N-deacetylase / N-sulfotransferase and application thereof

Abstract

The invention provides an N-deacetylase / N-sulfotransferase and an application of the N-deacetylase / N-sulfotransferase. According to the invention, systematic stability design and surface electrostatic optimization are carried out on the N-deacetylation structural domain of NDST, and multiple engineering strategies such as structural domain fusion and linker peptide optimization are combined to synergistically improve the specific activity, substrate affinity and bifunctional catalytic synergy of enzyme. Experimental results show that the specific enzyme activity of the modified NDST mutant is obviously higher than that of natural enzyme while the difunctional catalytic capability is maintained.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and enzyme engineering technology, and in particular to an N-deacetylase / N-sulfotransferase and its applications. Background Technology

[0002] Four NDST isoforms exist in mammals, among which NDST1 is the most widely expressed and most thoroughly studied isoform. This enzyme catalyzes N from the non-reducing end to the reducing end of the polysaccharide chain sequentially. Deacetylation and N The sulfation reaction produces discrete GlcNS (N) molecules of variable length. The GlcNS clusters are composed of sulfated glucosamine. These GlcNS clusters are further recognized and modified by downstream enzymes (such as C5 epimerases, 2-O-, 6-O-, and 3-O-sulfotransferases) to generate highly sulfated and epimerized heparin. Therefore, the catalytic efficiency of NDST directly determines the degree of modification of the heparin polysaccharide chain and its final biological activity.

[0003] NDST is a bifunctional enzyme, mainly composed of two relatively independent catalytic domains: an N-terminal N-deacetylation domain (NDAc) and a C-terminal N-sulfonyltransfer domain (NST). Existing research indicates that both the NAc and NST domains can independently catalyze their respective reaction steps under certain conditions. However, in natural NDST molecules, the two catalytic functions are coupled through spatial synergy between the domains, enabling continuous modification of polysaccharide substrates. Further research revealed differences in the kinetics of different NDST isoforms in the two catalytic steps; some isoforms exhibit higher catalytic rates in the N-deacetylation reaction, while others show higher catalytic efficiency in the N-sulfonyltransfer reaction, suggesting that the overall activity of NDST is limited by the synergistic efficiency between its domains.

[0004] Recent structural biology studies have revealed that the NAc and NST domains of NDST1 are spatially aligned in opposite directions, with their respective catalytically active sites facing in opposite directions. This structural feature, to some extent, limits the efficient transfer of polysaccharide substrates between the two catalytic sites, reducing the synergistic efficiency of sequential reactions. Furthermore, the binding of heparin precursor polysaccharides to NDST1 spatially tends to preferentially occur within the NST domain, potentially limiting the initiation step of the N-deacetylation reaction and further affecting overall catalytic efficiency. These structural characteristics are considered a significant reason for the limited synergistic catalytic efficiency of natural NDST.

[0005] Currently, research on the molecular modification of NDST remains relatively limited. Existing related technologies mainly focus on improving the stability, solubility, or expression level of the enzyme protein, or on local optimization of single catalytic domains. While these methods have improved the enzyme's performance to some extent, they have not yet significantly enhanced the specific enzyme activity of NDST from the perspective of overall structural synergy. In particular, systematic design and optimization schemes targeting the synergistic mechanism of the N-deacetylation domain and the N-sulfonyl transfer domain have not been publicly reported. Therefore, how to improve the synergistic catalytic efficiency and specific enzyme activity of NDST through rational design or modification at the domain level remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes an N-deacetylase / N-sulfotransferase and its applications.

[0007] This invention provides an N-deacetylase / N-sulfonyltransferase, wherein the N-deacetylase / N-sulfonyltransferase comprises a domain 1 and a domain 2, and the N-deacetylase / N-sulfonyltransferase is any one of the following: (1) The amino acid sequence of the structural domain 2 is shown in SEQ ID No. 3, and the amino acid sequence of the structural domain 1 is shown in SEQ ID No. 2; (2) The amino acid sequence of the structural domain 2 is shown in SEQ ID No. 4, and: (2-1) The amino acid sequence of domain 1 is shown in SEQ ID No. 1; or (2-2) The amino acid sequence of domain 1 is shown in SEQ ID No. 2; or (2-3) Compared with SEQ ID No. 2, the amino acid sequence of domain 1 is as follows: position 11 is glutamine, position 31 is lysine, position 43 is proline, position 146 is valine, position 155 is glutamine, position 184 is leucine, position 298 is aspartic acid, position 471 is asparagine, and position 481 is glutamine; or (2-4) Compared with SEQ ID No. 2, the amino acid sequence of the first domain 1 is as follows: glutamine at position 11, lysine at position 31, proline at position 43, arginine at position 79, lysine at position 113, valine at position 146, glutamine at position 155, leucine at position 184, aspartic acid at position 200, aspartic acid at position 298, glutamic acid at position 419, asparagine at position 471, and glutamine at position 481. (2-5) Compared with SEQ ID No. 2, the amino acid sequence of the structural domain 1 is arginine at position 155, arginine at position 227, lysine at position 242, arginine at position 316, arginine at position 335, arginine at position 385, lysine at position 411, arginine at position 499, and arginine at position 511.

[0008] In some embodiments, domain 1 and domain 2 are linked by a linker peptide, the linker peptide being 2 to 50 amino acids in length.

[0009] In some embodiments, (2-1) the amino acid sequence of the linker peptide is shown in SEQ ID No. 5-9.

[0010] In some embodiments, (2-2) the amino acid sequence of the linker peptide is shown in SEQ ID No. 10-14.

[0011] In some embodiments, domain 1 is located at the N-terminus of N-deacetylase / N-sulfotransferase; and domain 2 is located at the C-terminus of N-deacetylase / N-sulfotransferase.

[0012] The present invention also provides a nucleic acid molecule encoding the N-deacetylase / N-sulfotransferase.

[0013] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0014] The present invention also provides a host cell containing the expression vector described above.

[0015] In some embodiments, the host cell is Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, insect cells, or mammalian cells.

[0016] The present invention also provides the use of the N-deacetylase / N-sulfotransferase in the preparation of heparin, heparan sulfate or heparin precursor.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention is the first to systematically design the stability of the N-deacetylation domain (NDAc) of NDST and optimize its surface electrostatics, which fundamentally improves the overall efficiency of NDST bifunctional catalysis.

[0018] (2) This invention achieves an overall improvement in enzyme specific activity, substrate affinity and catalytic synergy between structural domains through the synergistic effect of multiple strategies such as domain fusion, stability design, surface electrostatic engineering and linker peptide optimization, thus breaking through the limitations of single modification methods.

[0019] (3) The highly active NDST mutant can directly use natural heparin precursor polysaccharide (containing N-acetyl) as substrate, eliminating the chemical N-deacetylation step, thereby reducing the cost of in vitro heparin synthesis and simplifying the process.

[0020] (4) NDST, as a bifunctional enzyme, can be directly used to construct microbial cell factories and realize the full biosynthesis of heparin, while the NST domain alone cannot achieve this function. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the structural domain combination of NDST in Embodiment 1 of the present invention.

[0023] Figure 2 This is a schematic diagram illustrating the principle of sulfonyltransferase catalytic activity detection in Example 1 of the present invention.

[0024] Figure 3 The specific enzyme activity of the NDST domain fusion variant in Example 1 of the present invention.

[0025] Figure 4 PROSS is used to predict NDST multisite mutations in Example 2 of this invention.

[0026] Figure 5 The specific enzyme activity of the NDST stability mutant in Example 2 of this invention.

[0027] Figure 6 This is a map showing the surface charge distribution of the NDST mutant predicted by Rosetta Supercharge in Example 3 of the present invention.

[0028] Figure 7 The specific enzyme activity of the NDST charge-engineered mutant in Example 3 of this invention.

[0029] Figure 8 The specific enzyme activity of the optimized variant of the NDAc1 and NSTopt linker peptide in Example 4 of this invention.

[0030] Figure 9 The specific enzyme activity of the optimized variant of the NDAc2 and NSTopt linker peptide in Example 4 of this invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0032] This invention mainly employs four approaches for the rational design of NDST: (I) Design and construction of NDST domain fusion: The catalytic rate of NDAc in NDST2 is higher than that of NDST1, and the catalytic rate of NST in NDST1 is higher than that of NDST2. Taking advantage of the difference in catalytic properties between different NDST subtypes, the highly active NDAc domain from NDST2 and the highly active NST domain from NDST1 are functionally fused to construct a chimeric enzyme with synergistic catalytic advantages.

[0033] (II) Design of NDAC domain stability mutants based on PROSS: There is currently no scheme for mutation optimization of the NDAC domain. This invention uses the PROSS computational tool to perform phylogenetic analysis and energy optimization on the NDAC domain, and predicts multi-point mutation combinations that can improve its thermal stability and folding efficiency.

[0034] (III) NDAc charge engineering based on Rosetta Supercharge: Using the Rosetta Supercharge tool, the net positive charge of the surface accessible region of the NDAc domain is designed. By introducing positively charged amino acid mutations, the electrostatic interaction between the enzyme and the negatively charged heparin precursor polysaccharide is improved, thereby increasing the affinity of NDAc for the substrate and improving the enzyme catalytic efficiency.

[0035] (iv) Optimization design of interdomain linker peptides based on iMARS: Use iMARS tools to simulate and predict linker peptides between two domains, rationally design or screen flexible / rigid linker peptide sequences connecting the NDAC and NST domains, optimize the relative conformation between the two catalytic domains, and improve the synergistic catalytic efficiency of the whole enzyme.

[0036] Example 1: Design and Construction of NDST Domain Fusion 1. Design and construction of NDST domain fusion Based on the native structure of NDST1 and the functions of its domains, a series of fusion proteins were designed and constructed to enhance sulfation and N-deacetyltransferase activity. These include combining domains with higher activity to form a series of NDST fusion proteins. 1. NDST1 wild type (NDST1).

[0037] 2. NDST1△83 is NDST1 with the N-terminal membrane-binding domain removed (NDAc1-NST).

[0038] 3. Replace the NDAC2 structural domain (NDAc2-NST) based on 2.

[0039] 4. Replace the optimized NSTopt structure field (NDAc1-NSTopt) with the one in step 2.

[0040] 5. Replace the NSTopt structure field (NDAc2-NSTopt) based on step 3.

[0041] like Figure 1 As shown in SEQ ID No. 1, the amino acid sequence of NDAC1 is shown in SEQ ID No. 2, the amino acid sequence of NDAC2 is shown in SEQ ID No. 3, and the amino acid sequence of NST is shown in SEQ ID No. 4.

[0042] 2. Protein expression, purification, and enzyme activity detection (1) Construction of Pichia pastoris expression system: The rationally designed NDST protein was expressed and purified using Pichia pastoris for in vitro characterization of enzyme activity. The designed NDST was constructed into the Pichia pastoris protein expression plasmid pPICZαA, and the α-factor signal peptide was removed from the plasmid to achieve intracellular expression of NDST. The plasmid was linearized by MssI digestion and integrated into the Pichia pastoris genome via homologous recombination.

[0043] (2) Preparation and transformation of Pichia pastoris competent cells: Pichia pastoris was streaked onto YPD plates and cultured at 30°C for 3 days. Single colonies were then inoculated into 10 mL of YPD liquid medium / 250 mL shake flask and cultured at 30°C and 220 rpm for 16-18 h as seed culture. A 1% seed culture was then inoculated into 50 mL of YPD liquid medium / 500 mL shake flask and cultured at 30°C and 220 rpm for 6-8 h until the OD600 reached 0.6-1. Pichia pastoris competent cell preparation and transformation were performed using the Coolaber Pichia pastoris competent cell preparation and transformation kit. 10 mL of the bacterial culture was centrifuged at 3000 rpm for 3 min, and the supernatant was removed. The cells were resuspended in 5 mL of B1 solution, centrifuged at 3000 rpm for 3 min, and the supernatant was removed. Resuspend the bacterial cells in 200 μL of B1 solution, transfer to a sterile 1.5 mL centrifuge tube, add 20 μL of linearized plasmid (5-50 μg) and 10 μL of carrier DNA (incubate at 100℃ for 10 min, then quickly place on ice), and mix by inversion. Add 1.4 mL of B2 solution and mix by inversion, then incubate at 30℃ for 60 min. Centrifuge at 3000 rpm for 3 min to remove the supernatant, and resuspend the bacterial cells in 1 mL of B3 solution. Centrifuge at 3000 rpm for 3 min to remove the supernatant, and resuspend the bacterial cells in 100 μL of B3 solution. Spread the mixture on Zeocin antibiotic resistance agar plates and incubate at 30℃ for 3-5 days.

[0044] (3) Screening of positive clones: A pair of primers designed based on the NDST sequence can be used to PCR a DNA fragment of about 2000 bp. Single colonies are picked from the plate culture medium and colony PCR is performed for verification. Colonies with the correct band size are selected for the next fermentation step.

[0045] (4) Fermentation expression of the target protein by engineered strains: Select a single colony that has been verified and inoculate it into 50 mL of YPD medium (50 mL medium / 500 mL Erlenmeyer flask). Incubate at 30℃ and 200 rpm for 24 h to obtain the seed culture. Inoculate the seed culture at a rate of 10% into liquid BMGY medium (50 mL medium / 500 mL Erlenmeyer flask) and incubate at 30℃ and 200 rpm for 15-16 h. Collect all cells by centrifugation at 4500 rpm for 5 min, wash twice with 20 mL of physiological saline, centrifuge at 4500 rpm for 5 min, discard the supernatant, and transfer the cells into liquid BMMY medium (50 mL medium / 500 mL Erlenmeyer flask). Incubate at 30℃ and 200 rpm for 96 h, adding 1% methanol by volume every 24 h.

[0046] (5) Protein extraction, purification, and concentration determination: Bacterial cells were collected by centrifugation at 4500 rpm for 5 min, resuspended in pre-chilled PBS, and homogenized twice using a high-pressure cell homogenizer at 4°C and 1000 Pa. The cells were then centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was added to a final concentration of 20 mM imidazole, and the mixture was incubated overnight on a shaker at 4°C with a 2.5 mL nickel column. After centrifugation at 4°C and 2200 rpm for 5 min, the supernatant was discarded. The nickel column was then added to a chromatography column, and 8 column volumes of pre-chilled 20 mM and 100 mM imidazole PBS solutions were sequentially passed through the column to remove contaminating proteins and elute the target protein. The eluent containing the target protein was collected and concentrated by centrifugation at 4°C and 4500 rpm using a 30 kD ultrafiltration tube. 20 mM HEPES pH 7.5 solution was added, and the mixture was centrifuged at 4°C and 4500 rpm. This process was repeated three times to replace the solution and remove the imidazole. The protein sample was stored on ice for later use. The concentration of the purified protein was determined using the YEASEN BCA Protein Assay Kit (Enhanced Version). BSA solutions with final concentrations of 0, 40, 200, 400, 600, 1000, 1200, 1400, and 2000 μg / mL were prepared as standards for plotting standard curves. The total required BCA working solution volume was calculated as (standards + test samples) × number of replicates × 200 μL. To prepare the BCA working solution: add 1 volume of reagent B to 50 volumes of reagent A and mix thoroughly. Add 25 μL of each standard and test sample to a microplate, add 200 μL of BCA working solution to each well, vortex for 30 s to mix, incubate at 37°C for 30 min, and measure the absorbance at 562 nm. Plot the standard curve and calculate the sample protein concentration based on the standard curve and dilution factor.

[0047] 3. Detection method and principle of N-sulfonyltransferase catalytic activity: AST IV (PAPS regenerase) can transfer the sulfate group of p-nitrophenyl sulfate (PNPS) to 3'-adenosine 5'-phosphate (PAP) to form a sulfonic acid group donor, PAPS, which participates in the sulfonate transfer reaction and the byproduct p-nitrophenol (PNP). The formation of PNP can be monitored by measuring the absorbance at 400 nm to detect the specific enzyme activity of NDST.

[0048] The enzyme activity reaction system consisted of: 50 mM PNPS, 20 mM HEPES, 50 μg / mL AST IV, 0.5 mM PAP, 100 mg / L heparin precursor polysaccharide, and 200 μg / mL NDST1, NDST1-NST, NDAc2-NST, NDAc1-NSTopt, or NDAc2-NSTopt, with a total volume of 200 μL. The reaction was incubated at 37℃ for 1 h, and the increase in OD400 before and after the reaction was detected. PNP solutions of 20, 40, 60, 80, and 100 μM were prepared using 20 mM pH 7.5 HEPES buffer. A standard curve of PNP was measured at OD400, and the NDST enzyme activity was calculated based on the standard curve.

[0049] The enzyme activity unit of a sulfonyltransferase is defined as the amount of enzyme required to release 1 μM PNP per hour at pH 7.5 and 37°C. Specific enzyme activity is defined as the number of micromoles of substrate conversion catalyzed by each milligram of enzyme protein per hour, expressed in μmol / mg / h. A schematic diagram illustrating the principle of sulfonyltransferase catalytic activity detection is shown below. Figure 2 As shown.

[0050] Test results are as follows Figure 3 As shown, the specific enzyme activity of the original NDST1 enzyme is 84.0 U / (mg·h). After removing the N-terminal transmembrane domain, the specific enzyme activity of NDAc1-NST is 72.1 U / (mg·h), a decrease of 14.2% compared to NDST1. After replacing the NDAc2 domain with NDAc1-NST, the specific enzyme activity of NDAc2-NST is 77.4 U / (mg·h), an increase of 7.3% compared to NDAc1-NST. After replacing the NSTopt domain with NDAc1-NST, the specific enzyme activity of NDAc1-NSTopt is 84.4 U / (mg·h), an increase of 17.1% compared to NDAc1-NST. After replacing the NSTopt domain with NDAc2-NST, the specific enzyme activity of NDAc2-NSTopt is 83.0 U / (mg·h), an increase of 7.3% compared to NDAc2-NST. In summary, replacing both the NDAc2 and NSTopt domains improved the specific enzyme activity of NDST.

[0051] Example 2: Design of a PROSS-based NDAC domain stability mutant: The amino acid sequence of NDAC2 was input into the PROSS website (https: / / pross.weizmann.ac.il / step / pross-terms / ), with the following parameter settings: coverage: 75; evalue: 0.0001; iddt_dist_cutoff: 5; iddt_threshold: 90; max_targets: 6000; min_id: 35; res_to_fix: 235A 236A 309A 305A 445A. Nine multimutation prediction sequences were obtained. Based on NDAC2-NSTopt (WT), the first six mutants were constructed for characterization and screening, as follows: Figure 4 As shown, they are respectively; Mutant 1 (M1): G113K, R471N, P481Q.

[0052] Mutant 2 (M2): S31K, T43P, R471N, P481Q.

[0053] Mutant 3 (M3): S31K, T43P, G113K, R471N, P481Q.

[0054] Mutant 4 (M4): S31K, T43P, G113K, I146V, Q184L, R471N, P481Q.

[0055] Mutant 5 (M5): A11Q, S31K, T43P, I146V, E155Q, Q184L, K298D, R471N, P481Q.

[0056] Mutant 6 (M6): A11Q, S31K, T43P, G113K, I146V, E155Q, Q184L, K298D, R471N, P481Q, V79R, R419E, G200D.

[0057] The specific enzyme activity of each mutant was tested according to the method in Example 1, and the results are as follows: Figure 5 As shown, the specific enzyme activity of NDAc2-NSTopt (WT type) is 47.4 U / (mg·h). Among the mutants, the specific enzyme activities of M1, M3, and M4 are 42.3, 40.9, and 41.1 U / (mg·h), respectively, which are slightly lower than those of WT; the specific enzyme activity of M2 is 47.4 U / (mg·h), which is basically consistent with WT. In contrast, M5 and M6 show significantly enhanced catalytic activity, with specific enzyme activities reaching 50.6 and 56.0 U / (mg·h), respectively. Among them, M6 has the highest specific enzyme activity, which is about 18.2% higher than that of WT, showing a significant activity enhancement effect.

[0058] It should be noted that, because the enzyme activity detection method used is highly sensitive to the initial kinetics of the reaction, the detection signal is mainly concentrated in the rapid change range at the beginning of the reaction. The time interval between the completion of the reaction system preparation and the start of detection is difficult to be completely consistent across different batches. Furthermore, differences in the storage time of enzyme samples under low-temperature conditions may also affect their instantaneous catalytic activity. Therefore, the absolute specific enzyme activity values ​​obtained from different batches may fluctuate. However, within the same batch of experiments, the control and experimental groups used completely identical operating conditions in terms of enzyme preparation, reaction system configuration, reaction initiation, and detection time. Therefore, the results can accurately reflect the relative catalytic performance differences between different enzyme configurations.

[0059] Example 3: NDAC charge engineering based on Rosetta Supercharge: Run the following code in Rosetta Supercharge: ~ / software / rosetta.binary.ubuntu.release-371 / main / source / bin / supercharge.static.linuxgccrelease -s NDAc2.pdb -use_input_sc -ignore_unrecognized_res -jd2:no_output -dont_mutate_glyprocys true -dont_mutate_correct_chargetrue -dont_mutate_hbonded_sidechains true -include_arg -include_lys -refweight_arg -0.98 -refweight_lys -0.65 -surface_residue_cutoff 25 -target_net_charge_active -target_net_charge 5 -jd2:no_output -nstruct 10>log.

[0060] By modifying the value of `target_net_charge` to 5, 10, and 20 in the code, mutants with net surface charges of 5, 10, and 20 for NDAC2 were designed, resulting in three optimized NDAC2 structures with net surface charges of 6, 10, and 19, respectively. The surface charge distribution of the NDST mutants predicted by RosettaSupercharge is shown in the figure below. Figure 6 As shown.

[0061] Based on the WT type, these three mutants were constructed and purified for in vitro characterization: Mutant 7 (M7): D227R, E242K, H335R, Y385R, Y411K, W511R; Mutant 8 (M8): E155R, D227R, E242K, H316R, H335R, Y385R, Y411K, Q499R, W511R; Mutant 9 (M9): S31R, E33K, E80R, Q148R, E155R, D227R, E242K, E252R, H316R, H335R, Y385R, H407K, Y411K, Q499R, W511R.

[0062] The specific enzyme activity of each mutant was tested according to the method in Example 1, and the results are as follows: Figure 7 As shown, the specific enzyme activity of each sample was measured, with the WT type showing 47.4 U / (mg·h). The specific enzyme activities of mutants M7 and M9 were 42.9 and 42.8 U / (mg·h), respectively, both lower than those of WT; while the specific enzyme activity of mutant M8 was significantly increased, reaching 59.2 U / (mg·h). Compared with WT, the specific enzyme activity of M8 increased by approximately 25.0%, demonstrating a significant activity enhancement effect.

[0063] Example 4: Optimized design of interdomain linking peptides based on iMARS The structures of NDAC1, NDAC2, and NST were predicted and generated using AlphaFold3 (https: / / alphafoldserver.com / ). The octasaccharide structure of the heparin precursor polysaccharide was drawn using ChemDraw and converted into PDB form. AutoDock was used to dock the domains and the heparin precursor polysaccharide, and the optimal docking structure was submitted to iMARS for linker peptide screening, resulting in 10 predicted linkers. These 10 linkers were then inserted between NDAC1 (or NDAC2) and NST to construct 10 optimized linker peptide variants for subsequent enzyme activity characterization.

[0064] Table 1. Linking peptides of NDAC1 and NST predicted by iMARS.

[0065] Table 2 iMARS-predicted linker peptides of NDAC2 and NST

[0066] Variant 1: NDAC1-L1-NSTopt, with the sequence of linker peptide 1 (L1) being GPGGS (SEQ ID No. 5).

[0067] Variant 2: NDAC1-L2-NSTopt, with the sequence of linker peptide 2 (L2) being SSS (SEQ ID No. 6).

[0068] Variant 3: NDAC1-L3-NSTopt, with the sequence of linker peptide 3 (L3) being MALEK (SEQ ID No. 7).

[0069] Variant 4: NDAC1-L4-NSTopt, with the sequence of linker peptide 4 (L4) being GGGD (SEQ ID No. 8).

[0070] Variant 5: NDAC1-L5-NSTopt, with the sequence of linker peptide 5 (L5) being GGGGAGGGGAGGGGA (SEQ ID No. 9).

[0071] Variant 6: NDAC2-L6-NSTopt, with the sequence of linker peptide 6 (L6) being GPGGA (SEQ ID No. 10).

[0072] Variant 7: NDAC2-L7-NSTopt, with the sequence of linker peptide 7 (L7) being GPGGDGPGGD (SEQ ID No. 11).

[0073] Variant 8: NDAC2-L8-NSTopt, with the sequence of linker peptide 8 (L8) being SS (SEQ ID No. 12).

[0074] Variant 9: NDAC2-L9-NSTopt, with the sequence of linker peptide 9 (L9) being GGGGSGGGGS (SEQ ID No. 13).

[0075] Variant 10: NDAC2-L10-NSTopt, with the sequence of linker peptide 10 (L10) being GS (SEQ ID No. 14).

[0076] The specific enzyme activity of each mutant was tested according to the method in Example 1, and the results are as follows: Figures 8-9 As shown, the specific enzyme activities of variants 1-5 were 55.2, 82.6, 52.7, 74.2, and 66.1 U / (mg·h), respectively. Among them, variant 2 (NDAc1-L2-NSTopt) exhibited the highest specific enzyme activity of 82.6 U / (mg·h), which was 40.4% higher than that of the control group NDAC1-NSTopt (specific enzyme activity 58.8 U / (mg·h)), showing a significant activity enhancement effect.

[0077] The specific enzyme activities of variants 6–10 were 55.1, 45.6, 56.2, 49.5, and 41.1 U / (mg·h), respectively. In this group, variant 8 (NDAc2-L8-NSTopt) had the highest specific enzyme activity at 56.2 U / (mg·h), which was 18.6% higher than the corresponding control group NDAC1-NSTopt (specific enzyme activity 47.4 U / (mg·h)).

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0079] sequence list SEQ ID No.1 MVLVFVESLYSQLGQEVVAILESSRFKYRTEIAPGKGDMPTLTDKGRGRFALIIYENILKYVNLDAWNRELLDKYCVAYGVGIIGFFKANENSLLSAQLKGFPLFLHSNLGLKDCSINPKSPLLYVTRP SEVEKGVLPGEDWTVFQSNHSTYEPVLLAKTRSSESIPHLGADAGLHAALHATVVQDLGLHDGIQRVLFGNNLNFWLHKLVFVDAVAFLTGKRLSLPLDRYILVDIDDIFVGKEGTRMKVEDVKALFDT QNELRAHIPNFTFNLGYSGKFFHTGTNAEDAGDDLLLSYVKEFWWFPHMWSHMQPHLFHNQSVLAEQMALNKKFAVEHGIPTDMGYAVAPHHSGVYPVHVQLYEAWKQVWSIRVTSTEEYPHLKPARYR RGFIHNGIMVLPRQTCGLFTHTIFYNEYPGGSSELDKIINGGELFLTVLLNPISIFMTHLSNYGNDRLGLYTFKHLVRFLHSWTNLRLQTLPPVQLAQKYFQIFSEEKDPLWQDPCEDKRHKDIWSKEK SEQ ID No.2 MEPVVLVFVESAYSQLGQEIVAILESSRFRYSTELAPGRGDMPTLTDNTHGRYVLVIYENLLKYVNLDAWSRELLDRYCVEYGVGIIGFFRAHEHSLLSAQLKGFPLFLHSNLGLRDYQVNPSAPLLHLTRPSRLEPGPLPGDDWTIFQSNHSTYEPVLLASLRPAEPAVPGPVLRRARLPTVVQDLGLHDGIQRVLFGHGLSFWLHKLIFVDAVAYLTGKRLCLDLDRYILVDIDDIFVGKEGTRMKVADVEALLTTQNKLRTLVPNFTFNLGFSGKFYHTGTEEEDAGDDMLLKHRKEFWWFPHMWSHMQPHLFHNRSVLADQMRLNKQFALEHGIPTDLGYAVAPHHSGVYPIHTQLYEAWKSVWGIQVTSTEEYPHLRPARYRRGFIHNGIMVLPRQTCGLFTHTIFYNEYPGGSRELDRSIRGGELFLTVLLNPISIFMTHLSNYGNDRLGLYTFESLVRFLQCWTRLRLQTLPPVPLAQKYFELFPQERSPLWQNPCDDKRHKDIWSKEK SEQ ID No.3 TCDRFPKLLIIGPQKTGTTALYLFLGMHPDLSSNYPSSETFEEIQFFNGHNYHKGIDWYMEFFPIPSNTTSDFYFEKSANYFDSEVAPRRAAALLPKAKVLTILINPADRAYSWYQHQRAHDDPVALKYTFHEVITAGSDASSKLRALQNRCLVPGWYATHIERWLSAYHANQILVLDGKLLRTEPAKVMDMVQKFLGVTNTIDYHKTLAFDPKKGFWCQLLEGGKTKCLGKSKGRKYPEMDLDSRAFLKDYYRDHNIELSKLLYKMGQTLPTWLREDLQNTR SEQ ID No.4 TCDRFPKLLIIGPQKTGTTALYLFLSMHPDLSSNYPSPETFEEIQFFNGHNYHKGIDWYMDFFPIPSNTTSDFYFEKSANYFDSEVAPKRAAALLPKAKILTILINPADRAYSWYQHQRAHNDPVALKYTFHEVITAGDDAPKELRALQNRCLVPGWYATHLERWLSYYHASQILVLDGKLLRTEPAKVMDMVQKFLGVTNTIDYHKTLAFDPKKGFWCQLLEGGKTKCLGKSKGRKYPPMDEESRKFLKDYYRDHNIELSKLLKKMGQPLPLWLREDLQNTR SEQ ID No.5 GPGGS SEQ ID No.6 SSS SEQ ID No.7 MALEK SEQ ID No.8 GGGD SEQ ID No.9 GGGGAGGGGAGGGGA SEQ ID No.10 GPGGA SEQ ID No.11 GPGGDGPGGD SEQ ID No.12 SS SEQ ID No.13 GGGGSGGGGS SEQ ID No.14 GS。

Claims

1. An N-deacetylase / N-sulfonyltransferase, characterized in that, The N-deacetylase / N-sulfonyltransferase comprises domain 1 and domain 2, and the N-deacetylase / N-sulfonyltransferase is any one of the following: (1) The amino acid sequence of the structural domain 2 is shown in SEQ ID No. 3, and the amino acid sequence of the structural domain 1 is shown in SEQ ID No. 2; (2) The amino acid sequence of the structural domain 2 is shown in SEQ ID No. 4, and: (2-1) The amino acid sequence of domain 1 is shown in SEQ ID No. 1; or (2-2) The amino acid sequence of domain 1 is shown in SEQ ID No. 2; or (2-3) Compared with SEQ ID No. 2, the amino acid sequence of domain 1 is as follows: position 11 is glutamine, position 31 is lysine, position 43 is proline, position 146 is valine, position 155 is glutamine, position 184 is leucine, position 298 is aspartic acid, position 471 is asparagine, and position 481 is glutamine; or (2-4) Compared with SEQ ID No. 2, the amino acid sequence of the first domain 1 is as follows: glutamine at position 11, lysine at position 31, proline at position 43, arginine at position 79, lysine at position 113, valine at position 146, glutamine at position 155, leucine at position 184, aspartic acid at position 200, aspartic acid at position 298, glutamic acid at position 419, asparagine at position 471, and glutamine at position 481. (2-5) Compared with SEQ ID No. 2, the amino acid sequence of the structural domain 1 is arginine at position 155, arginine at position 227, lysine at position 242, arginine at position 316, arginine at position 335, arginine at position 385, lysine at position 411, arginine at position 499, and arginine at position 511.

2. The N-deacetylase / N-sulfonyltransferase according to claim 1, characterized in that, Domain 1 and domain 2 are linked by a linker peptide, which is 2 to 50 amino acids in length.

3. The N-deacetylase / N-sulfonyltransferase according to claim 2, characterized in that, In (2-1), the amino acid sequence of the linker peptide is shown in SEQ ID No. 5~9.

4. The N-deacetylase / N-sulfonyltransferase according to claim 2, characterized in that, In (2-2), the amino acid sequence of the linker peptide is shown in SEQ ID No. 10~14.

5. The N-deacetylase / N-sulfotransferase according to claim 1, characterized in that, Domain 1 is located at the N-terminus of N-deacetylase / N-sulfotransferase; domain 2 is located at the C-terminus of N-deacetylase / N-sulfotransferase.

6. A nucleic acid molecule encoding the N-deacetylase / N-sulfotransferase according to any one of claims 1 to 5.

7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. The host cell according to claim 8, characterized in that, The host cell is Escherichia coli, Pichia pastoris, insect cells, Saccharomyces cerevisiae, or mammalian cells.

10. The use of the N-deacetylase / N-sulfotransferase according to any one of claims 1 to 5 in the preparation of heparin, heparan sulfate or its precursor.

Images