A method for increasing the catalytic activity of heparin 3-o-sulfotransferase 1

Abstract

The application discloses a method for improving catalytic activity of heparin 3-O-sulfotransferase 1, and belongs to the technical field of bioengineering. The method for improving catalytic activity of heparin 3-O-sulfotransferase 1 comprises the following steps: firstly, selecting Escherichia coli as a host bacterium; and then using a method for expressing and optimizing 3-O-sulfotransferase 1 by means of N-terminal fusion of a solubility-promoting tag, further mutating and transforming the optimized 3-O-sulfotransferase 1 sequence by rational design, and obtaining a high-activity production strain. The modification of the enzyme plays a crucial role in the anticoagulant activity of heparin, and the improvement of the enzyme activity has a positive significance for the enzymatic synthesis rate and the anticoagulant activity of heparin.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1. Background Technology

[0002] Heparin is a polysaccharide synthesized and secreted into the extracellular space by the Golgi apparatus of eukaryotic cells in nature. It is named after the liver, where it was first discovered. It is also found in animal tissues such as the lungs, blood vessel walls, and intestinal mucosa. Functionally, it interacts with various proteins in the extracellular matrix and plays an important role in anticoagulation, viral invasion, tumor spread, and signal transduction. It has excellent anticoagulant effects both in vivo and in vitro and is widely used in clinical practice.

[0003] Chemically, it is a linear polysaccharide with a straight chain and a negative charge, formed by alternating polysaccharide chains composed of glucosamine, L-iduronide, N-acetylglucosamine and D-glucuronic acid, which are sulfonated in multiple places. It is a heparin precursor composed of alternating glucuronic acid and N-acetylglucosamine units, which undergoes a series of deacetylation, isomerization and sulfation processes.

[0004] Heparin's mechanism of action involves its specific pentasaccharide structure binding to antithrombin III (AT-III), thereby enhancing its activity, inhibiting the formation of prothrombin kinase, and interfering with thrombin's function by forming a complex with thrombin and antithrombin. This also prevents the activation of coagulation factors and platelet aggregation. Clinically, it is mainly used for thromboembolic diseases, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, and hemodialysis. With advancements in pharmacology and clinical medicine, the application of heparin continues to expand.

[0005] Currently, the main methods for obtaining heparin include biological extraction, chemical synthesis, chemoenzymatic synthesis, and enzymatic synthesis. Biological extraction primarily involves extracting heparin from the small intestinal mucosa of animals. However, this method often fails to completely remove other glycosaminoglycan impurities, such as chondroitin sulfate. These impurities can trigger immune responses in humans, leading to certain deficiencies in the biosafety of the resulting product. There have been cases of heparin contamination that have harmed patients using the drug. Chemical synthesis of heparin is very complex and therefore relatively expensive. The main difference between chemoenzymatic and enzymatic synthesis lies in the first step of N-deacetylation, which uses alkaline reagents or deacetylases. Heparin synthesized via biological enzymatic synthesis has a more uniform structure, higher biosafety, and is simpler and easier to perform than chemical synthesis. Biological enzymatic synthesis of heparin is the best way to obtain heparin with better structural uniformity and higher biosafety.

[0006] There are seven subtypes of 3-O-sulfotransferases in nature. They can be classified according to function into the gD type, which catalyzes the formation of the glycoprotein binding receptor site of herpes simplex virus type I (HSV-1), and the AT type, which catalyzes the formation of heparan sulfate with anticoagulant function. The AT type includes two subtypes: 3-O-sulfotransferase 1 and 3-sulfotransferase 5. These enzymes preferentially modify glucosamine whose non-reducing terminus is a glucuronic acid that has not undergone modification by C5 epimerase and 2-O-sulfotransferase. 3-O-sulfotransferase 5 possesses the functions of both gD and AT type 3-O-sulfotransferases, while 3-O-sulfotransferase 1 specifically catalyzes the formation of heparin with anticoagulant activity from substrate polysaccharides. The activity of 3-O-sulfotransferase 1 in forming the anticoagulant structural unit is more than 250 times higher than that of 3-O-sulfotransferase 5. Human heparin 3-O-sulfotransferase 1 (HTS1) is a biological enzyme composed of 307 amino acids with a nearly spherical spatial structure and a distinct substrate-binding pocket for binding to the substrate heparin polysaccharide. HTS1 catalyzes the sulfonation of the 3-OH group of the N-acetylglucosamine unit to form an N-acetylglucosamine unit with a 3-sulfonic acid group. In the chemical structure of heparin, this sulfonic acid group plays a crucial role in the anticoagulant function of heparin. Currently, the reported sulfonate transfer activity of HTS1 expressed in *E. coli* Rosetta 2 cells is 14 pmol / µg. Given that the degree of sulfonation at the 3-OH site has a near-deterministic effect on heparin activity, the enzymatic preparation efficiency of heparin under the catalysis of the low-activity wild-type 3-OST-1 enzyme is limited, hindering industrial production.

[0007] Therefore, providing a method to improve the catalytic activity of heparin 3-O-sulfotransferase 1 is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A heparin 3-O-sulfotransferase 1 mutant is obtained by fusing a solubilizing tag MBP to the N-terminus of heparin 3-O-sulfotransferase 1, followed by molecular modification; the molecular modification includes any one of (1)-(3): (1) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) The amino acid at position 144 of heparin 3-O-sulfotransferase 1 was mutated from histidine to arginine; (3) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine, and the amino acid at position 144 was mutated from histidine to arginine. The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.7.

[0010] Furthermore, the gene encoding the heparin 3-O-sulfotransferase 1 mutant is described.

[0011] Furthermore, a plasmid containing the aforementioned gene.

[0012] Furthermore, genetically engineered bacteria expressing the heparin 3-O-sulfotransferase 1 mutant.

[0013] Furthermore, a method for producing heparin 3-O-sulfotransferase 1 involves using the aforementioned genetically engineered bacteria as the production strain to induce fermentation and produce heparin 3-O-sulfotransferase 1.

[0014] Furthermore, a method for enhancing the catalytic activity of heparin 3-O-sulfotransferase 1 involves first fusing a solubilizing tag MBP to the N-terminus of heparin 3-O-sulfotransferase 1, and then obtaining a heparin 3-O-sulfotransferase 1 mutant through molecular modification; the molecular modification includes any one of (1)-(3): (1) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) The amino acid at position 144 of heparin 3-O-sulfotransferase 1 was mutated from histidine to arginine; (3) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine, and the amino acid at position 144 was mutated from histidine to arginine. The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.7.

[0015] Furthermore, the application of the heparin 3-O-sulfotransferase 1 mutant in enhancing the activity of heparin 3-O-sulfotransferase 1.

[0016] Furthermore, the heparin 3-O-sulfotransferase 1 mutant has applications in the food, chemical, or pharmaceutical fields.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, which has the following beneficial effects: This invention is the first to use Escherichia coli Origami B(DE3) microbial cells to co-express the chaperone protein particle pGro7 with the pMal-c5E vector carrying the human heparin 3-O-sulfotransferase 1 gene. The resulting enzyme catalytic activity was 15.60 U / L, the specific enzyme activity was 1.68 U / mg, and the sulfotransfer rate was 12.98 pmol / µg.

[0018] This invention is the first to use Escherichia coli Origami B(DE3) microbial cells to co-express chaperone protein and 3-O-sulfotransferase 1 with amino acid 72 changed from tryptophan to arginine. The resulting enzyme catalytic activity was 26.40 U / L, which is 69% higher than before the mutation.

[0019] This invention is the first to use Escherichia coli Origami B(DE3) microbial cells to co-express chaperone protein and 3-O-sulfotransferase 1 with amino acid 144 changed from histidine to arginine. The resulting enzyme catalytic activity was 17.21 U / L, which is 11% higher than before the mutation.

[0020] This invention is the first to use Escherichia coli Origami B(DE3) microbial cells to co-express chaperone protein and 3-O-sulfotransferase 1, which has its amino acid position 72 mutated from tryptophan to arginine and its amino acid position 144 mutated from histidine to arginine. The resulting enzyme catalytic activity was 34.41 U / L, which is 121% higher than before mutation; the specific enzyme activity was 2.40 U / mg, which is 43% higher than wild type; and the sulfotransfer rate was 23.61 pmol / µg, which is 82% higher than wild type.

[0021] This invention is the first to integrate the linearized plasmid of the pPICZαA expression vector carrying the 3-O-sulfotransferase 1 gene into the genome of Pichia pastoris GS115 microbial cells, and the resulting enzyme catalytic activity is 66.77 U / L.

[0022] This invention is the first to express 3-O-sulfotransferase 1, which has a 72-amino acid position mutated from tryptophan to arginine, in Pichia pastoris GS115 microbial cells. The resulting enzyme catalytic activity was 70.27 U / L, which is 5% higher than before the mutation.

[0023] This invention is the first to express 3-O-sulfotransferase 1, which is mutated from histidine to arginine at position 144, in Pichia pastoris GS115 microbial cells. The resulting enzyme catalytic activity was 68.86 U / L, which was 3% higher than that before the mutation.

[0024] This invention is the first to express 3-O-sulfotransferase 1 of MBP protein with codon optimization fused to the N-terminus of Saccharomyces cerevisiae in Pichia pastoris GS115 microbial cells, and the resulting enzyme catalytic activity is 75.41 U / L, which is 13% higher than that before the fusion tag.

[0025] This invention is the first to express 3-O-sulfotransferase 1, an N-terminal MBP protein fused with codon-optimized Saccharomyces cerevisiae, with amino acid 72 modified from tryptophan to arginine, in Pichia pastoris GS115 microbial cells. The resulting enzyme catalytic activity was 88.51 U / L, which is 17% higher than the wild-type enzyme with the fusion tag and 33% higher than the wild-type without the fusion tag. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the catalysis of heparin 3-O-sulfotransferase 1 in this invention; Figure 2 This is a plasmid map of the heparin 3-O-sulfotransferase 1 N-terminus fused with the solubilizing tag MBP of the present invention; Figure 3 This is an SDS-PAGE of heparin 3-O-sulfotransferase 1 with an N-terminal MBP tag fused to it, expressed in Escherichia coli Origami B(DE3). Wherein, M: maker; Lane 1: intracellular supernatant of control strain; Lane 2: intracellular supernatant of wild-type heparin 3-O-sulfotransferase 1 in Escherichia coli; Lane 3: intracellular supernatant of heparin 3-O-sulfotransferase 1 (W72R) in Escherichia coli; Lane 4: intracellular supernatant of heparin 3-O-sulfotransferase 1 (H144R) in Escherichia coli; Lane 5: intracellular supernatant of heparin 3-O-sulfotransferase 1 (W72R / H144R) in Escherichia coli; Lane 6: purified wild-type heparin 3-O-sulfotransferase 1; Lane 7: purified heparin 3-O-sulfotransferase 1 (W72R / H144R). Figure 4 This describes the interaction between the 3-OST-1 mutation site and the corresponding 3-OST-3 site with the substrate in this invention; Figure 5 The enzyme activity of heparin 3-O-sulfotransferase 1 and its mutant expressed in Escherichia coli Origami B(DE3) according to the present invention; Figure 6 The enzyme activity of heparin 3-O-sulfotransferase 1 and its mutant expressed in Pichia pastoris GS115 is shown in this invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0029] Method for determining the enzyme activity of crude 3-OST-1 enzyme solution: Using heparin sodium derived from porcine small intestine as a substrate, a catalytic reaction system was constructed. The system included: 50 mM Tris-HCl (pH=7.4), 50 mM MES, 50 mM PNPS (p-nitrobenzenesulfonic acid), 0.5 mMPAP (3',5'-adenosine diphosphate), 2 mg / mL ASTIV purified enzyme (this gene was synthesized by the company), 1 mM CaCl2, 500 μL / mL crude 3-OST-1 (heparin 3-O-sulfotransferase 1) enzyme solution prepared in each example, and 2 mg / mL porcine small intestine heparin sodium (purchased from Sangon Biotech Co., Ltd.). The reaction was carried out at 40℃ for 2 h, and then heated at 90℃ for 10 min to terminate the reaction. The absorbance was measured at a wavelength of 410 nm.

[0030] The enzyme activity of 3-OST-1 is defined as the amount of enzyme required to generate 1 μmol of PNP (p-nitrophenol) per minute in a reaction system at 40°C and pH 7.4.

[0031] A schematic diagram of the reaction catalyzed by the 3-OST-1 enzyme to modify the substrate polysaccharide into heparin is shown below. Figure 1 .

[0032] Example 1: Preparation of 3-OST-1 and ASTIV (acylsulfatase IV) purification enzymes from Escherichia coli The nucleotide sequence of the gene encoding the codon-optimized heparin 3-O-sulfotransferase 1 in Escherichia coli is shown in SEQ ID NO. 1.

[0033] CGTCCTGCAGAACTGGGCCAGCAGGAACTGCTGCGTAAAGCAGGTACCCTGCAGGATGATGTGCGTGATGGTGTTGCCCCGAATGGCAGTGCACAGCAGCTGCCGCAGACCATTATTATTGGTGTGCGTAAAGGTGGCACCCGCGCACTGCTGGAAATGCTGAGTCTGCATCCGGATGTTGCAGCAGCAGAAAATGAAGTTCATTTCTTTGATTGGGAAGAACATTATAGCCATGGTCTGGGTTGGTATCTGAGCCAGATGCCGTTTAGCTGGCCGCATCAGCTGACCGTGGAAAAAACCCCGGCCTATTTTACCAGTCCGAAAGTGCCGGAACGTGTTTATAGTATGAATCCGAGCATTCGTCTGCTGCTGATTCTGCGCGATCCGAGCGAACGTGTGCTGAGTGATTATACCCAGGTGTTTTATAATCATATGCAGAAACATAAGCCGTATCCGAGCATTGAAGAATTTCTGGTGCGCGATGGTCGTCTGAATGTGGATTATAAAGCACTGAATCGCAGTCTGTATCATGTTCATATGCAGAATTGGCTGCGTTTCTTTCCGCTGCGTCATATTCATATTGTTGATGGCGATCGCCTGATTCGCGATCCGTTTCCGGAAATTCAGAAAGTTGAACGCTTTCTGAAACTGAGTCCGCAGATTAATGCAAGCAATTTTTATTTTAACAAGACCAAGGGCTTCTATTGTCTGCGTGATAGTGGCCGTGATCGTTGTCTGCATGAAAGTAAAGGTCGCGCACATCCGCAGGTGGATCCGAAACTGCTGAATAAGCTGCATGAATATTTTCATGAACCGAATAAGAAGTTCTTCGAACTGGTTGGCCGCACCTTTGATTGGCATTAA;SEQ ID NO. 1.

[0034] Heparan 3-O-sulfotransferase 1 is derived from Homo sapiens (Homo), and the amino acid sequence is shown in SEQ ID NO. 2.

[0035] RPAELGQQELLRKAGTLQDDVRDGVAPNGSAQQLPQTIIIGVRKGGTRALLEMLSLHPDVAAAENEVHFFDWEEHYSHGLGWYLSQMPFSWPHQLTVEKTPAYFTSPKVPERVYSMNPSIRLLLILRDPSERVLSDYTQVFYNHM SEQ IDNO.2.

[0036] 1) Construction of recombinant Escherichia coli expressing ASTIV and 3-OST-1 The synthesized ASTIV gene (Gene ID: 83783) was ligated with the pET-19b vector, which had been linearized with NdeI and BamHI restriction enzymes, using T4 ligase (Thermo Scientific) according to the manufacturer's instructions to obtain the recombinant plasmid pET-19b-ASTIV; the pET-19b-ASTIV plasmid was then transformed into... E. coli Recombinant strains expressing ASTIV were obtained from the BL21(DE3) host bacterium. E. coli BL21(DE3)-pET-19b-ASTIV.

[0037] The gene for the codon-optimized 3-OST-1 enzyme of *E. coli* synthesized by the company (gene sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2) was ligated with the pMal-c5E linear vector using a seamless cloning method to obtain the recombinant plasmid pMal-c5E-3-OST-1. The plasmid map is shown below. Figure 2 .

[0038] The pMal-c5E linear vector was prepared as follows: pMal-c5E linear vector was obtained by PCR amplification using a primer pair and a template containing MBP; the primer sequences are as follows: F1: 5'-GATTGGCATTAAAAGCTTCAAATAAAACGAAAGGCTCAG-3'; SEQ ID NO.3; R1: 5'-TCTGCAGGACGATGATGATGATGATGATGCATATGCG-3'; SEQ ID NO.4.

[0039] PCR reaction program: 98 ℃ for 3 min; 98 ℃ for 10 s, 56 ℃ for 15 s, 72 ℃ for 80 s, 34 cycles; 72 ℃ for 10 min; 12 ℃ hold). PCR was performed using Primestar enzyme (Takara), and the PCR system was followed according to the reagent instructions. Ligation was performed by mixing the linear vector with the 3-OST-1 gene and then ligating using a seamless cloning kit (Beijing TransGen Biotech Co., Ltd.), with conditions and ratios following the instructions. 500 ng of recombinant plasmid pMal-c5E-3-OST-1 and 500 ng of chaperone protein plasmid pGro7 (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) were added to... E. coli OrigamiB(DE3) competent cells (Beijing Huayueyang Biotechnology Co., Ltd.) were co-transformed with two plasmids according to the method described in the chemical transformation instructions. E. coli In OrigamiB(DE3), recombinants expressing 3-OST-1 were obtained. E. coli OrigamiB(DE3)-pMal-c5E-3-OST-1, and co-transfer pGro7 and pMal-c5E empty plasmids according to the same procedure described above. E. coli OrigamiB(DE3) competent cells were used to obtain control strains.

[0040] 2) Inducible expression of recombinant Escherichia coli expressing ASTIV and 3-OST-1 Select Recombination E. coli A single colony of BL21(DE3)-pET-19b-ASTIV was cultured overnight at 37°C and 220 rpm in 4 mL LB medium containing 100 μg / mL ampicillin. 1 mL of the bacterial culture was then transferred from the test tube to 50 mL LB medium at the same antibiotic concentration and incubated at 37°C and 200 rpm for approximately 3 hours until OD500. 600 With a concentration of nm between 0.8 and 1.0, IPTG was added to a final concentration of 0.5 mM, and the mixture was induced at 16 °C for 22 h.

[0041] Select recombinants separately E. coli Single colonies of OrigamiB(DE3)-pMal-c5E-3-OST-1 and control strains were cultured overnight at 37°C and 200 rpm in 4 mL LB medium containing 100 μg / mL ampicillin, 30 μg / mL chloramphenicol, and 50 μg / mL kanamycin. 1 mL of the bacterial culture was then transferred from the test tube to 50 mL LB medium with the same antibiotic concentration as the test tube, and cultured at 37°C and 200 rpm for approximately 3 hours until OD500. 600With a concentration of nm between 0.8 and 1.0, IPTG was added to a final concentration of 0.5 mM, and the mixture was induced at 16 °C for 22 h.

[0042] The above cultures were centrifuged at 4000 rpm and 4°C for 5 min to collect bacterial cells. The cells were resuspended in 1 / 4 volume of Tris-HCl buffer and homogenized using an autoclave at 800-1000 Bar. After centrifugation at 4000 rpm for 10 min, cell debris was removed, and the supernatant crude enzyme solution was collected. The SDS-PAGE of the supernatant crude enzyme solution of the control strain is shown in the figure. Figure 3 Middle lane 1, wild-type 3-OST-1 supernatant crude enzyme solution SDS-PAGE (see lane 1) Figure 3 Lane 2 of the middle swimming pool. The crude enzyme solution containing 3-OST-1 was used directly for 3-OST-1 enzyme activity testing, or for purifying 3-OST-1 to obtain pure enzyme for SDS-PAGE testing; the crude enzyme solution containing ASTIV was purified to obtain ASTIV purified enzyme, which was added to the 3-OST-1 activity testing reaction system to complete the activity test. The enzyme catalytic activity of wild-type 3-OST-1 expressed in *E. coli* Origami B(DE3) was 15.605 U / L.

[0043] 3) Purification of ASTIV and 3-OST-1 enzymes Pass the crude enzyme solution through a pre-chilled Ni affinity chromatography column using Tris-HCl buffer (50 mM Tris, 300 mM NaCl, 20% glycerol, pH=7.4). After repeatedly penetrating the agarose packing with the crude enzyme solution, gently wash the column with 5 column volumes of Tris-HCl buffer. Wash away contaminating proteins with 6-7 column volumes of Wash Buffer (50 mM Tris, 300 mM NaCl, 30 mM imidazole, pH=7.4). Elute the target protein with Elution buffer (50 mM Tris, 300 mM NaCl, 250 mM imidazole, 20% glycerol, pH=7.4) and collect. 1 L E. coli BL21(DE3)-pET-19b-ASTIV bacterial culture can be purified and concentrated to obtain 10 mL of pure enzyme with a concentration of 8-12 mg / mL. E. coli OrigamiB(DE3)-pMal-c5E-3-OST-1 bacterial culture can be purified and concentrated to obtain 1 mL of 0.35 mg / mL pure enzyme. SDS-PAGE of the 3-OST-1 wild-type pure enzyme is shown below. Figure 3 Middle lane 6.

[0044] In the enzyme activity assay of 3-OST-1 crude enzyme solution, the 500 µL / mL crude enzyme solution was replaced with 8 µg / mL of pure 3-OST-1 enzyme. The sulfonyl transfer rate was determined by calculating the amount of PNP generated per µg of enzyme sulfonyl transfer, and the specific enzyme activity was determined by calculating the sulfonyl transfer activity per mg of 3-OST-1 enzyme. The sulfonyl transfer rate of the wild-type pure 3-OST-1 enzyme was 12.98 pmol / µg, and the specific enzyme activity was 1.68 U / mg.

[0045] Example 2 Preparation of heparin 3-OST-1 enzyme from Pichia pastoris The nucleotide sequence of the gene encoding the Pichia pastoris codon-optimized heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO. 5.

[0046] AGACCAGCTGAATTGGGTCAACAAGAATTGTTGAGAAAGGCCGGTACTTTGCAAGATGATGTTAGAGATGGTGTTGCTCCAAACGGTTCTGCTCAACAATTGCCACAAACTATTATTATCGGTGTCCGTAAGGGTGGTACTCGTGCTTTGTTGGAAATGTTGTCTTTGCATCCAGATGTCGCCGCTGCTGAAAACGAAGTTCATTTTTTTGATTGGGAGGAGCACTACTCCCACGGTTTGGGTTGGTACTTGTCTCAAATGCCATTTTCTTGGCCACATCAATTGACTGTTGAAAAGACTCCAGCTTACTTTACCTCTCCAAAGGTTCCAGAAAGAGTTTACTCTATGAACCCATCTATTCGCTTGTTGTTGATCTTGCGTGACCCATCTGAGAGAGTTTTGTCTGACTACACTCAAGTTTTTTACAACCACATGCAGAAGCACAAGCCATACCCATCTATTGAAGAATTTTTGGTCCGTGACGGTCGTTTGAACGTTGATTACAAGGCTTTGAACAGATCCTTGTACCATGTTCATATGCAGAACTGGTTGCGTTTTTTTCCATTGAGACATATCCACATCGTCGATGGTGACAGATTGATTCGTGATCCATTTCCAGAGATCCAAAAGGTTGAAAGATTTTTGAAGTTGTCCCCACAGATCAACGCTTCCAACTTTTACTTTAACAAGACCAAGGGCTTCTACTGCTTGCGTGATTCTGGTAGAGATAGATGTTTGCATGAATCCAAGGGTAGAGCTCATCCACAAGTTGATCCAAAGTTGTTGAACAAGTTGCACGAATACTTTCACGAGCCAAACAAGAAGTTCTTTGAATTGGTTGGTCGTACCTTCGACTGGCAT; SEQ ID NO: 5.

[0047] The nucleotide sequence of the codon-optimized MBP gene for Saccharomyces cerevisiae is shown as SEQ ID NO. 6.

[0048]

[0049] MBP is derived from Escherichia coli (strain K12) and its amino acid sequence is shown in SEQ ID NO.7.

[0050] MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQS GLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYE SEQ ID NO.7.

[0051] 1) Construction of recombinant Pichia pastoris expressing 3-OST-1 The codon-optimized 3-OST-1 gene (SEQ ID NO.5) of *Pichia pastoris* synthesized by the company was ligated to the pPICZαA vector using a seamless cloning method. The pPICZαA vector was pre-digested with EcoRI and XbaI restriction endonucleases. The ligation method was the same as in Example 1, yielding the recombinant plasmid pPICZαA-3-OST-1. The AOX1 region of the recombinant plasmid was linearized by single digestion with PmeI and then electroporated into *Pichia pastoris* GS115. Successful transfection of the target fragment was verified by PCR. High copy numbers of the target strain were obtained by screening at different BleoR resistance concentrations (5 mg / mL, 8 mg / mL, 12 mg / mL, and 20 mg / mL), thus obtaining recombinant strains capable of growth under high resistance concentrations. P. pastoris GS115 -pPICZαA-3-OST-1.

[0052] The codon-optimized MBP (maltose-binding protein) gene (gene sequence shown in SEQ ID NO. 6, amino acid sequence shown in SEQ ID NO. 7) synthesized by the company was ligated upstream of the 3-OST-1 gene (SEQ ID NO. 5) and then ligated to the pPICZαA vector. The method involved assembling the MBP, 3-OST-1, and pPICZαA fragments using a seamless cloning method. The pPICZαA vector was pre-digested with EcoRI and XbaI restriction endonucleases to obtain the recombinant plasmid pPICZαA-MBP-3-OST-1. The AOX1 region of the recombinant plasmid pPICZαA-MBP-3-OST-1 was linearized by PmeI single-restriction digestion and then electroporated into Pichia pastoris GS115. Successful transfection of the target fragment was verified by PCR. High copy numbers of the target strain were obtained by screening at different BleoR resistance concentrations of 5 mg / mL, 8 mg / mL, 12 mg / mL, and 20 mg / mL, thus obtaining recombinant strains capable of growth under high resistance concentrations. P. pastoris GS115-pPICZαA-MBP-3-OST-1.

[0053] 2) Expression of 3-OST-1 and MBP-3-OST-1 in recombinant Pichia pastoris Select recombinant Pichia pastoris P. pastoris GS115 -pPICZαA-3-OST-1 or P. pastoris A single colony of GS115-pPICZαA-MBP-3-OST-1 was cultured overnight in 5 mL YPD medium at 30°C and 200 rpm. The bacterial culture was then transferred from the test tube to 50 mL BMGY medium to allow initial OD. 600 With a nm value of 0.1, incubate at 30℃ and 200 rpm for approximately 12-14 h until OD reaches zero. 600 The number of cells was approximately 12. The cells were collected and transferred to BMMY medium and cultured at 28°C for 96 h, with 1% methanol added every 24 h.

[0054] The culture was centrifuged at 4000 rpm and 4℃ for 10 min to collect the cells. The cells were resuspended in Tris-HCl Buffer and homogenized using a high-pressure homogenizer at 1600-1800 Bar. The cells were then centrifuged at 4000 rpm for 20 min to remove cell debris. The supernatant crude enzyme solution was collected. The crude enzyme solution containing 3-OST-1 was directly used for 3-OST-1 enzyme activity testing.

[0055] Example 3 Molecular modification of 3-OST-1 enzyme 1) Screening method for 3-OST-1 mutants In the molecular three-dimensional structure display software PyMOL, the spatial homology overlap of the crystal structures of the human 3-OST-1 / PAP (PDB ID: 1ZRH) binary complex and the human 3-OST-3 / PAP / tetrasaccharide heparin substrate ternary complex (PDB ID: 1T8U) enzyme molecules was analyzed. The important residues for the interaction between the two subtypes of 3-OST enzyme and the substrate were identified. Single-point saturation mutagenesis was performed on the sites in BIOVIA Discovery Studio. The stability of the mutation was determined based on the mutation energy, and the mutation strategy was designed in combination with the spatial structure-activity relationship.

[0056] The specific method involves performing homologous overlap of the two enzyme-substrate crystal structure models in the molecular 3D structure visualization software PyMOL. The aim is to identify residues where the two enzymes exhibit significant differences in substrate interactions, with 3-OST-1 showing weak interaction with the substrate glycan at that position while 3-OST-3 shows strong interaction. From 20 common amino acids, the most suitable mutation strategy is selected for each residue in the 3-OST-1 enzyme structure to be optimized. The strategy design must consider factors such as the spatial size of the location, the spatial orientation and polarity of the polar groups, and their interactions with other functional groups of the substrate. Interactions between polar atoms within 3 Å are considered strong, within 5 Å weak, and beyond 5 Å are considered negligible.

[0057] like Figure 4 In the 3-OST-1 enzyme, Trp72 (corresponding to amino acid number Trp59 in 1ZRH) has no interaction with the 2-O-sulfonic acid group on the idole ring 2 due to its large distance from the 2-O-sulfonic acid group. In contrast, the corresponding 3-OST-3 enzyme, Arg190, has a longer spatial morphology and stronger polarity, with an interaction distance of 3.8 Å with this group. Therefore, this position could be modified to Arg, similar to the 3-OST-3 enzyme, to increase its spatial length and polarity. In the 3-OST-1 enzyme, His144 (corresponding to amino acid number His131 in 1ZRH), the imidazole group has no interaction with the 1-hydroxyl group of the glucosamine unit 1 due to its large distance from the 1-hydroxyl group. In the 3-OST-3 enzyme, Arg260, the interaction distance with this group is 3.5 Å. Therefore, it is considered to mutate this site to Arg, which has greater steric hindrance and stronger polarity.

[0058] 2) Preparation method of 3-OST-1 mutant The method for mutating the 72 site in Escherichia coli was to use primers F2, R2 and F3, R3 to perform PCR amplification on the codon-biased 3-OST-1 gene (SEQ ID NO.1) of Escherichia coli. The procedure and system were the same as in Example 1, and gene fragments gene1 and gene2 were obtained.

[0059] F2: 5'-CATCATCATCGTCCTGCAGAACTGGGC-3'; SEQ ID NO.8; R2: 5'-CCAGACCATGGCTATAATGTTCTTCACGATCAAAGAAAT-3'; SEQ ID NO.9; F3: 5'-GAAGAACATTATAGCCATGGTCTGGGT-3'; SEQ ID NO.10; R3: 5'-AAGCTTTTTAATGCCAATCAAAGGTGCG-3'; SEQ ID NO. 11.

[0060] Following the system (primestar 25 µL, gene1 1 µL, gene2 1 µL, ddH2O 23 µL) and program (98℃ 3 min; 98℃ 10 s, 56℃ 15 s, 72℃ 80 s, 5 cycles; 72℃ 10 min 12℃ hold), 2 µL each of primers F2 and R3 were added to the PCR product, and the PCR program in Example 1 was followed to obtain gene 3-OST-1 (W72R).

[0061] The method for mutation at site 144 in Escherichia coli was to use primers F2, R4 and F4, R3 to perform PCR amplification on the codon-biased 3-OST-1 gene (SEQ ID NO.1) of Escherichia coli. The procedure and system were the same as in Example 1, and gene fragments gene3 and gene4 were obtained.

[0062] F4: 5'-TGCAGAAACATAAGCCGTATCCGAG-3'; SEQ ID NO. 12; R4: 5'-CGGATACGGCTTATGTTTCTGCATACGATTATAAAACACC-3'; SEQ ID NO. 13.

[0063] Following the system (primestar 25 µL, gene3 1 µL, gene4 1 µL, ddH2O 23 µL) and the above-described reaction procedure, 2 µL each of primers F2 and R3 were added to the PCR product, and the gene 3-OST-1 (H144R) was obtained by reacting according to the PCR procedure in Example 1.

[0064] The method for double-site mutation at sites 72 and 144 in Escherichia coli is to use the codon-preferred gene 3-OST-1 (W72R) as a template and perform mutations according to the same steps as the method for mutation at site 144 to obtain gene 3-OST-1 (W72R / H144R).

[0065] Plasmid construction, induced expression, and activity assay methods were the same as in Example 1. SDS-PAGE of the crude enzyme solution of mutant 3-OST-1 (W72R) is shown in [reference needed]. Figure 3 Middle lane 3; Crude enzyme solution SDS-PAGE of mutant 3-OST-1 (H144R) is shown in [reference needed]. Figure 3 Middle lane 4; Crude enzyme solution SDS-PAGE of mutant 3-OST-1 (W72R / H144R) is shown in [reference needed]. Figure 3 Middle lane 5; SDS-PAGE of mutant 3-OST-1 (W72R / H144R) pure enzyme is shown in [link to SDS-PAGE]. Figure 3 Middle lane 7.

[0066] The above-mentioned mutant gene in Pichia pastoris was constructed by PCR using the Pichia pastoris codon-biased 3-OST-1 gene (SEQ ID NO. 5) as a template. The operation steps were the same as those for Escherichia coli, but the primers were different. The specific primer sequences are as follows: F2': 5'-AGAAAGAGAGGCTGAAGCTAGACCAGCTGAATTGGGTCAAC-3'; SEQ ID NO.14; R2': 5'-CGTGGGAGTAGTGCTCTCTCTATCAAAAAAATGAACTTCGTTTTCAGCAGC-3'; SEQ IDNO.15; F3': 5'-GAGGAGCACTACTCCCACGG-3'; SEQ ID NO.16; R3': 5'-CAAACTCAATGATGATGATGATGATGATGCCAGTCGAAGGTACGAC-3'; SEQ ID NO. 17; F4': 5'-ATGCAGAAGCACAAGCCATACC-3'; SEQ ID NO.18; R4': 5'-TATGGCTTGTGCTTCTGCATTCTGTTGTAAAAAACTTGAGTGTAGTCAGACAAAAC-3'; SEQ ID NO. 19.

[0067] The plasmid construction, induced expression, and activity testing methods are the same as in Example 2.

[0068] The results are as follows Figure 5and Figure 6 As shown, the enzyme catalytic activity of wild-type (WT) 3-OST-1 expressed in *E. coli* Origami B(DE3) was 15.605 U / L; the enzyme catalytic activity of 3-OST-1 (W72R) with amino acid 72 changed from tryptophan to arginine was 26.398 U / L, an increase of 69% compared to the unmutated form; the enzyme catalytic activity of 3-OST-1 (H144R) with amino acid 144 changed from histidine to arginine was 17.214 U / L, an increase of 10% compared to the unmutated form; and the enzyme catalytic activity of 3-OST-1 (W72R / H144R) with amino acid 72 changed from tryptophan to arginine and amino acid 144 changed from histidine to arginine was 34.411 U / L, an increase of 121% compared to the unmutated form. 1 L of bacterial culture expressing 3-OST-1(W72R / H144R) enzyme was purified and concentrated to obtain 2 mL of 0.3 mg / mL pure enzyme solution. The specific enzyme activity and sulfonyl transfer rate of the 3-OST-1(W72R / H144R) pure enzyme were determined according to the method in Example 1. The specific enzyme activity was 2.40 U / mg, which was 43% higher than that of the wild type, and the sulfonyl transfer rate was 23.61 pmol / µg, which was 82% higher than that of the wild type.

[0069] The enzyme catalytic activity of wild-type 3-OST-1 (3-OST-1(WT)) expressed in Pichia pastoris GS115 was 66.773 U / L; the enzyme catalytic activity of 3-OST-1 (W72R) with amino acid 72 modified by mutation of tryptophan to arginine was 70.271 U / L, an increase of 5% compared to the unmutated form; the enzyme catalytic activity of 3-OST-1 (H144R) with amino acid 144 modified by mutation of histidine to arginine was 68.857 U / L, an increase of 3% compared to the unmutated form; and the enzyme catalytic activity of wild-type 3-OST-1 with N-terminal MBP tag fused in Pichia pastoris GS115 (MBP-3-OST-1(WT)) was 75.408 U / L. The activity of 3-OST-1 was 88.509 U / L, which was 13% higher than that of the wild type without the fusion tag. The enzyme catalytic activity of 3-OST-1 after the N-terminal fusion of MBP protein was expressed by Pichia pastoris GS115 with amino acid 72 changed from tryptophan to arginine was 17% higher than that of the wild type with the fusion tag and 33% higher than that of the wild type without the fusion tag.

[0070] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A heparin 3-O-sulfotransferase 1 mutant, characterized in that, A solubilizing tag MBP is fused to the N-terminus of heparin 3-O-sulfotransferase 1, and then a heparin 3-O-sulfotransferase 1 mutant is obtained by molecular modification; the molecular modification includes any one of (1)-(3): (1) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) The amino acid at position 144 of heparin 3-O-sulfotransferase 1 was mutated from histidine to arginine; (3) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine, and the amino acid at position 144 was mutated from histidine to arginine. The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.

7.

2. The gene encoding the heparin 3-O-sulfotransferase 1 mutant as described in claim 1.

3. A plasmid containing the gene of claim 2.

4. Genetically engineered bacteria expressing the heparin 3-O-sulfotransferase 1 mutant as described in claim 1.

5. A method for producing a heparin 3-O-sulfotransferase 1 mutant, characterized in that, Using the genetically engineered bacteria described in claim 4 as the production strain, heparin 3-O-sulfotransferase 1 mutant was induced to ferment and produce.

6. A method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, characterized in that, First, a solubilizing tag MBP is fused to the N-terminus of heparin 3-O-sulfotransferase 1, and then a heparin 3-O-sulfotransferase 1 mutant is obtained through molecular modification; the molecular modification includes any one of (1)-(3): (1) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) The amino acid at position 144 of heparin 3-O-sulfotransferase 1 was mutated from histidine to arginine; (3) The amino acid at position 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine, and the amino acid at position 144 was mutated from histidine to arginine. The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.

7.

7. The use of the heparin 3-O-sulfotransferase 1 mutant according to claim 1 in improving the activity of heparin 3-O-sulfotransferase 1.

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