A chondroitin synthase and its high-expression, high-activity, and high-thermal-stability mutants and their applications

Abstract

This invention relates to a chondroitin synthase and its high-expression, high-activity, and high-thermal-stability mutants and their applications. The amino acid sequence of the chondroitin synthase McCS is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence of the chondroitin synthase mutant tMcCS is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3. The amino acid sequence of the chondroitin synthase mutant McCS-M12 is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.5. The chondroitin synthase McCS disclosed in this invention is a novel chondroitin synthase derived from *Morackella canis*, possessing both GalNAc-T and GlcA-T transferase activities. Compared to the wild-type chondroitin synthase McCS, the chondroitin synthase mutants tMcCS and McCS-M12 exhibit higher enzyme activity and thermostability, greatly promoting the biomimetic synthesis and application of chondroitin and opening a new chapter in the research and development of glycosaminoglycans.

Description

Technical Field

[0001] This invention relates to a novel chondroitin synthase derived from Moraxella canis, its high expression level, high activity, high thermostability mutants, and their applications, belonging to the field of biotechnology. Background Technology

[0002] Chondroitin sulfate (CS) is an important polysaccharide abundant on the cell surface and in the extracellular matrix. It is a copolymer composed of a backbone of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) disaccharide repeating units and various sulfate group modifications. The unique sulfate monosaccharide sequence provides biological function specificity by regulating the polysaccharide's affinity for various chemokines and their receptors. The diverse CS chain structure allows it to interact with many proteins, thereby participating in the regulation of many biological and pathological processes, including cell differentiation, proliferation, migration, morphogenesis, intercellular recognition, inflammation regulation, neural network development, regeneration, and plasticity. Chondroitin (Chn) is the capsular polysaccharide backbone of some pathogenic bacteria and a precursor for CS synthesis in vertebrates, both in vivo and in vitro, with the structure: (-GlcA-1,3-GalNAc-1,4-). n .

[0003] Compared with traditional chemical synthesis methods, the chemoenzymatic synthesis of glycosaminoglycans (CS) has advantages such as relatively simple steps and high efficiency. The first step in the chemoenzymatic synthesis of CS is to synthesize and extend the Chn backbone using chondroitin synthase. Chondroitin synthase (ChnS, EC 2.4.1.175) is a member of the CAZy (Carbohydrate Active Enzyme Database) GT2 family and plays a key role in the biosynthesis of CS and its derivatives. They are bifunctional glycosyltransferases with β1-4-N-acetylgalactosyltransferase (β1-4-GalNAc-T) and β1-3-D-glucuronyltransferase (β1-3-GlcA-T) activities, located in two different catalytic domains, each containing a conserved UDP-glyco-binding DXD motif. This enzyme uses UDP-activated glyconucleotides as donor substrates and catalyzes the formation of chondroitin glycan chains by alternating addition of GalNAc and GlcA.

[0004] Currently, five highly homologous chondroitin synthases derived from microorganisms have been reported: PmCS isolated from Pasteurella Type F, KfoC from Escherichia coli K4, CpCS from Thiobacillus aeruginosa, ApCS from Avianobacterium paragallinarum, and AuCS from Actinobacillus urealyticum. Previous data indicate that these bacterial-derived chondroitin synthases exhibit varying degrees of performance limitations during in vitro enzymatic synthesis. Besides issues such as low expression levels (KfoC produces only about 15 mg of purified enzyme per liter of E. coli culture), limitations in receptor glycan length (PmCS recognizes the shortest chondroitin 3-glycan), and a limited range of donor types (most ChnS only recognize UDP-GalNAc), their GlcA transferase catalytic activity is also relatively low, only about one-tenth the activity of their coexisting GalNAc transferases. Particularly in reactions using GlcA-pNP as the initiating receptor for oligosaccharide synthesis, the shorter receptor glycan chain makes the low GlcA transferase activity the rate-limiting step. The rate-limiting catalytic capacity makes it difficult for the initial reaction to proceed to completion, significantly increasing the difficulty of purification and potentially leading to impurities in subsequent products. Its narrow donor-substrate adaptability restricts its potential application in the synthesis of non-natural CS derivatives.

[0005] Therefore, developing novel chondroitin synthases with high expression levels and good catalytic activity, as well as artificially modifying enzyme molecules through protein engineering techniques to improve their substrate adaptability for catalytic activity, are among the current research focuses in the chemical enzymatic synthesis of chondroitin. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a chondroitin synthase and its high-expression, high-activity, and high-thermal-stability mutants and their applications.

[0007] The technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides a novel chondroitin synthase McCS, the amino acid sequence of which is shown in SEQ ID NO.2 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1;

[0009] The novel chondroitin synthase McCS is derived from Moraxella canis.

[0010] In a second aspect, the present invention provides a chondroitin synthase mutant tMcCS, the amino acid sequence of which is shown in SEQ ID NO.4 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3;

[0011] The chondroitin synthase mutant tMcCS is based on chondroitin synthase McCS with 263 amino acids deleted from the C-terminus.

[0012] A third aspect of the present invention provides a chondroitin synthase mutant McCS-M12, the amino acid sequence of which is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5;

[0013] The chondroitin synthase mutant McCS-M12 is a chondroitin synthase mutant tMcCS with mutations in 7 amino acids; the specific mutations are: D444N, T445Y, K447Q, R480A, S602A, M641L, and I650V.

[0014] Compared with the chondroitin synthase mutant tMcCS, the chondroitin synthase mutant McCS-M12 has the following mutations: aspartic acid at position 444 is mutated to asparagine; threonine at position 445 is mutated to tyrosine; lysine at position 447 is mutated to glutamine; arginine at position 480 is mutated to alanine; serine at position 602 is mutated to alanine; methionine at position 641 is mutated to leucine; and isoleucine at position 650 is mutated to valine.

[0015] A fourth aspect of the present invention provides a recombinant vector constructed by inserting the nucleotide sequence of chondroitin synthase McCS, the nucleotide sequence of the chondroitin synthase mutant tMcCS, or the nucleotide sequence of the chondroitin synthase mutant McCS-M12 into a plasmid vector. The recombinant vector is not particularly limited to a starting vector and can be any vector known in the art, as long as it can replicate in a host. For example, the vector includes, but is not limited to, plasmids and bacteriophages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

[0016] More preferably, the plasmid vector is pET28a(+).

[0017] A fifth aspect of the present invention provides a recombinant cell obtained by transforming the aforementioned recombinant vector into a host cell. The term "host cell" has the meaning commonly understood in the art; it refers to a host cell capable of introducing the coding gene of the mutant of the present invention, and is called a recombinant host cell after introduction. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli BL21(DE3).

[0018] A sixth aspect of the present invention provides a method for preparing chondroitin synthase McCS, chondroitin synthase mutant tMcCS, or chondroitin synthase mutant McCS-M12 by fermentation, the method comprising culturing the host cells described above and isolating and obtaining chondroitin synthase and its truncated form and mutant.

[0019] According to a preferred embodiment of the present invention, the specific steps of the preparation method are as follows: recombinant host cells containing the coding genes of the enzyme and the mutant are cultured in LB liquid medium until the OD600 is 0.6-0.8, and IPTG is added to induce expression for 16-18 h; the bacterial cells are collected, sonicated, centrifuged and filtered, and purified by His tag to obtain chondroitin synthase McCS, chondroitin synthase mutant tMcCS, or chondroitin synthase mutant McCS-M12.

[0020] A seventh aspect of the present invention provides the use of the above-mentioned chondroitin synthase McCS, chondroitin synthase mutant tMcCS, or chondroitin synthase mutant McCS-M12 in the synthesis of chondroitin oligosaccharides or chondroitin polysaccharides.

[0021] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.

[0022] Beneficial effects

[0023] 1. The chondroitin synthase McCS disclosed in this invention is a novel chondroitin synthase derived from *Morakella canis*, possessing both GalNAc-T and GlcA-T transferase activities. Furthermore, under optimal conditions, its efficiency in utilizing the substrate UDP-GalNAc is higher than that of existing chondroitin synthases KfoC (derived from *Escherichia coli* K4), ApCS (derived from *Avianobacterium paragallinarum*), and AuCS (derived from *Actinomyces urealyticum*).

[0024] Compared to the most widely used KfoC, McCS showed the following: Chondroitin synthase expression levels increased to 3.6 times that of KfoC (McCS: 55 mg / L; KfoC: 15 mg / L); the total GalNAc transferase activity of McCS protein per liter of culture medium was nearly 10 times higher than that of KfoC (McCS: 19.97 IU / L; KfoC: 1.84 IU / L); and the total GlcA transferase activity of McCS was approximately 3 times that of KfoC (McCS: 1.95 IU / L; KfoC: 0.66 IU / L). Under the same reaction conditions, McCS also demonstrated a higher ability to utilize non-natural glycosyl donors (such as UDP-GalNAz, UDP-GalNTFA, etc.) than other existing chondroitin synthases. Furthermore, McCS can utilize the glycosyl donor UDP-GalNAz to transfer GalNAz to the chondroitin glycan chain with a GlcA non-reducing terminal, synthesizing a non-natural chondroitin oligosaccharide with an azido group in the glycan structure. The azido group in this glycan chain can rapidly undergo a bio-orthogonal reaction (Click reaction) with fluorescent dyes containing alkynyl groups or other types of dyes, thereby labeling the glycan chain for convenient detection. This provides a suitable detection method for the simple and stable detection of chondroitin sulfate.

[0025] 2. The chondroitin synthase mutant tMcCS provided by this invention, compared to wild-type chondroitin synthase McCS, involves the deletion of a meaningless fragment at the C-terminus of the wild-type McCS protein, resulting in the deletion of 263 amino acids from the C-terminus. This chondroitin synthase mutant tMcCS retains the activities of both GalNAc-T and GlcA-T transferases. Compared to wild-type chondroitin synthase McCS, the chondroitin synthase mutant tMcCS exhibits increased expression levels, enhanced activity, and improved thermostability.

[0026] Specifically, the expression level of the chondroitin synthase mutant tMcCS increased to 215 mg / L, which is 3.9 times that of the wild-type chondroitin synthase McCs (55 mg / L); the GalNAc-T activity of the chondroitin synthase mutant tMcCS reached 78.78 IU / L, which is 3.94 times that of the wild-type chondroitin synthase McCs (19.97 IU / L), and the GlcA-T activity of the chondroitin synthase mutant tMcCS reached 8.91 IU / L, which is 4.6 times that of the wild-type chondroitin synthase McCs (1.95 IU / L); the half-life (T1 / 2) of the chondroitin synthase mutant tMcCS at 37°C was extended by 20 hours compared with the wild-type chondroitin synthase McCs (tMcCS: 21.68 h, McCs: 1.56 h), and its substrate tolerance was also more extensive.

[0027] 3. The chondroitin synthase mutant McCS-M12 provided by this invention is a novel artificial chondroitin synthase. Compared with wild-type chondroitin synthase McCS, it not only deletes 263 amino acid residues at the C-terminus, but also has mutations at 7 amino acid sites (D444N, T445Y, K447Q, R480A, S602A, M641L, I650V). Compared with wild-type chondroitin synthase McCS, the expression level of chondroitin synthase mutant McCS-M12 is significantly improved, its activity is significantly enhanced, and its thermostability is significantly enhanced.

[0028] Specifically, the expression level of the chondroitin synthase mutant McCS-M12 increased to 415 mg / L, which is 7.5 times that of the wild-type chondroitin synthase McCS; the GalNAc-T activity of the chondroitin synthase mutant McCS-M12 reached 949 IU / L, which is 54 times that of the wild-type chondroitin synthase McCS (19.97 IU / L), and the GlcA-T activity of the chondroitin synthase mutant McCS-M12 reached 646 IU / L, which is 331 times that of the wild-type chondroitin synthase McCS (1.95 IU / L); the catalytic efficiency increased. The chondroitin synthase mutant McCS-M12 exhibits high Kcat / Km (GalNAc-T: 8.8 times, GlcA-T: 27 times). After incubation at 37°C for 12 hours, it retained over 50% of its activity, while the wild-type chondroitin synthase McCS lost almost half its activity within 2 hours at 37°C. The stability half-life (T1 / 2) of the chondroitin synthase mutant McCS-M12 at 37°C was 14.52 hours, while the wild-type chondroitin synthase McCS (McCS: 1.567 hours) was extended by 13 hours. Combined with its low-temperature characteristics and outstanding catalytic activity, the chondroitin synthase mutant McCS-M12 can be recycled multiple times at lower temperatures to synthesize the chondroitin backbone, greatly promoting the biomimetic synthesis and application of chondroitin and opening a new chapter in the research and development of glycosaminoglycans. Attached Figure Description

[0029] Figure 1 SDS-PAGE electrophoresis images of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, and chondroitin synthase mutant McCS-M12.

[0030] Figure 2 High-performance liquid chromatography (HPLC) curves validating the GalNAc transferase activity of chondroitin synthase McCS.

[0031] In the figure: the horizontal axis represents retention time, and the vertical axis represents the intensity of the electrical signal absorbed by the reaction product at 310 nm.

[0032] Figure 3Mass spectrometry analysis of the reaction products of chondroitin synthase McCS with GalNAc transferase activity.

[0033] In the figure: the horizontal axis represents the mass-to-charge ratio m / z, and the vertical axis represents the signal strength.

[0034] Figure 4 High-performance liquid chromatography (HPLC) curves validating the GlcA transferase activity of chondroitin synthase McCS.

[0035] In the figure: the horizontal axis represents retention time, and the vertical axis represents the intensity of the electrical signal absorbed by the reaction product at 310 nm.

[0036] Figure 5 Mass spectrometry analysis of the reaction products of chondroitin synthase McCS GlcA transferase activity.

[0037] In the figure: the horizontal axis represents the mass-to-charge ratio m / z, and the vertical axis represents the signal strength.

[0038] Figure 6 Results of basic enzymatic properties detection of chondroitin synthase McCS;

[0039] In the figure, A represents the determination of the optimal pH value of the buffer system during the synthesis reaction; B represents the determination of the optimal metal ions in the buffer system during the in vitro synthesis reaction; C represents the temperature curve of the in vitro synthesis reaction; where: the horizontal axis represents the corresponding condition changes, and the vertical axis represents the relative yield of the reaction products (with the optimal reaction group as 100%).

[0040] Figure 7 The results show the donor substrate specificity comparison of chondroitin synthase McCS and chondroitin synthases KfoC and AuCS in this invention.

[0041] In the figure, the horizontal axis represents different chondroitin synthases, and the vertical axis represents the relative yield of the reaction products (with the optimal reaction group as 100%).

[0042] Figure 8 BLAST and domain prediction results for chondroitin synthase McCS homologous proteins;

[0043] In the figure, A represents the search results of the chondroitin synthase McCS template in the NCBI database BLAST, and B represents the domain prediction results of chondroitin synthase McCS.

[0044] Figure 9 Results of amino acid sequence conservation analysis for chondroitin synthase McCS;

[0045] In the figure, the horizontal axis represents the amino acid sequence number, and the vertical axis represents the conservation analysis score. The higher the score, the higher the degree of conservation.

[0046] Figure 10The virtual saturation screening scores and three-dimensional structural simulations of chondroitin synthase McCS EV Couplings;

[0047] In the figure, A represents the scoring results of the EV Couplings virtual saturation screening. The more orange the color, the more beneficial the mutation is to the activity, and the more blue the color, the more detrimental the mutation is to the activity. B represents the corresponding positions of the 14 amino acid sites with the highest scores in the McCS three-dimensional structure.

[0048] Figure 11 Results of the first-round unit point mutation GlcA transferase activity of chondroitin synthase tMcCS;

[0049] In the figure, the horizontal axis represents the name of the mutant, and the vertical axis represents the multiple by which the GlcA transferase activity of the mutant is compared to that of the wild-type GlcA transferase.

[0050] Figure 12 Results of the second round of combined mutant GlcA transferase activity of chondroitin synthase tMcCS;

[0051] In the figure, the horizontal axis represents the name of the mutant, and the vertical axis represents the multiple by which the GlcA transferase activity of the mutant is compared to that of the wild-type GlcA transferase.

[0052] Figure 13 Results of the third round of iterative combined mutant GlcA transferase activity of chondroitin synthase tMcCS;

[0053] In the figure, the horizontal axis represents the name of the mutant, and the vertical axis represents the multiple by which the GlcA transferase activity of the mutant is compared to that of the wild-type GlcA transferase.

[0054] Figure 14 The soluble expression levels of chondroitin synthase McCS and its mutants;

[0055] In the figure, the horizontal axis represents the name of the mutant, and the vertical axis represents the soluble expression level of the mutant in the E. coli expression system.

[0056] Figure 15 The reaction kinetics curves of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12 are shown.

[0057] In the figure, A is the kinetic curve of GalNAc transferase activity, and B is the kinetic curve of GlcA transferase activity; where the horizontal axis represents the concentration of the donor substrate, and the vertical axis represents the degree of reaction.

[0058] Figure 16 The results show the comparison of the enzyme catalytic activities of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, chondroitin synthase mutant McCS-M12, and chondroitin synthase KfoC.

[0059] In the figure, the horizontal axis represents the name of the enzyme, and the vertical axis represents the catalytic activity per unit mass and per unit time of the protein.

[0060] Figure 17 Comparison of total enzyme activities of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, chondroitin synthase mutant McCS-M12, and chondroitin synthase KfoC.

[0061] In the figure, the horizontal axis represents the name of the enzyme, and the vertical axis represents the total catalytic activity of the protein obtained from a one-liter E. coli culture system.

[0062] Figure 18 The results show the donor substrate specificity comparison between chondroitin synthase McCS and the chondroitin synthase mutant McCS-M12.

[0063] In the figure, the horizontal axis represents chondroitin synthase from different sources, and the vertical axis represents the relative yield of the reaction products (with the optimal reaction group as 100%).

[0064] Figure 19 Thermostability curves of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, and chondroitin synthase mutant McCS-M12 at 37℃.

[0065] In the figure, the horizontal axis represents the pre-incubation time of the protein at 37°C, and the vertical axis represents the relative reaction rate. The relative reaction rate is calculated with the activity of the untreated protein as 100%. Detailed Implementation

[0066] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.

[0067] Example 1: Heterologous expression, activity detection, and enzymatic property determination of a novel chondroitin synthase McCS

[0068] 1. During a bioinformatics database search using the BLASTp algorithm, the inventors discovered that the amino acid sequence of a gene in *Moraxella canis* shared significant homology with the previously reported amino acid sequence of chondroitin synthase. Therefore, the inventors speculated that the protein product expressed by this gene might have chondroitin synthase activity and named this gene McCS. They then expressed the McCS gene in an *E. coli* expression system, named the expressed protein product McCS, whose amino acid sequence is shown in SEQ ID NO.2, and whose nucleotide sequence encoding the gene is shown in SEQ ID NO.1.

[0069] The specific methods for heterologous expression and purification of chondroitin synthase McCS are as follows:

[0070] The McCS gene was artificially synthesized by Nanjing Genscript Biotech Co., Ltd. according to the nucleotide sequence shown in SEQ ID NO.1, and cloned into the pET28a(+) vector. Then, it was chemically transformed into E.coli BL21(DE3) competent cells and cultured on LB solid medium containing kanamycin (50 μg / mL) for 12 h. Transformants were screened (a negative control experiment was performed at the same time) and positive transformants were obtained.

[0071] Single colonies of chondroitin synthase McCS-positive transformants were picked and activated in 20 mL of sterile LB liquid medium (containing 50 μg / mL kanamycin) at 37 °C, 225 rpm. The overnight activated culture was inoculated at a 1% inoculum into 1 L of LB liquid medium (containing 50 μg / mL kanamycin) for further culture. The culture was incubated at 37 °C, 225 rpm for 4 hours with shaking until the OD600 reached approximately 0.8. IPTG was added to a final concentration of 0.2 mM, and expression was induced for 16 h at 22 °C, 225 rpm. The cells were collected by centrifugation and resuspended in 1× wash buffer. The cells were then sonicated on ice (3 s on, 5 s off, 33% amplitude, 1500 KJ energy, 4 °C) for 30 min. The disrupted cells were centrifuged at 12000 rpm for 20 min (4 °C), and the supernatant was filtered through a 0.22 μm filter. Purification was performed using a Ni ion chromatography column. After loading the sample, it was washed with 1× washing buffer and finally eluted with elution buffer containing 200 mM imidazole to obtain the target protein chondroitin synthase McCS.

[0072] The purified chondroitin synthase McCS was identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown.

[0073] Depend on Figure 1 As can be seen, a clear band of approximately 110 kDa was observed in the lane of chondroitin synthase McCS, indicating that heterologous expression of chondroitin synthase McCS was successful.

[0074] Following the above method, single colonies of chondroitin synthase McCS positive transformants were cultured in 1L of LB liquid medium. Then, the protein concentration of chondroitin synthase McCS in the E. coli culture medium was determined using a BCA protein concentration assay kit (Beyotime P0011). The results showed that the soluble expression level of chondroitin synthase McCS was 55 mg / L.

[0075] 2. Assay of chondroitin synthase McCS activity

[0076] (1) Verification of β1-4-GalNAc transferase activity of chondroitin synthase McCS

[0077] The reaction was carried out using commercial GlcA-pNP (final concentration 0.2 mM) as the acceptor substrate and UDP-GalNAc (final concentration 0.3 mM) as the donor substrate. The reaction system is shown in Table 1.

[0078] The reaction system was incubated in a water bath at 25°C for 4 hours, and then heated in boiling water for 5 minutes to inactivate the enzyme and terminate the reaction. The reaction solution was filtered through a 0.22 μm filter membrane, and the reaction rate was detected by liquid chromatography. A YMC-Pack Polyamine II column was used, and the liquid chromatography system was a Shimadzu HPLC system. The UV detection system was an SPD-20A. The pNP group of the monosaccharide receptor showed specific absorption at a UV detection wavelength of 310 nm. The HPLC mobile phase flow rate was 0.5 mL / min, and the mobile phase gradient is shown in Table 2.

[0079] Table 1. Reaction system for verifying the β1-4-GalNAc transferase activity of chondroitin synthase McCS

[0080]

[0081] Table 2. HPLC analytical procedure used for detecting chondroitin oligosaccharides

[0082]

[0083] The high-performance liquid chromatography (HPLC) curve for verifying the GalNAc transferase activity of chondroitin synthase McCS is shown below. Figure 2 As shown, this indicates that chondroitin synthase McCS has GalNAc transferase activity, which can transfer the GalNAc group to the non-reducing end of GlcA-pNP to generate chondroitin disaccharide GalNAc-GlcA-pNP.

[0084] (2) Mass spectrometric confirmation of chondroitin disaccharide GalNAc-GlcA-pNP

[0085] To confirm the product structure of the above-described reactive reaction as GalNAc-GlcA-pNP, electrospray ionization mass spectrometry (ESI-MS) analysis was subsequently performed. The reaction was carried out on a large scale to obtain sufficient disaccharide product. The product was purified by P2 column analysis and then analyzed by MS on a Thermo LCQ-Deca. All MS samples were prepared by dissolving in 50% methanol. MS experiments were performed in negative ion mode with a spray voltage of 5 kV and a capillary temperature of 275 °C.

[0086] Mass spectrometry analysis results as follows Figure 3As shown, the molecular weight determined in the MS spectrum is consistent with the calculated molecular weight of chondroitin disaccharide, proving the formation of the product GalNAc-GlcA-pNP.

[0087] (3) Verification of β1-3-GlcA transferase activity of chondroitin synthase McCS

[0088] The product GalNAc-GlcA-pNP (final concentration 0.2 mM) obtained in step (2) was used as the acceptor substrate, and UDP-GlcA (final concentration 0.3 mM) was used as the donor substrate. The reaction system is shown in Table 3. The reaction system was reacted in a water bath at 25 °C for 4 h, and the reaction was terminated by heating with boiling water for 5 min to inactivate the enzyme. The reaction solution was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography according to the method described in Table 1.

[0089] Table 3. Reaction system for verifying the β1-3-GlcA transferase activity of chondroitin synthase McCS

[0090]

[0091] The high-performance liquid chromatography (HPLC) curve for verifying the GlcA transferase activity of chondroitin synthase McCS is shown below. Figure 4 As shown, a new trisaccharide peak (Trisaccharide-1) was generated in the reaction group, indicating that chondroitin synthase McCS has GlcA transferase activity, which can transfer the GlcA group to the non-reducing end of GalNAc-GlcA-pNP to generate chondroitin trisaccharide GlcA-GalNAc-GlcA-pNP.

[0092] (4) Mass spectrometry confirmation of chondroitin trisaccharide GlcA-GalNAc-GlcA-pNP: The method is the same as above. Chondroitin trisaccharide is generated on a larger scale (more than 20 mg of product). After purification, the product trisaccharide is analyzed by mass spectrometry.

[0093] Mass spectrometry analysis results as follows Figure 5 As shown, the molecular weight determined in the MS spectrum is consistent with the calculated molecular weight of chondroitin trisaccharide, proving the formation of the product GalNAc-GlcA-pNP.

[0094] 3. Enzymatic properties of chondroitin synthase McCS and its mutants

[0095] 1) Determination of the optimal pH for the in vitro reaction of the enzyme: The reaction system, except for the pH of the buffer solution, is as shown in Table 1. The Tris-HCl buffer was replaced with Tris-HCl / PBS / CH3COONa buffer with different pH values. A total of 13 pH gradient points were set: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.3, 5.9, 6.4, 7.0, 7.5, 8.0, and 8.5. Each gradient was repeated in triplicate. All other treatment conditions were the same as in step (1) of point 2 of this embodiment. The measurement results are as follows: Figure 6 As shown in Figure A.

[0096] 2) Determination of the optimal metal ion for in vitro enzyme reaction: The reaction system, except for the metal ion, is shown in Table 1. Mn 2+ Replace with the same concentration of Mg ions 2+ Mn 2+ Ca 2+ For each ion, three parallel experimental groups were set up, with a blank control included. All other treatment conditions were the same as in step (1) of point 2 of this embodiment. The measurement results are as follows: Figure 6 As shown in B.

[0097] 3) Study on the effect of reaction temperature on enzyme activity: The reaction system is shown in Table 1. Eight temperature gradients were set: 4℃, 10℃, 20℃, 25℃, 30℃, 37℃, 45℃, and 55℃, with three parallel sets for each gradient. The effect of reaction temperature on enzyme activity was determined by measuring the substrate conversion in each reaction. The results are shown in Table 1. Figure 6 As shown in C.

[0098] Depend on Figure 6 From A to C, we can see that the optimal pH for chondroitin synthase McCS is 5.0–5.3, and the optimal metal ion is Mn. 2+ The optimal reaction temperature is 20℃.

[0099] Example 2: Comparison of donor-substrate specificity of chondroitin synthase McCS and chondroitin synthases KfoC and AuCS

[0100] To determine the donor-substrate specificity of the chondroitin synthase McCS of this invention, commercially available GlcA-pNP was used as the initiator acceptor, and UDP-GalNAc and five structurally similar UDP-sugars (UDP-GalNAz, UDP-GalNTFA, UDP-GlcNAc, UDP-Gal, and UDP-Glc) were used as donors. Following the method described in point 2 of Example 1, the activities of chondroitin synthase McCS, chondroitin synthase KfoC, and chondroitin synthase AuCS were detected. The reaction system is shown in Table 1. All reactions were incubated in a 25°C water bath for 4 hours, and the degree of reaction was detected by HPLC. All groups were performed in triplicate. The results are shown in Table 1. Figure 7 As shown.

[0101] Among them, chondroitin synthases KfoC and AuCS are existing conventional chondroitin synthases, and their amino acid and nucleotide sequences have been disclosed. They can be obtained commercially or prepared according to the heterologous expression method described in point 1 of Example 1.

[0102] Depend on Figure 7 It is known that when the receptor is the monosaccharide GlcA-pNP, among the five monosaccharide donors, chondroitin synthase McCS can effectively transfer four glycosyl groups (GalNAc, GalNTFA, GalNAz, and GlcNAc) to the non-reducing end of GlcA-pNP. However, chondroitin synthase KfoC can only recognize two donor substrates, GalNAc and GalNAz, and its reaction rate is much lower than that of chondroitin synthase McCS.

[0103] Example 3: Chondroitin synthase mutant tMcCS with increased expression and enhanced thermostability

[0104] 1. Using wild-type chondroitin synthase McCS as a template sequence, the NCBI database was searched using the BLASTp method. The top 100 homologous sequences found by BLASTp were aligned, and the results are as follows: Figure 8 As shown in Figure A. Furthermore, the inventors of this application also used InterPro to predict the active domain of wild-type chondroitin synthase McCS, with the results shown below. Figure 8 As shown in B.

[0105] Depend on Figure 8 As can be seen from A, most homologous proteins lack significant homology in the last 263 amino acid sequences of wild-type chondroitin synthase McCS.

[0106] Depend on Figure 8 As shown in B, the C-terminal 263 amino acids of the wild-type chondroitin synthase McCS do not participate in the formation of any active domain, and are therefore a meaningless sequence. The inventors of this application decided to remove the non-essential C-terminal 263 amino acid residues of McCS to construct an engineered chondroitin synthase mutant tMcCS, the amino acid sequence of which is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0107] 2. The tMcCS gene was artificially synthesized by Nanjing GenScript Co., Ltd. according to the nucleotide sequence shown in SEQ ID NO.3. Then, the chondroitin synthase mutant tMcCS was prepared according to the heterologous expression and purification method described in point 1 of Example 1.

[0108] The purified chondroitin synthase mutant tMcCS was identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown.

[0109] Depend on Figure 1 As can be seen, a clear band with a size of approximately 78.9 kDa was observed in the lane of the chondroitin synthase mutant tMcCS, indicating that the heterologous expression of the chondroitin synthase mutant tMcCS was successful.

[0110] Following the heterologous expression method described in point 1 of Example 1, single colonies of chondroitin synthase McCS positive transformants were cultured in 1L of LB liquid medium. Then, the protein concentration of chondroitin synthase McCS in the E. coli culture medium was determined using a BCA protein concentration assay kit (Beyotime P0011). The results showed that the soluble expression level of chondroitin synthase McCS was 55 mg / L.

[0111] Meanwhile, the soluble expression level of chondroitin synthase KfoC was determined using the same method, and the result showed that the soluble expression level of chondroitin synthase McCs was 15 mg / L.

[0112] As can be seen, the soluble expression level of chondroitin synthase McCS is 3.6 times that of chondroitin synthase KfoC (McCS: 55 mg / L; KfoC: 15 mg / L), while the soluble expression level of the chondroitin synthase mutant tMcCS is 3.9 times that of chondroitin synthase McCS (McCS: 55 mg / L; tMcCS: 215 mg / L) and 14.3 times that of chondroitin synthase KfoC (tMcCS: 215 mg / L; KfoC: 15 mg / L). This indicates that the soluble expression levels of both chondroitin synthase McCS and the chondroitin synthase mutant tMcCS are significantly increased in the E. coli expression system, with the increase in the chondroitin synthase mutant tMcCS being greater.

[0113] Example 4: Hotspot identification of GlcA transferase domain amino acid sequence modification in chondroitin synthase mutant tMcCS (McCS-M1)

[0114] 1. The chondroitin synthase mutant tMcCS obtained in Example 3 was renamed chondroitin synthase mutant McCS-M1, and then the set of sites that can be modified was determined based on the conservation of its amino acid sequence.

[0115] The conservation of amino acid sequences in homologous proteins is typically calculated using methods such as multiple sequence alignment (MSA) and conservation scoring, aiming to assess the evolutionary stability of amino acid sites across different species or protein families. It can reflect the importance of certain sites during evolution; generally, highly conserved sites are crucial for protein function and structure, while sites with greater variation may allow for significant functional adjustments or adaptive evolution.

[0116] Homologous sequences of wild-type chondroitin synthase McCS were retrieved using NCBI homology search. Over 500 homologous sequences were manually selected and aligned using Jalview software. Gap regions were manually removed, and the conservation of each amino acid residue was calculated (with 10 as the maximum value). Higher values ​​indicate higher conservation at that site. This invention only modifies non-conserved regions; therefore, amino acid sites with scores <8 were the focus of subsequent mutation studies. The conservation analysis results are as follows: Figure 9 As shown.

[0117] This modification aims only to enhance the GlcA transferase activity of wild-type chondroitin synthase McCS. Therefore, the mutation should be performed near the GlcA transferase domain (Q433-567 of the McCS amino acid sequence). Furthermore, to minimize the impact on the N-terminal GalNAc transferase domain, the mutation site is chosen to start at amino acid 433. Based on protein secondary structure prediction, the sequence after amino acid 680 is disordered and does not participate in the formation of the active domain. Therefore, the final amino acid mutation site selection range for this invention is determined to be 433-680.

[0118] Then based on the information of homologous sequences ( Figure 8 ), 43 amino acid residues with a conservation score <8 in the range of 433-680 were selected, namely: 443, 444, 445, 447, 462, 473, 476, 479, 480, 481, 484, 485, 493, 494, 496, 500, 504, 507, 508, 533, 536, 539, 553, 554, 557, 559, 561, 566, 586, 590, 593, 598, 600, 602, 616, 624, 632, 638, 640, 641, 644, 648 and 652.

[0119] 2. According to the PROSS algorithm, the enhanced thermostability of the chondroitin synthase mutant McCS-M1 is beneficial to the mutation site.

[0120] The inventors used the PROSS algorithm to calculate and design chondroitin synthase McCS mutation sites that enhance stability within the above 42 sites. The mutation sites and mutation schemes located within the GlcA transferase domain from the PROSS project output were selected, specifically: T445Y, Q462E, N467D, D476E, I478L, N479E, L481H, G483S, and Q4. 84N, K489R, N504A, K507R, H508L, A509C, S510R, G515A, A533K, L536F, N548Y, A559S, S556N, T573M, N598S, E600T, S602A, I607M, M641K, Q655R, V659I, have 29 amino acid mutations compared to McCS-M1.

[0121] Based on the above results, considering both amino acid conservation and mutation sites beneficial to protein stability, a total of 18 amino acid sites were selected compared to McCS-M1, specifically: T445Y, Q462E, D476E, L481H, Q484N, N504A, K507R, H508L, A533K, L536F, A559S, N598S, E600T, S602A, I607M, M641K, Q655R, and V659I.

[0122] Example 5: Determination of the mutation site that enhances GlcA transferase activity in the chondroitin synthase mutant tMcCS (McCS-M1)

[0123] 1. The McCS-M1 mutant, modeled using SWISS-MODEL for homology, was input into the HotSpot Wizard 3.0 server. Mutation sites targeting GlcA transferase activity were calculated and designed within the 433-680 amino acid range, specifically: 442, 443, 444, 448, 496, and 522. Based on sequence conservation principles, sites 442, 448, and 522 were removed. The calculated hotspots were amino acids 443, 444, and 496.

[0124] 2. The amino acid sequence of the chondroitin synthase mutant McCS-M1 was input into the EV Couplings server (https: / / v2.evcouplings.org). The GlcA transferase domain was selected as the focus of analysis. By searching the database, the server performed multiple sequence alignment and evolutionary covariance analysis on approximately 106,306 homologous protein sequences with the chondroitin synthase mutant McCS-M1, providing evolutionary dependencies between amino acid residues. Based on the amino acid epistasis model, the mutational effects (i.e., virtual saturation mutations at each site) within the GlcA transferase domain were predicted, and three-dimensional structural models before and after mutation were provided, as detailed below. Figure 10 As shown.

[0125] Based on the scores of virtual saturation mutations at each point (higher scores indicate more favorable mutations), a total of 17 mutation hotspots were selected, namely: A533K, M641L, Q462E, Y661N, L605Y, I650V, R480A, K447Q, S602A, S510R, I478L, Q484N, R652K, V659I, N504A, A509C, and H508L.

[0126] Based on the results of the virtual saturation mutation, sites that would cause significant damage to the protein after mutation were further removed from the previously selected sites. In summary, the following 16 sites were selected as the first round of mutation hotspots: D444N, T445Y, K447Q, Q462E, I478L, R480A, Q484N, H508L, S510R, A533K, S602A, L605Y, M641L, I650V, V659I, and Y661N.

[0127] Example 6: Obtaining the chondroitin synthase mutant McCS-M12

[0128] 1. First round of mutations in the chondroitin synthase mutant McCS-M1

[0129] Using the nucleotide sequence of the chondroitin synthase mutant McCS-M1 (SEQ ID NO. 3) as a template and the sequences in Table 4 as primers, the first round of mutation amplification was performed according to the mutation hotspots selected in Example 5 to obtain the nucleotide sequence of the chondroitin synthase mutant McCS-M2. Then, following the heterologous expression and purification method described in point 1 of Example 1, the chondroitin synthase mutant McCS-M2 was prepared. The purified chondroitin synthase mutant McCS-M2 was then subjected to GlcA transferase activity detection and comparison according to the method described in point 2 of Example 1, with wild-type chondroitin synthase McCS as a control. The results are as follows: Figure 11 As shown.

[0130] The reaction system for PCR amplification of the McCS site-directed mutant encoding gene is as follows:

[0131]

[0132] PCR amplification conditions were performed according to the standard operating procedure of Novizan 2*Phanta Flash Master Mix (Dye Plus) instructions. After PCR amplification, 4.5 μL of the amplification product was added to 0.5 μL of 10*Loading Buffer, and the PCR results were detected by electrophoresis on a 1% agarose gel containing Goldview. The gel was diluted with 50*TAE buffer and used at 150V. After electrophoresis, the gel was observed and photographed using a gel imaging system.

[0133] Template digestion and recovery: 2 μL of DpnI was added to the PCR product to digest the template. After gently mixing the reaction solution, the mixture was briefly centrifuged and incubated at 37°C for 30 min. The reaction was then stopped by placing the mixture on ice. 2 μL of the DpnI-treated PCR product was collected for gel extraction and recovery, following the standard procedure of the Omega gel extraction / DNA purification kit instructions. The recovered target gene product was directly transformed into the expression host E. coli BL21(DE3). Six transformants from each mutant were selected, activated, and sequenced. Mutants with accurate sequencing results were then used for strain preservation and subsequent protein expression.

[0134] Table 4. First-round mutation primers

[0135]

[0136]

[0137] Depend on Figure 11 It is known that the first round of mutation predictions for the chondroitin synthase mutant McCS-M1 hit the following 15 sites of enhanced activity: D444N, T445Y, K447Q, Q462E, I478L, R480A, Q484N, H508L, S510R, A533K, S602A, M641L, I650V, V659I, and Y661N.

[0138] 2. Second round of mutations in the chondroitin synthase mutant McCS-M1

[0139] From the 15 single-point mutants with significantly enhanced activity obtained in point 1 of this embodiment, the most effective sites were selected for combination to form 5 combined mutants, specifically:

[0140] M2(M1-D444N / T445Y / K447Q), M3(M1-I478L / R480A / Q484N), M4(M1-H508L / S510R), M5(M1-M641L / I650V), M6(M1-V659I / Y661N).

[0141] Using the nucleotide sequence of the chondroitin synthase mutant McCS-M1 (SEQ ID NO.3) as a template and the sequences in Table 5 as primers, a second round of mutation amplification was performed on the five combined mutants selected in this step, yielding the nucleotide sequences of chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6. Then, following the heterologous expression and purification method described in point 1 of Example 1, the chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6 were prepared. The GlcA transferase activities of the purified chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6 were then detected and compared according to the method described in point 2 of Example 1, with wild-type chondroitin synthase McCS as a control. The results are as follows: Figure 12 As shown.

[0142] Table 5. Second Round of Mutation Primers

[0143]

[0144] Depend on Figure 12 It can be seen that each combination has different degrees of activity increase. Among them, the D444N / T445Y / K447Q triple mutant (McCS-M2) is the best mutation combination, followed by the 641L / I650V double mutant (McCS-M5). Other combination mutants all have different degrees of iterative activity improvement compared with the single point mutant.

[0145] 3. Third round of mutations in the chondroitin synthase mutant McCS-M1

[0146] Further iterations of the superior combination mutants selected in point 2 of this embodiment involve combining multiple mutants with enhanced catalytic activity in pairs or superimposing them with the S602A mutation site that has the greatest effect on activity gain in point 1 of this embodiment, forming the following 5 combination mutants:

[0147] M7(M1-D444N / T445Y / K447Q / S602A), M8(M1-M641L / I650V / S602A),

[0148] M9(M1-D444N / T445Y / K447Q / M641L / I650V),

[0149] M10(M1-D444N / T445Y / K447Q / S602A / M641L / I650V)

[0150] M11(M1-D444N / T445Y / K447Q / H508L / S510R / S602A / M641L / I650V)

[0151] Using the nucleotide sequences of chondroitin synthase mutants McCS-M1, McCS-M5, McCS-M7, and McCS-M10 as templates and the sequences in Table 6 as primers, a third round of mutation amplification was performed based on the five combined mutants selected in this step, resulting in the nucleotide sequences of chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11. Then, following the heterologous expression and purification method described in point 1 of Example 1, the chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11 were prepared. Following the method described in point 2 of Example 1, the GlcA transferase activities of the purified chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11 were detected and compared, with wild-type chondroitin synthase McCS used as a control. The results are as follows: Figure 13 As shown.

[0152] Table 6. Third-round mutation primers

[0153]

[0154] Depend on Figure 13It can be seen that the D444N / T445Y / K447Q triple mutant (McCS-M2) and S602A shed together produce the D444N / T445Y / K447Q / S602A quad mutant (McCS-M7), which has improved activity compared to either McCS-M2 or McCS-S602A. Similarly, the M641L / I650V double mutant (McCS-M5) and S602A shed together produce the D444N / T445Y / K447Q / S602A quad mutant (McCS-M7), which has improved activity compared to either McCS-M2 or McCS-S602A. The M641L / I650V / S602A triple mutant (McCS-M8) showed enhanced 602A activity. Further iterations of the McCS-M7 and McCS-M8 mutation sites yielded the optimal D444N / T445Y / K447Q / S602A / M641L / I650V six-mutant McCS-M10, exhibiting a 21-fold increase in GlcA transferase catalytic activity compared to the wild-type McCS. Adding McCS-M4 mutations to the McCS-M10 mutation site resulted in a decrease in activity, thus McCS-M10 was ultimately determined as the optimal mutant in this round.

[0155] 4. Fourth-round mutation of chondroitin synthase mutant McCS-M1

[0156] The optimal combination McCS-M10, obtained from the iteration in point 3 of this embodiment, is then combined with the site McCS-R480A, which has the greatest expression gain.

[0157] Combined with the mutation site R480A alone to form

[0158] M12(M1-D444N / T445Y / K447Q / R480A / S602A / M641L / I650V),

[0159] Together with the active gain combination mutation M3 (M1-I478L / R480A / Q484N) containing R480A, it forms

[0160] M13(M1-D444N / T445Y / K447Q / I478L / R480A / Q484N / S602A / M641L / I650V).

[0161] Using the nucleotide sequence of the chondroitin synthase mutant McCS-M10 as a template and the sequences in Table 7 as primers, a fourth round of mutation amplification was performed based on the two combined mutants selected in this step to obtain the nucleotide sequences of the chondroitin synthase mutants McCS-M12 and McCS-M13, respectively. Then, following the heterologous expression and purification method described in point 1 of Example 1, the chondroitin synthase mutants McCS-M12 and McCS-M13 were prepared, respectively.

[0162] Table 7. Fourth Round of Mutation Primers

[0163]

[0164] 5. The soluble expression levels of wild-type chondroitin synthases McCS, McCS-M1, M1-D444N, M1-T445Y, M1-K447Q, M2, M1-Q642E, M1-I478L, M1-R480A, M1-Q484N, M3, M1-H508L, M1-S510R, M4, M1-A533K, M1-S602A, M1-L605Y, M1-M641L, M1-I605V, M5, M1-V659I, M1-Y661N, M6, M7, M8, M9, M10, M11, M12, and M13 were determined according to the method described in point 1 of Example 1. The results are as follows: Figure 14 As shown.

[0165] Depend on Figure 14 It can be seen that the chondroitin synthase mutant McCS-M12 has the highest soluble expression level, reaching 415 mg / L, which is 7.5 times that of the wild-type chondroitin synthase McCS (McCS: 55 mg / L; McCS-M12: 415 mg / L). Therefore, the chondroitin synthase mutant McCS-M12 is determined to be the optimal mutant.

[0166] The purified chondroitin synthase mutant McCS-M12 was identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown.

[0167] Depend on Figure 1 It can be seen that a clear band with a size of approximately 78.9 kDa was observed in the lane of the chondroitin synthase mutant McCS-M12, indicating that the heterologous expression of the chondroitin synthase mutant McCS-M12 was successful.

[0168] Example 7: Enzymatic property analysis of chondroitin synthase mutant McCS-M12

[0169] 1. Determination of kinetic constants for the reaction catalyzed by chondroitin synthase McCS and the chondroitin synthase mutant McCS-M12

[0170] The kinetic parameters of chondroitin synthase McCS and the chondroitin synthase mutant McCS-M12 were measured by performing enzymatic reactions with different concentrations of UDP-GalNAc in parallel. The specific methods are as follows:

[0171] The kinetic constant of McCS was calculated by measuring the relationship between the donor substrate concentration (0.6 mmol / L) and the substrate concentration (UDP-GalNA or UDP-GlcA) from 0.1 to 0.5 mmol / L for each reaction group within a certain reaction time. The kinetic constants of McCS were calculated by fitting this curve to the Michaelis-Menten equation. The kinetic curves of chondroitin synthase McCS and the chondroitin synthase mutant McCS-M12 are shown below. Figure 15 As shown.

[0172] The kinetic parameters of chondroitin synthase KfoC and chondroitin synthase tMcCS were determined again using the same method. The results of the determination of chondroitin synthase McCS, chondroitin synthase mutant McCS-M12, chondroitin synthase KfoC and chondroitin synthase tMcCS are shown in Table 8.

[0173] Table 8. Calculated kinetic constants of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12

[0174]

[0175] From Table 8 and Figure 15 It can be seen that, compared with the wild-type chondroitin synthase McCS, the catalytic efficiency (Kcat / Km) of the chondroitin synthase mutant McCS-M12 is significantly improved. The catalytic efficiency of GalNAc-T of the chondroitin synthase mutant McCS-M12 is increased by 8.8 times, and the catalytic efficiency of GlcA-T is increased by 27 times (GalNAc-T: 8.8 times, GlcA-T: 27 times).

[0176] 2. The enzyme catalytic activities of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, chondroitin synthase mutant McCS-M12, and chondroitin synthase KfoC were determined according to the method described in point 1 of Example 1. The results are as follows: Figure 16 As shown.

[0177] The specific reaction system is the same as in Example 1, the reaction time is 1 hour, and the reaction temperature is 20°C. Enzyme activity is measured in micromoles of substrate transferred per milligram of protein per minute.

[0178] Depend on Figure 16 It can be seen that the GalNAc-T activity of the chondroitin synthase mutant McCS-M12 reaches 2.29 IU / mg, which is 7.15 times that of the wild-type chondroitin synthase McCS (0.33 IU / mg), and the GlcA-T activity of the chondroitin synthase mutant McCS-M12 reaches 1.56 IU / mg, which is 44.48 times that of the wild-type chondroitin synthase McCS (0.036 IU / mg).

[0179] 3. The total enzyme activities of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, chondroitin synthase mutant McCS-M12, and chondroitin synthase KfoC were determined, and the results are as follows: Figure 17 As shown.

[0180] The specific reaction system is the same as in Example 1, with a reaction time of 1 hour and a reaction temperature of 20°C. The total enzyme activity is calculated as the product of the enzyme activity and the protein expression level in one liter of culture system, representing the total number of micromoles of substrate that the purified protein in each liter of culture system can transfer.

[0181] Depend on Figure 17 It can be seen that, since the expression level of the chondroitin synthase mutant McCS-M12 is increased to 415 mg / L, which is 7.5 times that of the wild-type chondroitin synthase McCS, the total GalNAc-T enzyme activity of the chondroitin synthase mutant McCS-M12 reaches 949 IU / L, which is 54 times that of the wild-type chondroitin synthase McCS (19.97 IU / L); the total GlcA-T enzyme activity of the chondroitin synthase mutant McCS-M12 reaches 646 IU / L, which is 331 times that of the wild-type chondroitin synthase McCS (1.95 IU / L).

[0182] 4. Substrate specificity comparison of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12 was performed according to the method described in Example 2. The results are as follows: Figure 18 As shown.

[0183] Depend on Figure 18 It can be seen that the chondroitin synthase mutant McCS-M12 has a further enhanced ability to recognize donor substrates. Under the same conditions, the chondroitin synthase mutant McCS-M12 showed a higher degree of response to each recognizable substrate than the wild type, and also showed recognition of UDP-Gal.

[0184] 5. Detection of the thermal stability of chondroitin synthase and its mutants

[0185] The thermostability of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, and chondroitin synthase mutant McCS-M12 was tested as follows: Chondroitin synthase McCS, chondroitin synthase mutant tMcCS, and chondroitin synthase mutant McCS-M12 were pre-incubated at 37°C for different times (0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours), respectively. Then, GalNAc transferase activity was performed according to the method described in point 1 of Example 1. The conversion rate of GlcA-pNP to GalNAc-GlcA-pNP was determined by HPLC. The stability half-life (T2) of each enzyme was also determined. 1 / 2Analysis and calculations were performed using GraphPad Prism 8 software. All groups underwent three parallel experiments, and the results are as follows: Figure 19 As shown.

[0186] Depend on Figure 19 It can be seen that the chondroitin synthase mutant McCS-M12 still retained more than 50% of its activity after being incubated at 37°C for 12 hours, while the wild-type chondroitin synthase McCS lost almost half of its activity in less than 2 hours after being incubated at 37°C. The stability half-life (T1 / 2) of the chondroitin synthase mutant McCS-M12 at 37°C was 14.52 hours, while that of the wild-type chondroitin synthase McCS (McCS: 1.567 hours) was extended by 13 hours.

Claims

1. A chondroitin synthase mutant tMcCS, characterized in that, The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.3; The chondroitin synthase mutant tMcCS is based on chondroitin synthase McCS with 263 amino acids deleted from the C-terminus; the amino acid sequence of chondroitin synthase McCS is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. A chondroitin synthase mutant McCS-M12, characterized in that, The amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.5; The chondroitin synthase mutant McCS-M12 is the chondroitin synthase mutant tMcCS of claim 1, in which 7 amino acids are mutated; the specific mutations are: D444N, T445Y, K447Q, R480A, S602A, M641L, and I650V.

3. A recombinant vector, characterized in that, The recombinant vector is constructed by inserting the nucleotide sequence of the chondroitin synthase mutant tMcCS of claim 1 or the nucleotide sequence of the chondroitin synthase mutant McCS-M12 of claim 2 into a plasmid vector.

4. The recombinant vector as described in claim 3, characterized in that, The plasmid vector is pET28a(+).

5. A recombinant cell, characterized in that, The recombinant cells are obtained by converting the recombinant vector of claim 3 into host cells.

6. The recombinant cell as described in claim 5, characterized in that, The host cell was Escherichia coli BL21 (DE3).

7. A method for preparing chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12 by fermentation, characterized in that, The method includes culturing the host cells of claim 5 and isolating and obtaining the chondroitin synthase mutant tMcCS or the chondroitin synthase mutant McCS-M12.

8. The preparation method according to claim 7, characterized in that, The specific steps of the preparation method are as follows: recombinant host cells containing the encoding gene of the chondroitin synthase mutant are cultured in LB liquid medium until the OD600 is 0.6-0.8, and IPTG is added to induce expression for 16-18 h; the cells are collected, sonicated, centrifuged and filtered, and purified by His tag to obtain chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12.

9. The use of the chondroitin synthase mutant tMcCS according to claim 1 in the synthesis of chondroitin oligosaccharides or chondroitin polysaccharides.

10. The use of the chondroitin synthase mutant McCS-M12 according to claim 2 in the synthesis of chondroitin oligosaccharides or chondroitin polysaccharides.

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