A heparin n-sulfotransferase mutant with high activity and high thermal stability, a coding gene thereof and application thereof

Abstract

The present application relates to a kind of high activity, high thermal stability heparin N-sulfate group transferase mutant and its coding gene and application.The amino acid sequence of the N-sulfate group transferase mutant M8 is as shown in SEQ ID NO.2, and the nucleotide sequence of coding gene is as shown in SEQ ID NO.1.The N-sulfate group transferase mutant M8 is 24 amino acids in wild-type N-sulfate group transferase Mutations.Determination of stability, compared with wild-type N-sulfate group transferase at 37 DEG C only incubate less than 1 day, all activity is lost, the N-sulfate group transferase mutant M8 of the present application has high activity and extremely strong high thermal stability, can be incubated at 37 DEG C 7 days still keep about 50% activity.The present application improves the efficiency of heparin chemical enzyme synthesis, expands the application range of N-sulfate group transferase mutant, makes it possible to synthesize heparin at high temperature, greatly promotes the application development of heparin biomimetic synthesis.

Description

Technical Field

[0001] The present invention relates to a highly active and highly thermally stable heparin N-sulfatase mutant and its encoding gene and application, belonging to the field of biotechnology. Background Art

[0002] Heparin (HP) is an important glycosaminoglycan composed of repeating disaccharide units of D-β-glucuronic acid (or L-α-iduronic acid) and N-acetylglucosamine. Heparin has significant medicinal value. In addition to its diverse anticoagulant applications, it is also used to treat angina pectoris, nephrotic syndrome, severe burns, rheumatoid arthritis, and other conditions. Its demand consistently ranks among the highest in the international biotechnology pharmaceutical sector.

[0003] Heparin is primarily produced by extracting it from animal tissues and organs, such as bovine lungs and porcine intestinal mucosa. However, natural extraction methods are characterized by low safety, low yield, high solvent consumption, and severe environmental pollution. Furthermore, product quality is difficult to control due to the wide variation in raw materials, and impurities (such as chondroitin sulfate) are easily introduced during the production process. The widespread heparin contamination incident that occurred in 2008, resulting in the deaths of nearly 100 patients, significantly promoted the development of non-animal heparin production. Chemoenzymatic synthesis strategies are of great significance for new drug development due to their strong stereoselectivity, high yield, mild reaction conditions, uniform and stable product quality, and ease of functional group derivatization and modification. They are expected to become an ideal new technology for the synthesis of heparin oligosaccharides. The use of the tool enzymes in chemoenzymatic methods is a limiting factor in the further development of this strategy.

[0004] In the in vivo heparin synthesis pathway, N-deacetyl / N-sulfotransferase (NDST) is a bifunctional enzyme with two enzymatic domains: an N-deacetylase N-deacetylase active domain and an N-sulfotransferase N-sulfotransferase (NST) N-sulfotransferase domain. The N-sulfotransferase domain converts GlcNAc residues into N-sulfated glucosamine (GlcNS) residues. The N-deacetylase domain removes the acetyl group from GlcNAc, forming N-unsubstituted glucosamine (GlcNH2). The NDST modification step is crucial for HS biosynthesis, as other HS biosynthetic enzymes, such as C5-epimerase, 2-O-sulfotransferase, and 3-O-sulfotransferase, require the presence of a GlcNS residue to complete their modification.

[0005] Because the degree of N-sulfation largely determines the pharmacological activity of heparin, the activity of N-sulfotransferases is crucial for enzymatic synthesis in vitro. However, wild-type N-sulfotransferases exhibit low activity and stability when expressed in prokaryotic systems due to a lack of protein glycosylation. To date, there have been no reports of systematic protein engineering efforts based on N-sulfotransferases. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a highly active and highly thermally stable heparin N-sulfatase mutant, its encoding gene, and its application.

[0007] The technical solutions of the present invention are as follows:

[0008] In a first aspect of the present invention, there is provided an N-sulfotransferase mutant M8, 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;

[0009] The N-sulfatase mutant M8 is a wild-type N-sulfatase in which 24 amino acids are mutated; the mutations are specifically: G625S, S637P, E660D, R688K, V699I, D721N, S738D, S742K, K743E, I761L, N771S, K779Q, E784D, T801I, T807H, L842E, D843E, S741P, A767Y, E839P, A846K, T869P, Y864H, and T872L.

[0010] Compared with the wild-type N-sulfotransferase, the N-sulfotransferase mutant M8 has the following mutations: glycine at position 625 is mutated to serine, serine at position 637 is mutated to proline, glutamic acid at position 660 is mutated to aspartic acid, arginine at position 688 is mutated to lysine, valine at position 699 is mutated to isoleucine, aspartic acid at position 721 is mutated to asparagine, serine at position 738 is mutated to aspartic acid, serine at position 742 is mutated to lysine, lysine at position 743 is mutated to glutamic acid, isoleucine at position 761 is mutated to leucine, and asparagine at position 771 is mutated to serine. , lysine at position 779 mutated to glutamine, glutamate at position 784 mutated to aspartic acid, threonine at position 801 mutated to isoleucine, threonine at position 807 mutated to histidine, leucine at position 842 mutated to glutamate, aspartic acid at position 843 mutated to glutamate, serine at position 741 mutated to proline, alanine at position 767 mutated to tyrosine, glutamate at position 839 mutated to proline, alanine at position 846 mutated to lysine, threonine at position 869 mutated to proline, tyrosine at position 864 mutated to histidine, and threonine at position 872 mutated to leucine.

[0011] Preferably, according to the present invention, the wild-type N-sulfotransferase is the N-Sulfotransferase domain (L557-R882) of the wild-type human N-deacetyl / N-sulfotransferase NDST-1, and the N-Sulfotransferase domain contains 325 amino acids; the NCBI accession number of the wild-type human N-deacetyl / N-sulfotransferase NDST-1 is P52848.1.

[0012] A second aspect of the present invention provides an expression cassette or recombinant vector containing the gene encoding the N-sulfotransferase mutant M8. The recombinant vector is not particularly limited to the starting vector and can be any vector known in the art, as long as it is capable of replicating in a host. For example, the vector includes, but is not limited to, a plasmid or a bacteriophage. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

[0013] Preferably, according to the present invention, the recombinant vector is obtained by connecting the coding gene of the N-sulfatase mutant M8 with an expression vector, and the expression vector is a plasmid.

[0014] More preferably, the plasmid is pGEX-4T-1.

[0015] A third aspect of the present invention provides a recombinant host cell containing the gene encoding the N-sulfotransferase mutant M8. The term "host cell" has the meaning commonly understood in the art, and is a host cell capable of introducing the gene encoding the mutant of the present invention. Following introduction, such a host cell is referred to as a recombinant host cell. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli OrigamiB (DE3).

[0016] A fourth aspect of the present invention provides a method for producing the N-sulfatase mutant M8 by fermentation, the method comprising culturing the above-mentioned host cell and isolating and obtaining the N-sulfatase mutant.

[0017] Preferably, according to the present invention, the method for producing the N-sulfatase mutant M8 by fermentation comprises the following specific steps: culturing the recombinant host cells containing the encoding gene of the N-sulfatase mutant M8 in LB liquid culture medium to an OD600 of 0.6 to 0.8, adding IPTG to induce expression for 16 to 18 hours; collecting the cells, ultrasonically disrupting them, centrifuging and filtering them, and purifying them through a GST tag to obtain the N-sulfatase mutant M8.

[0018] The fifth aspect of the present invention provides the use of the above-mentioned N-sulfatase mutant M8 in the synthesis of N-sulfated heparin oligosaccharides and polysaccharides.

[0019] According to the present invention, the application is preferably to use 3'-phosphoadenosine-5'phosphosulfate (PAPS) as a donor and heparin oligosaccharides and polysaccharides with a -GlcNH2-GlcA- structure as starting acceptors to catalyze the reaction to generate N-sulfated oligosaccharides and polysaccharides with a structure of -GlcNS-GlcA-.

[0020] Experimental operations not described in detail in the present invention can be carried out according to conventional experimental operations in this technical field.

[0021] Beneficial effects

[0022] 1. The N-sulfotransferase mutant M8 provided by the present invention is a novel artificial heparin N-sulfotransferase. Compared to the wild-type N-sulfotransferase (the L557-R882 portion of NCBI accession number P52848.1), it has 24 amino acid mutations and possesses the activity of transferring sulfate groups to the -GlcNH2-GlcA- group of the heparin structure. Stability tests have shown that, while the wild-type N-sulfotransferase loses all activity after less than one day of incubation at 37°C, the N-sulfotransferase mutant M8 of the present invention has high activity and extremely strong thermal stability, maintaining approximately 50% of its activity even after incubation at 37°C for seven days.

[0023] 2. The N-sulfatase mutant M8 provided by the present invention effectively improves the efficiency of chemoenzymatic synthesis of heparin, expands the application range of N-sulfatase, makes it possible to synthesize heparin at high temperature, greatly promotes the development of biomimetic synthesis of heparin, and opens a new chapter for the research and development of glycosaminoglycans. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 RMSF analysis of each amino acid of wild-type N-sulfotransferase and simulated glycosylated N-sulfotransferase obtained from 20 ns molecular dynamics simulation at 37°C.

[0025] In the figure: the horizontal axis is the amino acid site, and the vertical axis is ΔRMSF.

[0026] Figure 2 This is an amino acid conservation analysis obtained by aligning more than a thousand homologous sequences of wild-type N-sulfotransferase.

[0027] In the figure: the horizontal axis is the amino acid site, and the vertical axis is the conservation score of the amino acid residue.

[0028] Figure 3 Comparison of the residual enzyme activity and expression levels of four N-sulfotransferase mutants after incubation at 37°C for 24 h calculated by PROSS.

[0029] In the figure: the horizontal axis represents the N-sulfatase mutant, and the vertical axis represents the enzyme activity.

[0030] Figure 4 The predicted results are those of virtual saturation screening.

[0031] Figure 5 Activity assay results of the N-sulfatase mutant M8 point mutation

[0032] In the figure: the horizontal axis represents different enzymes or mutants, and the vertical axis represents the residual enzyme activity after being incubated at 50°C for 0.5h, which is used to evaluate its stability.

[0033] Figure 6 Activity assay results of iterative mutants of N-sulfatase mutant M8

[0034] In the figure: the horizontal axis represents different enzymes or mutants, and the vertical axis represents the residual enzyme activity after being incubated at 50°C for 0.5h, which is used to evaluate its stability.

[0035] Figure 7 Comparison of the activities and stabilities of wild-type N-sulfotransferase, N-sulfotransferase mutant M1, and N-sulfotransferase mutant M8.

[0036] In the figure: the horizontal axis represents different enzymes or mutants, and the vertical axis represents the enzyme activity and stability.

[0037] Figure 8 The residual enzyme activities of wild-type N-sulfotransferase and N-sulfotransferase mutant M8 after incubation at 37°C for different time periods.

[0038] In the figure: the horizontal axis is the time of keeping at 37°C, and the vertical axis is the conversion rate of the substrate. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are further described below in conjunction with the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0040] The expression and purification methods of the N-sulfatase mutant recombinant proteins involved in the following examples are as follows:

[0041] The amino acid sequences of the N-sulfotransferase mutants were codon-optimized in Escherichia coli and then commissioned to Nanjing GenScript for synthesis or mutation. They were cloned into the pGEX-4T-1 vector and then chemically transformed into OrigamiB (DE3) competent cells. The cells were cultured on LB solid medium containing carbenicillin (50 μg / mL) for 12 h, and transformants were screened (negative control experiments were performed simultaneously) to obtain positive transformants.

[0042] Single colonies of N-sulfotransferase mutant transformants were selected and inoculated into 20 mL of sterile LB liquid medium (containing 50 μg / ml carbenicillin) for activation culture (37°C, 225 rpm). A 1% inoculum of the overnight activated culture was inoculated into 1 L of LB liquid medium (containing 50 μg / ml carbenicillin) for expansion. The culture was shaken at 37°C, 225 rpm for 4 hours until the OD600 was approximately 0.8. IPTG was added to a final concentration of 0.2 mM, and expression was induced at 22°C, 225 rpm for 16-18 hours. The cells were harvested by centrifugation and resuspended in 1× PBS buffer. Ultrasonic disruption was performed on ice (3 s on, 5 s off, 33% amplitude, 1500 kJ energy, 4°C) for 30 min. The disrupted cells were centrifuged at 12,000 rpm for 20 min (4°C), and the supernatant was filtered through a 0.22 μm filter. Purification was performed using a GST chromatography column. After loading, the protein was washed with 1× PBS buffer and eluted with 1× PBS buffer containing 10 mM reduced glutathione to obtain the target protein. The purified protein was stored in 20% glycerol and stored in a -80°C refrigerator. The wild-type N-sulfotransferase was expressed and purified using the same method. The purified N-sulfotransferase mutant protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration of NST and mutants was determined using the BCA protein concentration assay kit (Biyuntian P0011).

[0043] Wild-type N-sulfotransferase was expressed and purified in the same manner.

[0044] The methods for determining the activity and stability of the N-sulfatase mutant recombinant proteins involved in the following examples are as follows:

[0045] 1. Determination of the Sulfate Transfer Activity of N-Sulfate Transferase Mutants

[0046] The reaction was conducted using the heparan deacetylase pentasaccharide GlcA-GlcNH2-GlcA-GlcNH2-GlcA-pNP as the acceptor substrate and PAPS as the donor substrate. The reaction system is shown in Table 1. The reaction was incubated in a water bath at 37°C for 0.5 h. The enzyme was inactivated by heating in boiling water for 5 min, thereby terminating the reaction. The reaction solution was filtered through a 0.22 μm filter and analyzed by HPLC according to the method described in Table 2. The pNP group of the monosaccharide acceptor has specific absorption at a UV wavelength of 310 nm. The mobile phase flow rate was 0.5 ml / min. The activity was defined as 1 IU per hour, representing the transfer of 1 μmol of sulfate to the acceptor substrate. The unit of activity was μmol / h.

[0047] Table 1. Reaction system for determination of sulfate transfer activity of N-sulfatase mutants

[0048]

[0049] Table 2. HPLC analysis method used for detection of heparin oligosaccharides

[0050]

[0051] 2. Stability Determination of Sulfate Transfer Activity of N-Sulfate Transferase Mutants

[0052] The reaction was conducted using the heparan deacetylase pentasaccharide GlcA-GlcNH2-GlcA-GlcNH2-GlcA-pNP as the acceptor substrate and PAPS as the donor substrate. The reaction system is shown in Table 1. The enzyme was first incubated at 50°C for 0.5 h, followed by the addition of the substrate. The reaction was then incubated in a 37°C water bath for 0.5 h. The enzyme was then inactivated by heating with boiling water for 5 min, thereby terminating the reaction. The reaction solution was filtered through a 0.22 μm filter and analyzed by HPLC according to the method described in Table 2. The pNP group of the monosaccharide acceptor has a specific absorption at a UV wavelength of 310 nm. The mobile phase flow rate was 0.5 ml / min. The activity was defined as 1 IU per hour, representing the transfer of 1 μmol of sulfate to the acceptor substrate. The unit of activity is μmol / h.

[0053] The activity and stability of the wild-type N-sulfotransferase were determined in the same manner.

[0054] Example 1: PROSS algorithm for calculating N-sulfatase mutants with enhanced thermal stability

[0055] The crystal structures of the glycosylated wild-type N-sulfotransferase (L557-R882 portion of NCBI accession number P52848.1, NST) and the wild-type N-sulfotransferase protein lacking glycosylation were input into the simulation software gromacs. Under default conditions, a 20 ns dynamics simulation was performed at 298 K. The amino acid RMSF values ​​were extracted, and the change in RMSF values ​​with and without glycosylation was calculated as ΔRMSF. The results are shown in Figure 2. Figure 1 As shown, then The more concentrated amino acid site regions serve as the focus of subsequent mutation studies.

[0056] NCBI homology search was used to search for homologous sequences of wild-type N-sulfotransferase from Homo sapiens (NCBI accession number P52848.1, L557-R882 part, NST). More than 1,000 homologous sequences were manually selected and aligned using Jalview software. Gap regions were manually deleted and the conservation of each amino acid residue was calculated (with 10 as the maximum value). The results are shown below. Figure 2The higher the value, the more conservative the site. Amino acid sites with a score < 8 are used as the focus of subsequent mutation studies.

[0057] According to the above dynamic simulation and homologous sequence information ( Figure 1 and Figure 2 ), select There are 39 amino acid residues with conservation scores <8, namely: G625, S637, E660, T669, D682, V685, R688, A691, K698, V699, T701, N705, D721, S738, S741, S742, K743, I761, A767, Y768, H769, A770, N771, Q772, K779, E784, K787, M791, T801, H805, T807, G823, E839, L842, D843, A846, Y864, T869, and T872.

[0058] The inventors used the PROSS algorithm to calculate and design four N-sulfatase (NST) mutants that are beneficial for enhancing stability within the above 39 sites, namely:

[0059] NST-design1: G625S, S637P, S741P, A767Y, E839P, T869P, 6 amino acids mutated compared to the wild-type N-sulfotransferase;

[0060] NST-design2: G625S, S637P, V699I, D721N, S738D, T807H, L842E, S741P, A767Y, E839P, T869P, 11 amino acid mutations compared to the wild-type N-sulfotransferase;

[0061] NST-design3: G625S, S637P, E660D, V699I, D721N, S738D, K743E, K779Q, E784D, T807H, L842E, S741P, A767Y, E839P, T869P, 15 amino acids mutated compared to the wild-type N-sulfotransferase;

[0062] NST-design4: G625S, Y634F, S637P, H649N, E660D, V699I, D721N, T729S, S738D, S742K, K743E, A746D, K779Q, M791E, H805S, E784D, T807H, L842E, S741P, A767Y, E839P, T869P, 22 amino acids are mutated compared to the wild-type N-sulfotransferase.

[0063] Then the N-sulfatase mutant NST-design1-4 was expressed and purified as a recombinant protein, and the activity and stability of the purified protein were tested. The test results are as follows: Figure 3 shown.

[0064] Depend on Figure 3 Among the N-sulfatase mutants NST-design 1-4, NST-design 3, with 15 mutations, exhibited the best activity, stability, and highest expression. We named it NST-M1, representing the first-generation mutant. Of the 39 hotspot amino acid sites identified by the PROSS algorithm, 15 were successfully iterated for mutation in NST-M1, leaving 24 hotspot amino acid sites.

[0065] Example 2. Activity determination of virtual saturation mutation and single-point mutation of N-sulfatase mutant M1

[0066] The NST-M1 mutant modeled using SWISS-MODEL was input into the molecular simulation software Discovery Studio. The virtual mutation function of the software was used to mutate the remaining 24 sites into another 19 amino acids. The change in the folding free energy ΔΔG after mutation was used as the main reference. The stability after mutation was evaluated using the ΔΔG value. Values ​​greater than 0 were predicted to be mutations that were not conducive to protein stability, and values ​​less than 0 were speculated to be mutations that might be conducive to protein stability. In addition, several obviously unfavorable amino acid mutations were manually deleted. The results are shown in the figure below. Figure 4 The boxed position is the designed site mutation.

[0067] Depend on Figure 4 It can be seen that 9 invalid sites were deleted from the remaining 24 sites, and the remaining 15 sites had single point mutations.

[0068] Then follow Figure 4 The predicted NST mutation was used to express and purify the N-sulfatase mutant recombinant protein, and the activity and stability of the purified protein were determined. The wild-type N-sulfatase was used as a control. The results were as follows: Figure 5shown.

[0069] Depend on Figure 5 It can be seen that the NST mutations predicted in this example hit the following 17 stability-enhancing points: T872L, T801I, T701I, S742K, S742R, D843E, A846K, I761L, K787D, M791I, Y864K, Y864H, R688K, K698V, Y768Q, Y768N and N771S.

[0070] Example 3. Iterative evolution of N-sulfatase mutants

[0071] The 17 single-point mutants with significantly improved stability obtained in Example 2 were divided into four groups according to their spatial positional relationships:

[0072] Group 1: T872L, T801I, and T701I;

[0073] Group 2: S742K, S742R, D843E, and A846K;

[0074] Group 3: I761L, K787D, M791I, Y864K, and Y864H;

[0075] Group 4: R688K, K698V, Y768Q, Y768N and N771S.

[0076] Then, using NST-M1 as the parent, NST mutations were set in each group, with each pairwise mutation or three superimposed combination mutants. Then, the N-sulfatase mutant recombinant protein was expressed and purified according to this mutation to obtain the N-sulfatase mutant. The activity and stability of the purified protein were then measured, and NST-M1 (design 3) was used as a control. The results are shown in Figure 2. Figure 6 As shown. Figure 6 The optimal mutants in each group can be obtained.

[0077] Subsequently, the optimal T872L / T801I double mutant (NST-M2) from group 1 iteration was combined with the optimal S742K / D843E / A846K triple mutant (NST-M3) from group 2 iteration to form a T872L / T801I / S742K / D843E / A846K quintuple mutant (NST-M6). The optimal I761L / Y864H double mutant (NST-M4) from group 3 iteration was combined with the optimal R688K / N771S double mutant (NST-M5) from group 4 iteration to form an I761L / Y864H / R688K / N771S quadruple mutant (NST-M7). Finally, the five mutants of T872L / T801I / S742K / D843E / A846K (NST-M6) and the four mutants of I761L / Y864H / R688K / N771S (NST-M7) were combined to obtain the nine mutants of T872L / T801I / S742K / D843E / A846K / I761L / Y864H / R688K / N771S (NST-M8) with the highest activity and stability. The mutant NST-M8 had mutations at 9 sites relative to the mutant NST-M1 and mutations at 24 sites compared to the wild-type N-sulfotransferase.

[0078] Then, NST-M8 was expressed and purified as a recombinant protein, and the activity and stability of the purified protein were determined. Wild-type N-sulfotransferase and NST-M1 were used as controls. The results were as follows: Figure 7 shown.

[0079] Depend on Figure 7 It can be seen that compared with the wild-type N-sulfotransferase, the activity of NST-M8 is increased by 2.5 times and the stability is increased by 11.3 times, indicating that the N-sulfotransferase mutant M8 can effectively improve the efficiency of chemoenzymatic synthesis of heparin and expand the application range of N-sulfotransferase.

[0080] Example 4: Determination of the temporal stability of the 8th generation mutant of N-sulfatase (NST-M8)

[0081] The purified wild-type N-sulfotransferase and NST-M8 protein were incubated at 37°C for 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days, and their residual enzyme activities were measured. Figure 8 shown.

[0082] Depend on Figure 8It can be seen that the wild-type N-sulfotransferase loses almost all its activity after one day of incubation at 37°C, while NST-M8 retains over 50% of its activity after seven days of incubation at 37°C. This demonstrates that the N-sulfotransferase mutant M8 is extremely stable compared to the wild-type N-sulfotransferase, indicating its high application value and potential in heparin synthesis. It makes heparin synthesis possible at high temperatures and greatly promotes the development of biomimetic heparin synthesis applications.

Claims

1. An N-sulfatase mutant M8, characterized in that Its amino acid sequence is shown in SEQ ID NO.2; The N-sulfatase mutant M8 is a wild-type N-sulfatase in which 24 amino acids are mutated; The wild-type N-sulfotransferase is the N-Sulfotransferase domain of the wild-type human N-deacetyl / N-sulfotransferase NDST-1, which contains 325 amino acids; the NCBI accession number of the wild-type human N-deacetyl / N-sulfotransferase NDST-1 is P52848.1; The mutations are specifically: G625S, S637P, E660D, R688K, V699I, D721N, S738D, S742K, K743E, I761L, N771S, K779Q, E784D, T801I, T807H, L842E, D843E, S741P, A767Y, E839P, A846K, T869P, Y864H, T872L.

2. The gene encoding the N-sulfatase mutant M8 according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence is shown in SEQ ID No.

1.

4. A recombinant vector containing the gene encoding the N-sulfatase mutant M8 according to claim 3.

5. A recombinant host cell containing a gene encoding the N-sulfatase mutant M8 according to claim 3.

6. A method for producing the N-sulfatase mutant M8 according to claim 1 by fermentation, characterized in that: The method comprises culturing the host cell according to claim 5 and isolating and obtaining the N-sulfatase mutant according to claim 1.

7. The method according to claim 6, wherein The recombinant host cells containing the gene encoding the N-sulfatase mutant M8 according to claim 3 were cultured in LB liquid medium to an OD600 of 0.6-0.8, and IPTG was added to induce expression for 16-18 hours; the cells were collected, ultrasonically disrupted, centrifuged, and purified via a GST tag to obtain the N-sulfatase mutant M8.

8. Use of the N-sulfatase mutant M8 according to claim 1 in the synthesis of N-sulfated heparin oligosaccharides and polysaccharides.

9. The use according to claim 8, characterized in that The application is to use 3'-phosphoadenosine-5'phosphosulfate (PAPS) as a donor and heparin oligosaccharides and polysaccharides with a -GlcNH2-GlcA- structure as starting acceptors to catalyze the reaction to generate N-sulfated oligosaccharides and polysaccharides with a structure of -GlcNS-GlcA-.

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