A method for constructing a chondroitin-4-o-sulfotransferase mutant

Abstract

The application discloses a chondroitin-4-O-sulfotransferase mutant construction method and belongs to the field of bioengineering technology. The application is characterized in that a wild-type chondroitin-4-O-sulfotransferase (C4ST) is precisely reformed through a calculation-guided semi-rational design, a SUMO label and an AE connecting peptide are fused at an N end to improve soluble expression, and on this basis, a three-point combined mutant V74L / M117T / Y235V is constructed through a site-directed mutagenesis technology, the enzyme activity of the mutant in a 5L fermenter is as high as 76328 U / L, the specific enzyme activity is 327 U / mg, and the enzyme activity and the specific enzyme activity are 16.6 times and 1.3 times of the wild type, respectively. The mutant is used for catalyzing preparation of chondroitin sulfate A, the sulfation degree can reach 97.0% after 48 h of reaction at 37 DEG C. The application significantly improves the catalytic efficiency of C4ST and provides a key technology of efficient and stable enzyme method for preparing CSA.

Description

Technical Field

[0001] This invention relates to a method for constructing a chondroitin-4-O-sulfotransferase mutant, belonging to the field of bioengineering technology. Background Technology

[0002] Chondroitin-4 O β-sulfonyltransferase (C4ST-1, also known as CHST11) is a key enzyme in the biosynthesis of chondroitin sulfate A (CSA), capable of specifically transferring the sulfate group from the donor molecule 3'-phosphoadenylate-5'-phosphate sulfate (PAPS) to... N The C4 hydroxyl group of the α-acetylgalactosamine (GalNAc) residue. CSA, as an important glycosaminoglycan, is widely used in the pharmaceutical and health product fields, particularly playing a crucial role in treating osteoarthritis, promoting cartilage repair, and regulating neural development. However, wild-type C4ST faces multiple challenges when applied to the industrial enzymatic production of CSA. First, C4ST is a eukaryotic enzyme, and its soluble expression efficiency is generally low in commonly used heterologous expression hosts (such as *Escherichia coli* and *Pichia pastoris*), easily forming insoluble inclusion bodies, which severely limits the scale of industrial enzyme production and cost control. Second, even when soluble C4ST is obtained through optimized expression conditions, its intrinsic catalytic activity (specific enzyme activity) is often insufficient, making it difficult to meet the requirements of high efficiency and high conversion rate for industrial production.

[0003] To address the aforementioned issues, existing technologies primarily focus on optimizing expression conditions or increasing the soluble expression level of the enzyme. For example, some studies have improved the soluble expression and activity of C4ST-1 by co-expressing it with molecular chaperones such as triggering factors (TF) and disulfide isomerases (PDI). Co-expression of the molecular chaperone TF increased the activity of C4ST-1 to 128.5 U / L. Similar optimization strategies exist for other sulfotransferases; for instance, heparin 3-O-sulfotransferase 1 (HS3ST1) achieved its catalytic activity to 88.5 U / L by fusing a solubilizing tag MBP and performing point mutations. Furthermore, addressing the common degradation problem of sulfotransferases, some studies have significantly reduced enzyme fragmentation and increased the activity to 1178.5 U / L by modifying the Kex2 protease cleavage site of chondroitin sulfotransferase (UST). However, most of these methods focus on increasing enzyme activity, expression levels, and stability, emphasizing the enhancement of expression levels rather than fundamentally improving the intrinsic catalytic efficiency of the enzyme molecule, i.e., increasing its specific enzyme activity. Therefore, developing a molecular modification strategy that can significantly improve the intrinsic catalytic activity of C4ST is of great significance for achieving efficient and low-cost biomanufacturing of CSA. Summary of the Invention

[0004] The technical problem to be solved by this invention is that wild-type C4ST has a low soluble expression level and insufficient intrinsic specific enzyme activity, which makes it difficult to meet the requirements of industrial production of chondroitin sulfate A.

[0005] To address the aforementioned technical problems, this invention provides a C4ST mutant with enhanced enzyme activity.

[0006] This invention provides a C4ST mutant, which has mutations at one or more of the following sites based on the wild-type C4ST amino acid sequence shown in SEQ ID NO.1: V74L, N102S, M117T, Y235V.

[0007] In one embodiment, the mutant is based on the wild-type C4ST shown in SEQ ID NO.1, by mutating valine at position 74 to leucine, asparagine at position 102 to serine, methionine at position 117 to threonine, or tyrosine at position 235 to valine.

[0008] In one embodiment, the mutant is based on the wild-type C4ST shown in SEQ ID NO.1, with valine at position 74 mutated to leucine and methionine at position 117 mutated to threonine.

[0009] In one embodiment, the mutant is based on the wild-type C4ST shown in SEQ ID NO.1, with valine at position 74 mutated to leucine and tyrosine at position 235 mutated to valine.

[0010] In one embodiment, the mutant is based on the wild-type C4ST shown in SEQ ID NO.1, with valine at position 74 mutated to leucine, methionine at position 117 mutated to threonine, and tyrosine at position 235 mutated to valine.

[0011] The present invention also provides a fusion enzyme, which is constructed by fusing a lysis-promoting tag to the N-terminus of the C4ST mutant; the lysis-promoting tag includes, but is not limited to, the SUMO tag (SEQ ID NO.6).

[0012] In one embodiment, to further improve expression efficiency and stability, the mutant is linked to the solubilization tag via a linker peptide, which includes, but is not limited to, the AE linker peptide.

[0013] In one embodiment, the amino acid sequence of the AE linker peptide is as shown in SEQ ID NO.7.

[0014] The present invention also provides a gene encoding the C4ST mutant or the fusion enzyme.

[0015] In one embodiment, the microbial cells are Escherichia coli or Pichia pastoris.

[0016] In one embodiment, the recombinant Escherichia coli is used as Escherichia coli Rosetta(DE3) was used as the expression host, and pET-32a was used as the expression vector.

[0017] In one embodiment, the Pichia pastoris is used as Komagataella phaffii GS115 was used as the expression host, and pPIC9K was used as the expression vector.

[0018] This invention also provides a method for preparing chondroitin sulfate A, which involves adding the C4ST mutant or the fusion enzyme to a reaction system containing chondroitin. The reaction system contains ATP, MgSO4, recombinant arylsulfonyltransferase ASAKS5, and the C4ST mutant.

[0019] In one embodiment, the reaction system contains ATP at a concentration of 5-20 mM, MgSO4 at a concentration of 10-30 mM, ASAKS5 at a concentration of 0.2-1.0 g / L, the amount of C4ST mutant added is 0.5-2.0 g / L (based on pure enzyme protein), and chondroitin at a concentration of 1-5 g / L. In one embodiment, the reaction system contains 10 mM ATP, 20 mM MgSO4, 0.5 g / L ASAKS5, 1.0 g / L C4ST mutant, and 2.0 g / L chondroitin.

[0020] In one embodiment, the reaction temperature is 35-40°C.

[0021] In one implementation, the reaction time is 24-72 h.

[0022] In one embodiment, the reaction is carried out at 37°C for 48 hours.

[0023] The present invention also provides the use of the C4ST mutant, fusion enzyme or recombinant microorganism in the preparation of chondroitin sulfate or chondroitin sulfate-containing products.

[0024] Beneficial effects: 1. This invention significantly improves the soluble expression level of C4ST mutant in a heterologous host by fusing the soluble tag SUMO and the linker peptide AE.

[0025] 2. This invention utilizes a semi-rational design strategy to precisely modify the distal region of C4ST, successfully screening multiple single-point mutants with significantly enhanced catalytic activity. Among them, the optimal single-point mutant M117T achieved an enzyme activity of 2840 U / L in Pichia pastoris shake-flask fermentation, which is 4.8 times that of the wild type.

[0026] 3. This invention further combines beneficial mutations to obtain combined mutants with synergistically enhanced catalytic activity. The three-point combined mutant V74L / M117T / Y235V exhibited an enzyme activity as high as 9733 U / L in Pichia pastoris shake flasks, 16.6 times that of the wild type; after scale-up culture in a 5 L fermenter, the enzyme activity reached 76328 U / L, with a specific enzyme activity of 327 U / mg. When this mutant was used to catalyze the preparation of CSA, under optimized conditions for 48 h, the sulfonation degree reached 97.0%.

[0027] This invention provides a highly efficient and stable key technology for the efficient preparation of CSA by enzymatic methods, significantly enhancing its potential for industrial application. Attached Figure Description

[0028] Figure 1 This is a schematic diagram showing the distribution of nine potential replacement sites on the C4ST structure identified by the HotSpot Wizard server.

[0029] Figure 2 This is a liquid phase detection image of the C4ST catalytic product CSA in Example 7.

[0030] Figure 3 This is an UPLC-MS image of CSA, a product of C4ST catalysis.

[0031] Figure 4 To determine the C4ST single-point mutant enzyme activity of Escherichia coli and Pichia pastoris in shake flasks.

[0032] Figure 5 To determine the C4ST single-point mutant enzyme activity of Escherichia coli and Pichia pastoris in a 5 L fermenter.

[0033] Figure 6 To determine the activity of C4ST multi-point combination mutant enzyme in Pichia pastoris in shake flasks. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Methods in the embodiments: 1. The following embodiments involve Escherichia coli Rosetta (DE3) Komagataella phaffiiGS115 was a commercially available bacterial strain, and pET-32a(+) and pPIC9K were commercially available plasmids. Plasmid construction reagents and sequencing validation were purchased and performed by Suzhou Genewiz Biotechnology Co., Ltd. All analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] 2. Culture medium LB seed medium (Luria-Bertani) (g / L): yeast extract 5; peptone 10; NaCl 10. Solid LB medium supplemented with 2% agar powder. During cultivation, LB resistance medium supplemented with 50 mg / L kanamycin.

[0037] TB fermentation medium (Terrific Broth) (g / L): yeast extract 24.0; peptone 12.0; K2HPO4·3H2O 16.43; KH2PO4 2.31; glycerol 5.0. During the culture process, 50 μg / mL kanamycin was added to the TB resistant medium.

[0038] BHIS medium (g / L): Brain heart extract 37.0; Sorbitol 91.0.

[0039] YPD medium (Yeast extract, peptone, dextrose) (g / L): yeast extract 10; peptone 20; glucose 20. The glucose stock solution was prepared at 10× and sterilized separately (115℃ for 20 min) before use.

[0040] BMGY medium (Buffered glycerol-complex) (g / L): Yeast extract 10; Peptone 20; 100 mM K₂HPO₄·3H₂O 3.01; KH₂PO₄ 11.81; Biotin 4 × 10⁻⁶ -4 Yeast-based nitrogen source (YNB) 13.4%; Glycerol 10%. Biotin stock solution is prepared at 500× concentration for use. YNB stock solution is prepared at 10× concentration for filtration and sterilization.

[0041] BMMY medium (Buffered methanol-complex) (g / L): Yeast extract 10; Peptone 20; 100 mM K₂HPO₄·3H₂O 3.01; KH₂PO₄ 11.81; Biotin 4 × 10⁻⁶ -4 Yeast nitrogen source (YNB) 13.4. Add 1% (v / v) methanol every 24 hours during cultivation. Biotin is generally prepared at 500× for use. YNB is generally prepared at 10× for use after filtration and sterilization.

[0042] 3. Detection Method C4ST enzyme activity assay: C4ST enzyme activity is determined by measuring the absorbance of PNPs formed during the reaction at 400 nm. The reaction system is 1.5 mL, including 50 mM PNPS, 0.5 mM PAP, 2 mg / mL AST IV, 20% (v / v) glycerol, 2 mg / mL chondroitin, and an appropriate amount of C4ST supernatant. The reaction is carried out at 37°C for 2 h. The reaction is terminated by boiling in a water bath for 10 min. After centrifugation at 10000×g for 10 min, the supernatant is discarded, and the absorbance at 400 nm is measured. The enzyme activity unit (U) of C4ST is defined as the amount of enzyme required to produce 1 µM PNP per hour under specific reaction conditions (37°C).

[0043] The method for detecting CSA sulfonation is as follows: The enzymatically catalyzed product is subjected to a boiling water bath for 10 min to terminate the reaction. The supernatant is centrifuged at 8000 × g for 10 min to obtain the sulfonated products. These products are then degraded into CS disaccharide by csABCI and analyzed by UPLC-MS. The degree of sulfonation is the molar ratio of sulfonated disaccharide (CSA disaccharide) to total disaccharides (chondroitin disaccharide, CSA disaccharide).

[0044] 4. Protein Purification Method: Collect the fermented cells, resuspend them in PBS buffer, and homogenize them under high pressure. Centrifuge the homogenate at 12000×g for 30 min at 4℃, and collect the supernatant as the crude enzyme solution. Filter the crude enzyme solution through a 0.22 μm filter membrane and purify it using a Ni-NTA affinity chromatography column. Elute impurities with equilibration buffer containing 20 mM imidazole (20 mM PBS, 500 mM NaCl, pH 7.5), and then elute the target protein with elution buffer containing 250 mM imidazole (20 mM PBS, 500 mM NaCl, pH 7.5). Collect the target protein peak, desalt it using a PD-10 desalting column to storage buffer (20 mM PBS, 150 mM NaCl, pH 7.5) to obtain pure enzyme for SDS-PAGE analysis and specific enzyme activity assay.

[0045] Example 1: Design of C4ST mutant and construction of recombinant bacteria (1) Target identification and single-point mutation design: Using the HotSpot Wizard 3.0 server, energy-kinetic coupling analysis was performed on the three-dimensional structural model of wild-type mouse C4ST (amino acid sequence as shown in SEQ ID NO.1, and its encoding nucleotide sequence as shown in SEQ ID NO.2 after codon optimization by Pichia pastoris), identifying 9 potential substitution sites located in the distal region of the protein ( Figure 1), including: V74, Y81, T100, N102, M117, S233, G234, Y235, L236.

[0046] (2) Construction of single-point mutant plasmids: Using the pUC57 plasmid containing the sequence shown in SEQ ID NO.2 as a template, site-directed mutagenesis primers were designed (Table 1), and single-point mutations were introduced by PCR. The PCR product was purified and recovered after digestion of the template with Dpn I. For the *E. coli* expression system, the mutated gene fragment was cloned into the pET-32a(+) vector linearized with BamHI and HindIII via homologous recombination to construct the recombinant plasmids pET32a-C4ST(WT) or pET32a-C4ST(Mutant). For the *Pichia pastoris* expression system, homologous arms were first introduced into the 5' and 3' ends of the mutant gene by PCR, and then cloned into the pPIC9K vector linearized with EcoRI and NotI via homologous recombination. The gene was then fused with the α-factor signal peptide on the vector to construct the recombinant plasmids pPIC9K-C4ST(WT) or pPIC9K-C4ST(Mutant). All constructed plasmids were verified by sequencing.

[0047] Table 1 Primer Construction

[0048] Example 2: Construction of a SUMO tag and AE-linked peptide fusion expression vector To further improve the soluble expression of C4ST and its mutants, based on the pPIC9K-C4ST(WT) or pPIC9K-C4ST(Mutant) plasmids constructed in Example 1, nucleotide sequences encoding the SUMO tag (SEQ ID NO. 6) and AE linker peptide (SEQ ID NO. 7), respectively, were inserted at the N-terminus of the C4ST gene (immediately after the α-factor signal peptide) via homologous recombination (SEQ ID NO. 3 and SEQ ID NO. 4), respectively, to construct the recombinant plasmids pPIC9K-SUMO-AE-C4ST(WT) and pPIC9K-SUMO-AE-C4ST(Mutant). Simultaneously, to facilitate purification, a 6×His tag encoding sequence (SEQ ID NO. 5) was fused to the C-terminus of the C4ST gene.

[0049] Example 3: Construction of recombinant Escherichia coli and preparation of C4ST by shake-flask fermentation The recombinant plasmids pET32a-C4ST(WT) and pET32a-C4ST(Mutant) constructed in Example 1 were transformed into [missing information - likely a specific plasmid or a specific plasmid]. E. coliRosetta (DE3) competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated into 3 mL of LB liquid medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C and 220 rpm. A 2% (v / v) inoculum was then transferred to 250 mL shake flasks containing 50 mL of TB medium (containing 50 μg / mL kanamycin) and incubated at 37°C and 220 rpm until OD500. 600 The concentration was approximately 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25℃ and 220 rpm for 16 h. After fermentation, the cells were collected, and the OD was measured. 600 The enzyme was resuspended in an appropriate amount of buffer solution and sonicated. The supernatant was collected by centrifugation and the C4ST enzyme activity was measured. The OD values ​​of the fermentation broths of wild-type (WT) and mutant were measured. 600 Approximately 8-10. The enzyme activities of each strain are as follows: Figure 4 As shown in the figure. The results showed that the enzyme activities of the single-point mutants V74L, N102S, M117T, and Y235V were 684 U / L, 568 U / L, 885 U / L, and 965 U / L, respectively, all higher than that of the wild type (368 U / L). However, the enzyme activities of the mutants T100R and L236V were significantly reduced.

[0050] Example 4: Construction of recombinant Pichia pastoris and preparation of C4ST by shake-flask fermentation The pPIC9K-SUMO-AE-C4ST(WT / Mutant) recombinant plasmid constructed in Example 2 was linearized with SalI and then electroporated to... K. phaffii GS115 competent cells were plated on MD plates and cultured at 30°C for 2-3 days until single colonies appeared. Positive transformants were verified by colony PCR. Positive single colonies were picked and inoculated into 25 mL of YPD medium and cultured at 30°C and 220 rpm for 16-20 h to prepare a seed culture. The seed culture was transferred to 50 mL of BMGY medium at a 10% (v / v) inoculation rate and cultured at 30°C and 220 rpm for 24 h. The cells were collected by centrifugation at 3000×g for 5 min at room temperature and washed twice with sterile 0.9% NaCl solution to remove residual glycerol. The cells were resuspended in 50 mL of BMMY medium and the initial OD was adjusted. 600 The culture medium was approximately 1.0. The cells were cultured at 25°C and 220 rpm, with methanol added every 24 h to a final concentration of 1% (v / v) for 96 h to induce expression. After fermentation, the cells were collected, and the OD was measured. 600 The mixture was then homogenized under high pressure, and the supernatant was collected by centrifugation to determine the C4ST enzyme activity. After 96 h of induction, the OD values ​​of the fermentation broths of wild-type and mutant strains were measured. 600Approximately 50-60. The enzyme activity of each strain is as follows: Figure 4 As shown in the figure. The results showed that the enzyme activities of the single-point mutants V74L, N102S, M117T, and Y235V were 2076 U / L, 1554 U / L, 2840 U / L, and 2572 U / L, respectively, which were 3.5 times, 2.6 times, 4.8 times, and 4.3 times that of the wild type (592 U / L). Among them, the M117T mutant had the highest enzyme activity.

[0051] Example 5: Scale-up culture of recombinant Escherichia coli and recombinant Pichia pastoris in a 5 L fermenter (1) 5 L fermentation of *E. coli*: Recombinant *E. coli* (expressing the C4ST single-point mutant) stored at -80℃ was streaked onto LB agar plates for activation and incubated overnight at 37℃. Single colonies were picked and inoculated into 50 mL of LB liquid medium and cultured at 37℃ and 220 rpm for 8-10 h as the primary seed culture. The primary seed culture was transferred at an inoculum rate of 4% (v / v) to a 5 L fermenter containing 1.8 L of TB medium. Fermentation process control: temperature 37℃, automatic cascade and dissolved oxygen linkage at 200-800 rpm, aeration rate of 2 vvm, and pH maintained at 7.0 by adding ammonia. OD 600 When the temperature reaches 20-30°C, lower the temperature to 25°C and add IPTG to a final concentration of 0.5 mM for induction. Continue culturing for 20 h after induction, and measure enzyme activity every 4 h. Results are as follows: Figure 5 As shown, the enzyme activity of each mutant reached its peak 16-20 h after induction. Among them, the highest enzyme activities of V74L, N102S, M117T and Y235V reached 3885 U / L, 3573 U / L, 5823 U / L and 6390 U / L, respectively.

[0052] (2) Pichia pastoris 5 L fermenter: A single colony of recombinant Pichia pastoris (expressing the C4ST single-point mutant) was inoculated into 50 mL of YPD medium and cultured at 30℃ and 220 rpm for 18 h as the primary seed culture. The primary seed culture was then transferred at a 10% (v / v) inoculation rate to a 5 L fermenter containing 1.35 L of BMGY medium (pH 6.8). Initial fermentation conditions: temperature 30℃, rotation speed 500 rpm, aeration rate 2 vvm, pH 6.8 maintained by adding ammonia, and dissolved oxygen (DO) controlled at 20%-30% by adjusting rotation speed and aeration rate in relation to glycerol feeding. After glycerol depletion (DO value rapidly rebounded), starvation culture was continued for 2 h. Subsequently, methanol was added for induction, while the temperature was lowered to 25℃. The methanol flow rate was gradually increased from 2 mL / h to 6 mL / h to maintain DO above 20%. Enzyme activity was measured every 12 h after induction. Results are as follows: Figure 5As shown, the enzyme activity of each mutant reached its peak 72-96 h after induction. Among them, V74L, N102S, M117T and Y235V reached the highest enzyme activity of 6831 U / L, 6462 U / L, 9337 U / L and 11273 U / L at 96 h, respectively.

[0053] Example 6: Construction and activity analysis of C4ST combined mutants Based on the pPIC9K-SUMO-AE-C4ST plasmid constructed in Example 2, three single-point mutation sites (V74L, M117T, Y235V) with the most significant enzyme activity enhancement were selected. Double-point mutants (V74L / M117T, V74L / Y235V, M117T / Y235V) and triple-point mutants (V74L / M117T / Y235V) were constructed using overlap extension PCR or sequential site-directed mutagenesis. The constructed recombinant plasmids were transformed into Pichia pastoris, and expression and enzyme activity were measured according to the shake-flask fermentation method described in Example 4. The results are as follows: Figure 6 As shown, the enzyme activities of the two-point mutants V74L / M117T, V74L / Y235V, and M117T / Y235V reached 4465 U / L, 7328 U / L, and 4022 U / L, respectively. Among them, V74L / Y235V showed a significant synergistic effect, with enzyme activity much higher than any single-point mutant. The enzyme activity of the finally obtained three-point mutant V74L / M117T / Y235V was further increased to 9733 U / L, which is 16.6 times that of the wild type.

[0054] Recombinant Pichia pastoris expressing the three-point mutant V74L / M117T / Y235V was cultured in a 5 L fermenter according to the method in Example 5(2). The results showed that after 96 h of induction, its highest enzyme activity reached 76328 U / L. The specific enzyme activity of the purified three-point mutant was 327 U / mg, and that of the wild type was 252 U / mg.

[0055] Example 7: Preparation of chondroitin A catalyzed by C4ST mutant (1) Effect of temperature on catalytic reaction: The three-point mutant V74L / M117T / Y235V purified in Example 6 was used to catalyze the preparation of CSA. The reaction system (1 mL) was prepared in 50 mM Tris-HCl buffer (pH 7.5) as follows: 10 mM ATP, 20 mM MgSO4, 0.5 g / L ASAKS5 (Xu, et al. Closed-Loop System Driven by ADPPhosphorylation from Pyrophosphate Affords Equimolar Transformation of ATP to 3'-Phosphoadenosine-5'-phosphosulfate), 1.0 g / L C4ST purified enzyme, and 2.0 g / L chondroitin. The reaction system was placed in water baths at 25℃, 30℃, 35℃, 37℃, and 40℃ for 48 h, respectively. After the reaction, the degree of sulfonation was determined according to the aforementioned method. The results showed that the highest degree of sulfonation, reaching 97.0%, was achieved at 37℃ for 48 h; the degrees of sulfonation at 35℃ and 40℃ were 91.2% and 94.5%, respectively; and the degrees of sulfonation at 25℃ and 30℃ were lower, at 52.3% and 78.6%, respectively.

[0056] (2) Preparation of CSA: Under optimal conditions (37℃, 48 h), the above reaction system was used to catalyze the reaction using crude enzyme solution (equivalent to 1.0 g / L C4ST protein) of wild-type C4ST and three-point mutant V74L / M117T / Y235V, and pure enzyme (1.0 g / L). The results showed that after 48 h, the sulfonation degree of the product could reach over 95% using both the mutant crude enzyme solution and the pure enzyme, with the sulfonation degree of the pure enzyme system remaining stable at around 97.0%. The kinetic parameters of the mutant are shown in Table 2. The liquid phase and liquid mass spectra of the CSA product are shown in Table 2. Figure 2 and Figure 3 .

[0057] Table 2 Kinetic parameters and specific enzyme activities of wild type and mutant.

[0058] Comparative Example 1: Effects of other distal site mutations on C4ST enzyme activity In Example 1, we simultaneously constructed and expressed several other distal site mutants (Y81C, T100R, S233P, G234D, and L236V) identified by HotSpot Wizard, and performed shake-flask fermentation and enzyme activity assays in Pichia pastoris according to the method in Example 4. The results are shown in Table 3. Compared with the wild type (relative enzyme activity value of 1.0), the enzyme activities of the Y81C, S233P, and G234D mutants did not change significantly, while the enzyme activities of the T100R and L236V mutants decreased significantly, only 0.52 times and 0.43 times that of the wild type, respectively. This indicates that not all predicted distal site mutations can bring positive effects, and the V74L, N102S, M117T, and Y235V sites screened in this invention are specific and unpredictable for enhancing C4ST enzyme activity.

[0059] Table 3. Relative enzyme activities of mutants at other distal sites.

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Chondroitin-4 O - Sulfonyltransferase mutant, characterized by Based on the wild-type C4ST amino acid sequence shown in SEQ ID NO.1, there are mutations at one or more of the following sites: V74L, N102S, M117T, Y235V.

2. The chondroitin-4- according to claim 1 O - Sulfonyltransferase mutant, characterized by The mutant is based on the wild-type C4ST shown in SEQ ID NO.1, with the following mutations: valine at position 74 is mutated to leucine, asparagine at position 102 is mutated to serine, methionine at position 117 is mutated to threonine, or tyrosine at position 235 is mutated to valine.

3. The mutant according to claim 1, characterized in that, The mutant is based on the wild-type C4ST shown in SEQ ID NO.1, with valine at position 74 mutated to leucine, methionine at position 117 mutated to threonine, and tyrosine at position 235 mutated to valine.

4. A fusion enzyme, characterized in that, A SUMO fusion tag is attached to the N-terminus of any of the mutants described in claims 1 to 3; the mutant and the SUMO fusion tag are linked via an AE linker peptide.

5. A gene encoding any of the mutants described in claims 1 to 3 or the fusion enzyme described in claim 4.

6. A recombinant microorganism expressing any one of the mutants described in claims 1 to 3, characterized in that, The microorganisms include Escherichia coli or Pichia pastoris.

7. The recombinant microorganism according to claim 6, characterized in that, by Escherichia coli Rosetta(DE3) was used as the expression host, and pET-32a was used as the expression vector; or... Komagataella phaffii GS115 was used as the host cell and pPIC9K was used as the expression vector.

8. A method for preparing chondroitin sulfate A, characterized in that, The mutant of any one of claims 1 to 3 is reacted in a reaction system containing chondroitin; the reaction system contains ATP, MgSO4, arylsulfonyltransferase and chondroitin.

9. The method according to claim 8, characterized in that, The reaction should be carried out at 35-40°C for at least 48 hours.

10. The use of any of the mutants described in claims 1 to 3, or any of the recombinant microorganisms described in claims 6 to 7, in the preparation of chondroitin sulfate or products containing chondroitin sulfate.

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