] Advantages:

[0028] 1. The present application realizes the active expression of heparin 2-O-sulfotransferase and improves the expression level by fusing the SUMO solubility tag to the N-terminus of heparin 2-O-sulfotransferase through a connecting peptide PAPAP, and optimizing the codons of the first ten amino acids of SUMO.

[0029] 2. The application uses relevant software to identify hot spot regions of heparin 2-O-sulfotransferase, adopts an evolutionary-based regression consensus strategy, and comprehensively analyzes a plurality of homologous sequences to carefully select 21 potential key amino acid sites. These sites not only cover amino acids that are crucial 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 mutation, and finally a mutant with high stability and catalytic activity is obtained.

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

[0031] Figure 1 is a catalytic schematic diagram of heparin 2-O-sulfotransferase.

[0032] Figure 2 is a gel map of heparin 2-O-sulfotransferase fused with different solubility tags; wherein the lanes from left to right are cell supernatant (left) and precipitate (right) of pET32a empty load (control), Ga2OST, SUMO-Ga2OST, MBP-Ga2OST, TrxA-Ga2OST, SET2-Ga2OST, NusA-Ga2OST; the red arrow points to Ga2OST with different fusion tags, and the blue arrow points to GroEL.

[0033] Figure 3 is a partial heparin 2-O-sulfotransferase mutant enzyme gel map; wherein the lanes from left to right are Marker, wild type (WT), A98K, Y145F, A98K / Y145F.

[0034] Figure 4 is a heparin 2-O-sulfotransferase mutant fed-batch sampling gel map; wherein the lanes from left to right are Marker and protein gel map of broken wall after sampling and inducing at different times. DETAILED DESCRIPTION

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

[0036] 2. Plasmid construction reagents and sequencing verification are purchased and completed in Shanghai Biosun Biotech Co., Ltd.

[0037] 3. Various analytical pure reagents are purchased from National Pharmaceutical Group.

[0038] 4. Culture medium:

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

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

[0041] 5. Enzyme activity detection method:

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

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

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

[0045] The heparin 2-O-sulfotransferase (shown in SEQ ID NO. 1) derived from the original chicken (Gallus gallus) was selected for codon optimization according to the codon bias of E. coli. The optimized nucleotide sequence (shown in SEQ ID NO. 2) was connected between the BamH I and Hind III restriction enzyme cutting sites of the plasmid pET32a(+), and the recombinant plasmid pET32a-Ga2OST was obtained.

[0046] The solubility-promoting tag genes SUMO, MBP, TrxA, SET2, 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 endonuclease by Gibson assembly to realize the fusion of the solubility-promoting tag with the N-terminus of heparin 2-O-sulfotransferase. Then, the same homologous recombination technique was used to insert the linker peptides PAPAP, G4S, (Gly)6 and EAAAK (nucleotide sequences are respectively as shown in SEQ ID NO. 8-11) between the solubility-promoting tag and heparin 2-O-sulfotransferase, respectively. The recombinant plasmids pET32a-SUMO-PAPAP-Ga2OST were constructed.

[0047] E. coli OrigamiB (DE3) was selected as the host to express the above-mentioned recombinant plasmid, and the chaperone plasmid pGro7 was introduced to promote the correct folding of the protein. The recombinant plasmid was transformed into E. coli OrigamiB (DE3), and after verification, it was streaked and cultured on a plate containing ampicillin (100 μg / L) and chloramphenicol (35 μg / L), and a single colony was inoculated in LB seed medium and cultured until the seed solution was obtained. Then, the seed solution was transferred into 50 mL TB fermentation medium at a volume fraction of 1 mL / 50 mL, and 1 g / L of arabinose and 0.4 mM of IPTG were added after 2 h of continuous culture at 25°C for induction culture for 12 h. After completion, the bacterial cells were collected, high-pressure homogenized for disruption, and then used for purification and analysis.

[0048] The collected bacterial cells were high-pressure homogenized for disruption, and then high-speed centrifugation was performed to remove cell debris. The supernatant was filtered using a 0.22 μm water-based membrane, and the target protein was purified using Ni-NTA affinity chromatography. After equilibrating the column with A solution, the crude enzyme solution was loaded, and then the column was equilibrated with A solution. Different concentrations of B solution were used to wash the column and collect the washing solution. SDS-PAGE was used to verify the purified components, and the purest component was desalted using a PD-10 desalting column. Low-salt buffer (20 mM PBS, 150 mM NaCl; pH 7.5) was used during desalting, and the purified and desalted protein was collected. The A solution is 20 mM PBS buffer pH 7.5, 500 mM NaCl; the B solution is 20 mM PBS buffer pH 7.5, 500 mM NaCl, 500 mM imidazole.

[0049] Example 2: Comparison of different solubility-promoting tags and linker peptides on the expression amount of heparin 2-O-sulfotransferase

[0050] The fermentation broth of the recombinant bacteria constructed in Example 1 was centrifuged, and the bacterial cells were collected, washed and resuspended to adjust the OD 600The crude enzyme solution was obtained after breaking the cells, and 0.1 g / L CDSNS heparin, 1.5 g / L pNPS, 1.0 g / L PAPS, 2.0 g / L AST IV and 0.5 mL of the crude enzyme extract were added (in terms of final concentration) to the crude enzyme solution, the catalytic system reaction temperature was 40°C, and the catalytic time was 4 h. The enzyme activity was determined by detecting the absorbance of pNP at 400 nm during the reaction using an enzyme-linked immunoassay instrument. The results showed that the enzyme activity of the heparin 2-O-sulfotransferase N-terminally fused with the SUMO tag and the PAPAP connecting peptide was higher. The protein gel map of heparin 2-O-sulfotransferase with different solubility-promoting tags is shown in Figure 2 : From left to right, the lanes are the intracellular supernatant (left) and the precipitate (right) of pET32a empty load (control), Ga2OST, SUMO-Ga2OST, MBP-Ga2OST, TrxA-Ga2OST, SET2-Ga2OST, NusA-Ga2OST; the red arrow points to Ga2OST with different fusion tags. The blue arrow points to GroEL (without connecting peptide).

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

[0052] Name Enzymatic 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 SUMO-G4S-Ga2OST 269.65±13.55 SUMO-(Gly)6-GalOST 192.28±8.83 SUMO-EAAAK-Ga2OST 361.81±14.13

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

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

[0055] The mutant strains with significantly enhanced fluorescence were screened from 5000 cells using fluorescence-activated cell sorting (FACS) technology. Subsequently, these mutant strains 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 a shake flask. Then, the enzyme activity of the sfGFP reporter gene-removed strain was measured. By comparison, the strain with the highest enzyme activity was determined, and the optimal nucleotide sequence corresponding to the N-terminal first 10 amino acids of SUMO is shown as SEQ ID NO: 13.

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

[0057] Based on the pET-32a-SUMO-PAPAP-Ga2OST plasmid containing the SUMO mutation shown in SEQ ID NO: 13 constructed in Example 3, single-point mutations were introduced into Ga2OST using specific primers (Table 2) by site-directed mutagenesis technology. The entire plasmid was PCR amplified using the specified primers to introduce the desired mutations. After amplification, the PCR product was digested with Dpn I restriction endonuclease and purified by column method. The obtained linearized fragment was assembled by Gibson assembly method, then transformed into E. coli Top 10 cells, and the cells were spread on culture medium containing ampicillin. Positive clones were selected for further culture, and the plasmid was extracted to confirm its purity, and then transformed into E. coli OrigamiB(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 measured after the protein was purified according to the method of Example 1, and the results are shown in Table 4, where the specific enzyme activities of mutants A98K and Y145F were 573.95 U / mg and 665.00 U / mg, respectively. Figure 3

[0058] Table 2 Site-directed mutagenesis primers

[0059]

[0060]

[0061] Table 3 Information of heparin 2-O-sulfotransferase single-point mutant

[0062]

[0063]

[0064] Example 5: Obtaining heparin 2-O-sulfotransferase multiple-point superimposed mutant ​

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

[0066] Table 4 Information of heparin 2-O-sulfotransferase superimposed mutants

[0067] Mutation site Relative value of enzymatic activity relative to wild type Wild type 1.00 A85G / A98K 1.85 A85G / Y145F 2.01 A85G / F316L 1.75 A98K / Y145F 2.50 A98K / F316L 1.94 Y145F / F316L 1.54 A85G / A98K / Y145F 0.90 A85G / A98K / F316L 1.59 A98K / Y145F / F316L 1.01

[0068] Example 6: Optimization of fermentation conditions of heparin 2-O-sulfotransferase to improve its yield

[0069] The fermentation conditions for expressing the heparin 2-O-sulfotransferase mutant A98K / Y145F constructed in Example 5 were optimized, including: the recombinant strain was cultured in TB medium at 37°C to OD 600 0.6-0.8, 1 g / L of arabinose was added to a final concentration, and the culture temperature was adjusted to 25°C. After half an hour, IPTG was added, and the induction culture was continued.

[0070] The culture temperature after induction, culture time, and inducer concentration during fermentation were optimized, respectively, including:

[0071] (1) Fermentation according to the above method, the difference is that the culture temperature after induction is adjusted to 20-30, respectively. The results show that induction at 25°C can increase the crude enzyme activity to 649.39 U / mL.

[0072] (2) Fermentation according to the above method, the difference is that the induction is carried out at 25°C, and the induction culture time is adjusted to 12-48 h, respectively. The results show that the crude enzyme activity of 12 h culture increases to 758.31 U / mL.

[0073] (3) Fermentation according to the above method, the difference is that the concentration of the inducer arabinose is adjusted to 0-1 g / L, the concentration of IPTG is adjusted to 0.2-0.8 mM, and the induction is carried out at 25°C for 12 h. The results show that when the concentrations of arabinose and IPTG are 1 g / L and 0.4 mM respectively, the crude enzyme activity is increased to 837.75 U / mL.

[0074] Example 7: Fermentation of heparin 2-O-sulfotransferase in a 5-L fermenter

[0075] Subsequently, in order to verify the applicability of the fermentation parameters optimized in Example 6 to the culture of heparin 2-O-sulfotransferase mutants on a larger scale, a fed-batch culture strategy was adopted for further verification in a 5-L fermenter. The specific steps are as follows:

[0076] (1) Preparation of seed liquid: a single colony was inoculated into LB seed culture medium, and cultured at 37°C, 220 rpm for 8-10 h to prepare a first-stage seed liquid; the first-stage seed liquid 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 a second-stage seed liquid.

[0077] (2) Fermentation condition control: the strain was inoculated into a fermenter containing 2 L of TB culture medium at a inoculation amount of 10%, and 20 g / L of glycerol was added as the initial concentration. First, the culture was incubated at 37°C for 2-3 h until the OD 600 was 8-10; then, the temperature was reduced to 25°C for induction, and arabinose and IPTG were added to a final concentration of 1 g / L and 0.4 mM, respectively, and the culture was continued for 30 h. During the culture, the aeration amount was maintained at 2 VVM. When the dissolved oxygen DO decreased to less than 50%, the feeding of glycerol was started, and the feeding was stopped when the DO reached 80%, and the above feeding operation was continued when the DO decreased to less than 50%.

[0078] The heparin 2-O-sulfotransferase in the fermentation broth was detected at the end of fermentation, and the results showed that the time yield reached 37.23 mg of protein / g DCW, and the enzyme activity reached 5.72 x 10 6 U / L at 22 h after induction.

[0079] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Heparin 2- O - Sulfonyltransferase mutant, characterized by Based on the parent shown in SEQ ID NO.1, the following mutations are performed: a single point mutation of A85G, A98K, Y145F or F316L, or a combination of any two of the above mutations, or simultaneous mutations of A85G, A98K and F316L.

2. A fusion protein, characterized in that, In the heparin 2- of claim 1 O The sulfotransferase mutant is constructed by linking a solubilizing tag to its N-terminus; the solubilizing tag includes SUMO, MBP, TrxA, or NusA.

3. The fusion protein according to claim 2, characterized in that, The solubilization tag is a SUMO mutant; the SUMO mutant optimizes the nucleotide sequence corresponding to the first 10 amino acids after the N-terminal start codon encoded by the SUMO sequence shown in SEQ ID NO.12, and the nucleotide sequence corresponding to the first 10 amino acids after the N-terminal start codon of the optimized SUMO is shown in SEQ ID NO.

13.

4. The fusion protein according to claim 2 or 3, characterized in that, The heparin 2- O The sulfotransferase mutant is linked to the solubilizing tag via a linker peptide.

5. Encoding the heparin 2- as described in claim 1 O - Sulfonyltransferase mutant, or the gene of any of the fusion proteins described in claims 2 to 4.

6. Expressing the heparin 2- as described in claim 1 O - A sulfotransferase mutant or a recombinant microorganism of any of the fusion proteins described in claims 2 to 4.

7. A recombinant Escherichia coli, characterized in that, by E. coli OrigamiB (DE3) or E. coli BL21(DE3) serves as the host cell for expressing heparin 2- as described in claim 1. O - Sulfonyltransferase mutant or any of the fusion proteins described in claims 2 to 4.

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

9. A method for preparing the heparin 2- of claim 1 O The method for using sulfonyltransferase mutants is characterized by... The recombinant Escherichia coli of claim 7 or 8 is cultured in a culture medium for a period of time, and the enzyme solution is collected; the culture process is induced with arabinose and / or IPTG.

10. The heparin 2- according to claim 1 O - The use of a sulfotransferase mutant, or the fusion protein of any one of claims 2 to 4, or the gene of claim 5, or the recombinant microorganism of claim 6, or the recombinant Escherichia coli of claim 7 or 8, or the method of claim 9 in the pharmaceutical field for the preparation of products containing heparin or heparan sulfate.

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