Chondroitin synthase as well as high-expression, high-activity and high-thermal-stability mutant and application thereof

Abstract

The invention relates to chondroitin synthase as well as a high-expression, high-activity and high-thermal-stability mutant and application thereof. The amino acid sequence of the chondroitin synthase McCS is shown as SEQ ID NO.2, and the nucleotide sequence of the chondroitin synthase McCS is shown as SEQ ID NO.1; the amino acid sequence of the chondroitin synthase mutant tMcCS is as shown in SEQ ID NO.4, and the nucleotide sequence of the chondroitin synthase mutant tMcCS is as shown in SEQ ID NO.3. The amino acid sequence of the chondroitin synthase mutant McCS-M12 is as shown in SEQ ID NO.6, and the nucleotide sequence of the chondroitin synthase mutant McCS-M12 is as shown in SEQ ID NO.5. The chondroitin synthase McCS disclosed by the invention is a brand-new chondroitin synthase derived from Murrakia canis, and has the activities of two transferases, namely GalNAc-T and GlcA-T, at the same time. Compared with wild type chondroitin synthase McCS, the chondroitin synthase mutants tMcCS and McCS-M12 have higher enzymatic activity and thermal stability, the biomimetic synthesis application development of chondroitin is greatly promoted, and a brand new page is opened for research and development of glycosaminoglycans.

Description

Technical Field

[0001] The invention relates to a novel chondroitin synthase derived from Moraxella canis and a mutant thereof with high expression amount, high activity and high thermal stability and application thereof, belonging to the field of biotechnology. Background Art

[0002] Chondroitin sulfate (CS) is one of the important polysaccharides that exist in large quantities on the cell surface and in the extracellular matrix. It is a copolymer composed of a skeleton composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) and sulfate modifications at various sites. The unique sulfated monosaccharide sequence provides specificity of biological functions by regulating the affinity of polysaccharides for various chemokines and their receptors. The diverse structure of CS chains allows it to interact with many proteins, thereby participating in the regulation of many biological and pathological processes such as cell differentiation, proliferation, migration, morphogenesis, mutual recognition between cells, inflammation regulation, development, regeneration and plasticity of neural networks, etc. Chondroitin (Chn) is the polysaccharide skeleton of the capsule of some pathogens and is also the precursor of CS synthesis in vivo and in vitro in vertebrates. Its structure is: (-GlcA-1,3-GalNAc-1,4-) n .

[0003] Compared with traditional chemical synthesis, the chemoenzymatic synthesis of glycosaminoglycans has the advantages of relatively simple steps and high efficiency. The first step in the chemoenzymatic synthesis of CS is to use chondroitin synthase to synthesize and extend the Chn skeleton. 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-acetylgalactosaminyltransferase (β1-4-GalNAc-T) and β1-3-D-glucuronosyltransferase (β1-3-GlcA-T) activities located in two different catalytic domains, each of which contains a conserved UDP-sugar-binding DXD motif. The enzyme uses UDP-activated sugar nucleotides as donor substrates and catalyzes the formation of chondroitin sugar chains by alternating the addition of GalNAc and GlcA.

[0004] There are five highly homologous chondroitin synthases reported from microorganisms: PmCS isolated from Pasteurella Type F, KfoC from Escherichia coli K4, CpCS from Thiobacillus chlororaphis, ApCS from Avibacterium paragallinarum, and AuCS from Actinobacillus urea. According to previous data, these bacterial-derived chondroitin synthases show different degrees of performance limitations when synthesized enzymatically in vitro. In addition to the low expression level (KfoC only produces about 15 mg of purified enzyme per liter of E. coli culture), the length of the receptor sugar chain (PmCS recognizes the shortest chondroitin trisaccharide) and the single type of donor recognition (most ChnS only recognize UDP-GalNAc), their GlcA transferase catalytic activity is relatively low, only about one-tenth of the activity of the GalNAc transferase that exists at the same time, especially when GlcA-pNP is used as the starting receptor to synthesize oligosaccharides, the short receptor sugar chain is the low activity of GlcA transferase and the rate-limiting step of the reaction. The rate-limiting catalytic capacity makes it difficult for the initial step reaction to proceed to completion, greatly increasing the difficulty of purification and possibly resulting in impure subsequent products. Its narrow donor substrate adaptability limits its potential for application in the synthesis of non-natural CS derivatives.

[0005] Therefore, one of the current research focuses of the chemoenzymatic synthesis of chondroitin is to develop new chondroitin synthases with high expression levels and good catalytic activity, and to artificially modify the enzyme molecules through protein engineering techniques to improve the substrate adaptability of its catalytic activity. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a chondroitin synthase and a high-expression, high-activity, high-thermal stability mutant and application thereof.

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

[0008] The first aspect of 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] The second aspect of 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 obtained by deleting 263 amino acids at the C-terminus of the chondroitin synthase McCS.

[0012] The third aspect of the present invention provides a chondroitin synthase mutant McCS-M12, whose amino acid sequence 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 in which 7 amino acids are mutated; the mutations are specifically: 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: the aspartic acid at position 444 is mutated to asparagine, the threonine at position 445 is mutated to tyrosine, the lysine at position 447 is mutated to glutamine, the arginine at position 480 is mutated to alanine, the serine at position 602 is mutated to alanine, the methionine at position 641 is mutated to leucine, and the isoleucine at position 650 is mutated to valine.

[0015] The fourth aspect of the present invention provides a recombinant vector, which is constructed by inserting the nucleotide sequence of the above-mentioned 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 has no particular restrictions on the starting vector, and can be any vector known in the art, as long as it can be replicated in the 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] The fifth aspect of the present invention provides a recombinant cell, which is obtained by transforming the above-mentioned recombinant vector into a host cell. The "host cell" has the meaning generally understood in the art, and it is a host cell into which the encoding gene of the mutant of the present invention can be introduced, and after the introduction, it is called a recombinant host cell. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli E.coli BL21 (DE3).

[0018] The 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 above-mentioned host cells and isolating and obtaining chondroitin synthase and its truncations and mutants.

[0019] Preferably, according to the present invention, the specific steps of the preparation method are: culturing the recombinant host cells containing the coding genes of the enzyme and the mutant in LB liquid culture medium until OD600 is 0.6-0.8, adding IPTG to induce expression for 16-18 hours; collecting the bacteria, ultrasonically disrupting, centrifuging and filtering, and purifying through the His tag to obtain chondroitin synthase McCS, chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12.

[0020] The 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 operations not described in detail in the present invention can be performed according to conventional experimental operations in the technical field.

[0022] Beneficial Effects

[0023] 1. The chondroitin synthase McCS disclosed in the present invention is a new chondroitin synthase derived from Moraxella canis, and has two transferase activities, GalNAc-T and GlcA-T. And under the optimal conditions, the efficiency of using the substrate UDP-GalNAc is higher than that of the existing chondroitin synthase KfoC (derived from Escherichia coli K4), chondroitin synthase ApCS (derived from Avibacterium paragallinarum) and chondroitin synthase AuCS (derived from Actinobacillus urea).

[0024] Compared with the most widely used KfoC, the expression level of chondroitin synthase McCS was increased to 3.6 times that of KfoC (McCS: 55mg / L; KfoC: 15mg / L); the total GalNAc transferase activity of McCS protein per liter of culture medium was nearly 10 times higher than that of KfoC (McCS: 19.97IU / L; KfoC: 1.84IU / L); for GlcA transferase activity, the total enzyme activity of McCS was about 3 times that of KfoC (McCS: 1.95IU / L; KfoC: 0.66IU / L). Under the same reaction conditions, the ability of McCS to utilize non-natural glycosyl donors (such as UDP-GalNAz, UDP-GalNTFA, etc.) is also higher than that of other existing chondroitin synthases. In addition, McCS can use the sugar donor UDP-GalNAz to transfer GalNAz to the chondroitin sugar chain with GlcA at the non-reducing end, and synthesize non-natural chondroitin oligosaccharides with azide groups in the sugar chain structure. The azide groups in the sugar chain can quickly undergo bioorthogonal reactions (Click reactions) with fluorescent dyes or other types of dyes with alkyne groups, so that the sugar chain is labeled for easy detection, which can provide a suitable detection method for the simple and stable detection of chondroitin sulfate.

[0025] 2. Compared with the wild-type chondroitin synthase McCS, the chondroitin synthase mutant tMcCS provided by the present invention deletes the meaningless fragment at the C-terminus of the wild-type McCS protein, and a total of 263 amino acids at the C-terminus are deleted. The chondroitin synthase mutant tMcCS retains the two transferase activities of GalNAc-T and GlcA-T. Compared with the wild-type chondroitin synthase McCS, the chondroitin synthase mutant tMcCS has an increased expression level, enhanced activity, and enhanced thermal stability.

[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); 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: 21.68h, McCS: 1.56h), and its substrate tolerance was also wider.

[0027] 3. The chondroitin synthase mutant McCS-M12 provided by the present invention is a new artificial chondroitin synthase. Compared with the wild-type chondroitin synthase McCS, not only the 263 amino acid residues at the C-terminus are deleted, but also mutations occur at 7 amino acid sites (D444N, T445Y, K447Q, R480A, S602A, M641L, I650V) on this basis. Compared with the wild-type chondroitin synthase McCS, the expression level of the chondroitin synthase mutant McCS-M12 is significantly improved, the activity is significantly enhanced, and the thermal stability 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); 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 was improved. High Kcat / Km (GalNAc-T: 8.8 times, GlcA-T: 27 times); after being incubated at 37℃ for 12 hours, the chondroitin synthase mutant McCS-M12 still maintained more than 50% of its activity, while the wild-type chondroitin synthase McCS lost half of its activity in less than 2 hours at 37℃; the stability half-life (T1 / 2) of the chondroitin synthase mutant McCS-M12 at 37℃ 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, it is possible to use the chondroitin synthase mutant McCS-M12 to synthesize the chondroitin skeleton multiple times at a lower temperature, which greatly promotes the application of chondroitin biomimetic synthesis and opens a new chapter for the research and development of glycosaminoglycans. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 High performance liquid chromatography (HPLC) curve for verifying the GalNAc transferase activity of chondroitin synthase McCS;

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

[0032] Figure 3It is the mass spectrometry analysis spectrum of the GalNAc transferase activity reaction product of chondroitin synthase McCS;

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

[0034] Figure 4 High performance liquid chromatography (HPLC) curve for verification of the GlcA transferase activity of chondroitin synthase McCS;

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

[0036] Figure 5 It is the mass spectrometry analysis spectrum of the GlcA transferase activity reaction product of chondroitin synthase McCS;

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

[0038] Figure 6 The results of the basic enzymatic properties test of chondroitin synthase McCS;

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

[0040] Figure 7 The results are the comparison of the donor substrate specificity of the chondroitin synthase McCS of the present invention and the chondroitin synthase KfoC and AuCS;

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

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

[0043] In the figure, A is the search result of BLAST in the NCBI database using chondroitin synthase McCS as a template, and B is the domain prediction result of chondroitin synthase McCS.

[0044] Fig. 9 The result of conservation analysis of the amino acid sequence of chondroitin synthase McCS;

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

[0046] Fig.10Scoring results and three-dimensional structure simulation of EV Couplings virtual saturation screening for chondroitin synthase McCS;

[0047] In the figure, A is the EV Couplings virtual saturation screening scoring result. The more orange the color, the more favorable the mutation is to the activity, and the more blue the color, the less favorable the mutation is to the activity. B is the corresponding positions of the 14 highest-scoring amino acid sites on the McCS three-dimensional structure.

[0048] Fig.11 The results of the GlcA transferase activity of the first round of single-site mutation of chondroitin synthase tMcCS;

[0049] In the figure, the abscissa represents the name of the mutant, and the ordinate represents the multiple of the GlcA transferase activity of the mutant compared to the wild-type GlcA transferase activity.

[0050] Fig.12 The results of the second round of combined mutations of chondroitin synthase tMcCS for GlcA transferase activity;

[0051] In the figure, the abscissa represents the name of the mutant, and the ordinate represents the multiple of the GlcA transferase activity of the mutant compared to the wild-type GlcA transferase activity.

[0052] Fig.13 The results of the GlcA transferase activity of the third round of iterative combination mutations of chondroitin synthase tMcCS;

[0053] In the figure, the abscissa represents the name of the mutant, and the ordinate represents the multiple of the GlcA transferase activity of the mutant compared to the wild-type GlcA transferase activity.

[0054] Fig.14 is the soluble expression level of chondroitin synthase McCS and its mutants;

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

[0056] Fig.15 The reaction kinetics curve of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12;

[0057] In the figure, A is the kinetic curve of GalNAc transferase activity, and B is the kinetic curve of GlcA transferase activity; wherein the abscissa represents the concentration of the donor substrate, and the ordinate represents the degree of reaction.

[0058] Fig.16 The results are 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 of the protein per unit mass and per unit time.

[0060] Fig.17 The results are the comparison of the 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 in one liter of Escherichia coli culture system.

[0062] Fig.18 The results are the comparison of donor substrate specificity of chondroitin synthase McCS and 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 product (with the best reaction group as 100%).

[0064] Fig.19 The thermal stability curves of chondroitin synthase McCS, chondroitin synthase mutant tMcCS and chondroitin synthase mutant McCS-M12 at 37°C;

[0065] In the figure, the abscissa represents the pre-incubation time of the protein at 37° C., and the ordinate represents the relative reaction rate, which is calculated by taking the activity of the untreated protein as 100%. DETAILED DESCRIPTION

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

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

[0068] 1. The inventors discovered that an amino acid sequence formed by a gene in Moraxella canis had considerable homology with the amino acid sequence of a previously reported chondroitin synthase by searching a bioinformatics database using the BLASTp algorithm. Therefore, the inventors speculated that the protein product expressed by this gene may have chondroitin synthase activity, and named this gene McCS. The McCS gene was then used to express the protein in an Escherichia coli expression system, and the protein product was named McCS. The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0069] The heterologous expression and purification method of chondroitin synthase McCS is as follows:

[0070] Nanjing GenScript was commissioned to artificially synthesize the McCS gene according to the nucleotide sequence shown in SEQ ID NO.1, and cloned into the pET28a(+) vector, and then chemically transformed into E. coli BL21(DE3) competent cells, cultured on LB solid medium containing kanamycin (50 μg / mL) for 12 h, and screened transformants (negative control experiments were performed at the same time) to obtain positive transformants.

[0071] Pick a single colony of chondroitin synthase McCS positive transformant in 20mL sterilized LB liquid medium (containing 50μg / ml kanamycin) for activation culture (37℃, 225r / min). The overnight activated cultured bacterial solution was inoculated into 1L LB liquid medium (containing 50μg / ml kanamycin) at a 1% inoculum for expansion culture, and cultured at 37℃, 225r / min for 4 hours until OD600 was about 0.8, and IPTG with a final concentration of 0.2mM was added to induce expression at 22℃, 225r / min for 16h. The bacteria were collected by centrifugation, resuspended with 1× washing buffer, and ultrasonically broken on ice (working 3s, resting 5s, amplitude 33%, energy 1500KJ, 4℃) for 30min, and the broken bacteria were centrifuged at 12000rpm for 20min (4℃), and the supernatant was filtered with a 0.22μm filter membrane. Purification was performed using a Ni ion chromatography column, and after loading, the sample was washed with a 1× washing buffer, and finally eluted with an elution buffer containing 200 mM imidazole to obtain the target protein chondroitin synthase McCS.

[0072] The purified chondroitin synthase McCS was identified by SDS-PAGE. Figure 1 shown.

[0073] Depend on Figure 1 It can be seen that a clear band with a size of about 110 KDa appeared in the lane of chondroitin synthase McCS, indicating that the heterologous expression of chondroitin synthase McCS was successful.

[0074] According to the above method, a single colony of chondroitin synthase McCS positive transformant was cultured in 1 L of LB liquid culture medium, and then the protein concentration of chondroitin synthase McCS in the Escherichia coli culture medium was determined using a BCA protein concentration determination kit (Biyuntian P0011). The measurement results showed that the soluble expression level of chondroitin synthase McCS was 55 mg / L.

[0075] 2. Activity determination of chondroitin synthase McCS

[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 of 0.2 mM) as the acceptor substrate and UDP-GalNAc (final concentration of 0.3 mM) as the donor substrate. The reaction system is shown in Table 1;

[0078] The reaction system was reacted in a water bath at 25°C for 4 hours, and the enzyme was inactivated by boiling water for 5 minutes to terminate the reaction. The reaction solution was filtered with a 0.22 μm filter membrane and the reaction rate was detected by liquid phase. The chromatographic column used was a YMC-Pack Polyamine II column, the liquid phase system was a Shimadzu liquid phase, and the ultraviolet detection system was SPD-20A. The pNP group of the monosaccharide receptor has specific absorption at an ultraviolet 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 verification of β1-4-GalNAc transferase activity of chondroitin synthase McCS

[0080]

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

[0082]

[0083] The high performance liquid chromatography (HPLC) curve of the GalNAc transferase activity of chondroitin synthase McCS is shown in Figure 2 As shown, it indicates that chondroitin synthase McCS has GalNAc transferase activity and 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] In order to confirm that the product structure of the above active reaction is GalNAc-GlcA-pNP, electrospray ionization mass spectrometry (ESI-MS) analysis was subsequently performed. The reaction was carried out on a larger scale to obtain sufficient disaccharide products. The product was purified by a P2 column and then subjected to MS analysis on a Thermo LCQ-Deca. All samples for MS 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] The results of mass spectrometry analysis are as follows Figure 3As shown, the molecular weight measured in the MS spectrum is consistent with the calculated molecular weight of chondroitin disaccharide, proving the generation 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 of 0.2 mM) obtained in step (2) was used as an acceptor substrate, and UDP-GlcA (final concentration of 0.3 mM) was used as a donor substrate for the reaction. 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 enzyme was inactivated by heating in boiling water for 5 min to terminate the reaction. The reaction solution was filtered with a 0.22 μm filter membrane and then liquid phase detection was performed according to the method described in Table 1.

[0089] Table 3. β1-3-GlcA transferase activity verification reaction system of chondroitin synthase McCS

[0090]

[0091] The HPLC curve of the GlcA transferase activity of chondroitin synthase McCS is shown in Figure 4 As shown, a new trisaccharide peak (Trisaccharide-1) was generated in the reaction group, indicating that the chondroitin synthase McCS has GlcA transferase activity and can transfer the GlcA group to the non-reducing end of GalNAc-GlcA-pNP to generate the 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, and chondroitin trisaccharide is produced on a larger scale (more than 20 mg of product). After purification, the product trisaccharide is subjected to mass spectrometry analysis.

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

[0094] 3. Study on the enzymatic properties of chondroitin synthase McCS and its mutants

[0095] 1) Determination of the optimal reaction pH of the enzyme in vitro reaction: The reaction system is as shown in Table 1 except for the buffer pH. The Tris-HCl buffer is replaced with Tris-HCl / PBS / CH3COONa buffers of different pH values. A total of 13 pH gradient points are set, namely 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 has three groups in parallel. The other treatment conditions are the same as those in step (1) of point 2 of this example. The measurement results are shown in FIG. Figure 6 As shown in A.

[0096] 2) Determination of the optimal metal ion for the in vitro reaction of the enzyme: The reaction system except the metal ion is as shown in Table 1. 2+ Replace with the same concentration of Mg ions 2+ , Mn 2+ , Ca 2+ Three parallel experimental groups were set for each ion, and a blank control was set. The other treatment conditions were the same as 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. The reaction temperature is set at 4°C, 10°C, 20°C, 25°C, 30°C, 37°C, 45°C, and 55°C, with a total of 8 temperature gradient points. Each gradient has three parallel groups. The effect of reaction temperature on enzyme activity was determined by measuring the conversion of the substrate 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 optimum pH for chondroitin synthase McCS is 5.0 to 5.3, and the optimum metal ion is Mn 2+ , the optimum reaction temperature is 20℃.

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

[0100] In order to determine the difference in donor substrate specificity of the chondroitin synthase McCS of the present invention, commercial GlcA-pNP was used as the starting acceptor, UDP-GalNAc and 5 UDP-sugars with similar structures were used as donors (UDP-GalNAz, UDP-GalNTFA, UDP-GlcNAc, UDP-Gal and UDP-Glc), and the activity of chondroitin synthase McCS, chondroitin synthase KfoC and chondroitin synthase AuCS was detected according to the method described in Example 1, point 2. The reaction system is as described in Table 1. All reactions were incubated in a water bath at 25°C for 4 hours, and the reaction extent was detected by HPLC. All groups were subjected to 3 parallel experiments. The results are shown in Table 1. Figure 7 shown.

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

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

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

[0104] 1. Using wild-type chondroitin synthase McCS as a template sequence, search the NCBI database using the BLASTp method. Align the homologous sequences with the top 100 scores found by BLASTp. The results are as follows: Figure 8 In addition, the inventors of the present application also used InterPro to predict the active domain of wild-type chondroitin synthase McCS. The results are as follows: Figure 8 As shown in B.

[0105] Depend on Figure 8 A shows that most homologous proteins lack obvious homology in the last 263 amino acid sequence of wild-type chondroitin synthase McCS.

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

[0107] 2. Nanjing GenScript Corporation was commissioned to artificially synthesize the tMcCS gene according to the nucleotide sequence shown in SEQ ID NO.3, and then the chondroitin synthase mutant tMcCS was prepared according to the heterologous expression and purification method described in Example 1, point 1.

[0108] The purified chondroitin synthase mutant tMcCS was identified by SDS-PAGE. Figure 1 shown.

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

[0110] According to the heterologous expression method described in Example 1, point 1, a single colony of chondroitin synthase McCS positive transformant was cultured in 1 L of LB liquid culture medium, and then the protein concentration of chondroitin synthase McCS in the Escherichia coli culture medium was determined using a BCA protein concentration determination kit (Biyuntian P0011). The measurement results showed that the soluble expression level of chondroitin synthase McCS was 55 mg / L.

[0111] At the same time, the soluble expression level of chondroitin synthase KfoC was determined by the same method. The results showed that the soluble expression level of chondroitin synthase McCS was 15 mg / L.

[0112] It can be seen that 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 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), indicating that the soluble expression levels of chondroitin synthase McCS and chondroitin synthase mutant tMcCS in the Escherichia coli expression system are significantly improved, and the improvement of chondroitin synthase mutant tMcCS is greater.

[0113] Example 4: Determination of hotspots for modification of the amino acid sequence of the GlcA transferase domain of the chondroitin synthase mutant tMcCS (McCS-M1)

[0114] 1. The chondroitin synthase mutant tMcCS obtained in Example 3 was renamed as 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 homologous protein amino acid sequences is usually calculated by multiple sequence alignment (MSA) and conservation scoring methods, aiming to evaluate the evolutionary stability of amino acid sites in different species or protein families. It can reflect the importance of certain sites in the evolutionary process. Usually, highly conserved sites are crucial to the function and structure of proteins, while sites with greater variation may allow greater functional adjustments or adaptive evolution.

[0116] The homologous sequences of wild-type chondroitin synthase McCS were searched using NCBI homology, and more than 500 homologous sequences were manually selected. After homologous sequence alignment using Jalview software, the gap region was manually deleted, and the conservation of each amino acid residue was calculated (with 10 as the maximum value). The higher the value, the higher the conservation of the site. The present invention only modified the non-conservative region, so the amino acid site region with a score <8 was used as the focus of subsequent mutation research. The conservative analysis results are as follows: Fig. 9 shown.

[0117] This modification is only aimed at improving the GlcA transferase activity of the wild-type chondroitin synthase McCS, so the mutation should be carried out near the GlcA transferase domain (the Q433-567 part of the McCS amino acid sequence), and in order to ensure that the GalNAc transferase domain at the N-terminus of the protein is not affected as much as possible, the selection of the mutation site starts from the 433th amino acid. According to the protein secondary structure prediction results, the sequence after the 680th amino acid is a disordered sequence and does not participate in the formation of the active domain, so the final selection range of the amino acid mutation site of the present invention is 433-680.

[0118] Based on the information of homologous sequences ( Figure 8 ), selected amino acid residues with amino acid conservation scores <8 in the range of 433-680, totaling 43, 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 mutation sites that are beneficial to the thermal stability enhancement of the chondroitin synthase mutant McCS-M1 are calculated.

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

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

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

[0123] 1. The McCS-M1 mutant modeled by SWISS-MODEL was input into the HotSpot Wizard3.0 server, and the mutation sites for improving GlcA transferase activity were calculated and designed within the amino acid range of 433-680, specifically: 442, 443, 444, 448, 496 and 522. According to the principle of sequence conservation, sites 442, 448 and 522 were eliminated. The hot spots calculated this time 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), and the GlcA transferase domain was selected as the analysis focus. By searching the database, the server performed multiple sequence alignment and evolutionary information co-variation analysis on approximately 106,306 homologous protein sequences of the chondroitin synthase mutant McCS-M1, and gave the evolutionary dependency relationship between amino acid residues. The mutation effect of each amino acid site within the GlcA transferase domain was predicted based on the amino acid epistasis model (i.e., virtual saturation mutation of each site) and the three-dimensional structural model before and after the mutation was given, as shown in the following figure. Fig.10 shown.

[0125] According to the scoring of virtual saturation mutations at each site (the higher the score, the more favorable the mutation), 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] According to the results of virtual saturation mutation, the sites that had obvious damage to the protein after mutation in the previously selected sites were further eliminated. 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. The first round of mutation of the chondroitin synthase mutant McCS-M1

[0129] The nucleotide sequence of the chondroitin synthase mutant McCS-M1 (SEQ ID NO.3) was used as a template, and the sequences in Table 4 were used 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, the chondroitin synthase mutant McCS-M2 was prepared according to the heterologous expression and purification method described in Example 1, point 1. The GlcA transferase activity of the purified chondroitin synthase mutant McCS-M2 was then detected and compared according to the method described in Example 1, point 2. At the same time, the wild-type chondroitin synthase McCS was used as a control. The results are as follows: Fig.11 shown.

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

[0131]

[0132] PCR amplification conditions were performed according to the standard operation of the 2*Phanta Flash Master Mix (Dye Plus) instruction manual of Novizan. After PCR amplification, 4.5 μL of amplified product was added to 0.5 μL 10*Loading Buffer, and the PCR results were detected by electrophoresis using a 1% agarose gel containing Goldview. 50*TAE buffer was used after dilution, and the voltage was 150V. After electrophoresis, the gel imager was used to observe and take pictures.

[0133] Digestion and recovery of gene template: Add 2 μL of DpnI digestion template to the PCR product, gently mix the reaction solution, centrifuge it instantly, keep it at 37°C for 30 minutes, and then put it on ice to stop the reaction. Take 2 μL of DpnI-treated PCR product for gel cutting and recovery. The operation is carried out according to the standard operation of the Omega gel recovery / DNA purification kit instructions. The recovered target gene product is directly transformed into the expression host E.coli BL21 (DE3). Six transformants are selected for each mutant and activated and sequenced. After the sequencing results are correct, the mutants are strained for strain preservation and subsequent protein expression.

[0134] Table 4. Primers for the first round of mutation

[0135]

[0136]

[0137] Depend on Fig.11 It can be seen that the chondroitin synthase mutant McCS-M1 mutations predicted by the first round of mutation hit the following 15 activity-enhanced points: D444N, T445Y, K447Q, Q462E, I478L, R480A, Q484N, H508L, S510R, A533K, S602A, M641L, I650V, V659I, and Y661N.

[0138] 2. Second round of mutation of chondroitin synthase mutant McCS-M1

[0139] From the 15 single-point mutants with significantly improved activities obtained in the first aspect of this example, the sites with more obvious effects were selected and combined nearby to form 5 combined mutants, which are:

[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, the second round of mutation amplification was performed according to the 5 combined mutants selected in this step to obtain the nucleotide sequences of the chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6, respectively. Then, according to the heterologous expression and purification method described in Example 1, point 1, chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6 were prepared, respectively. Then, according to the method described in Example 1, point 2, the purified chondroitin synthase mutants McCS-M2, McCS-M3, McCS-M4, McCS-M5, and McCS-M6 were tested for GlcA transferase activity and compared, and the wild-type chondroitin synthase McCS was used as a control. The results are as follows: Fig.12 shown.

[0142] Table 5. Second round of mutation primers

[0143]

[0144] Depend on Fig.12 It can be seen that each combination has different degrees of increased activity, among which the D444N / T445Y / K447Q triple mutant (McCS-M2) is the optimal mutation combination, followed by the 641L / I650V double mutant (McCS-M5), and the other combination mutants have different degrees of iterative activity improvement compared with the single-site mutants.

[0145] 3. The third round of mutation of the chondroitin synthase mutant McCS-M1

[0146] Further iteration of the advantageous combination mutants selected from point 2 of this example, combining multiple mutants with improved catalytic activity in pairs or superimposing them with the S602A mutation site with the greatest effect on activity gain in point 1 of this example, to form the following five 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, the third round of mutation amplification was performed according to the 5 combined mutants selected in this step to obtain the nucleotide sequences of chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11, respectively. Then, according to the heterologous expression and purification method described in Example 1, point 1, chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11 were prepared, respectively. The GlcA transferase activity of the purified chondroitin synthase mutants McCS-M7, McCS-M8, McCS-M9, McCS-M10, and McCS-M11 was then tested and compared according to the method described in Example 1, point 2, while the wild-type chondroitin synthase McCS was used as a control. The results are as follows: Fig.13 shown.

[0152] Table 6. Primers for the third round of mutation

[0153]

[0154] Depend on Fig.13It can be seen that the D444N / T445Y / K447Q triple mutant (McCS-M2) and S602A iteratively produced the D444N / T445Y / K447Q / S602A quadruple mutant (McCS-M7) with improved activity compared with McCS-M2 or McCS-S602A, and the M641L / I650V double mutant (McCS-M5) and S602A iteratively produced the D444N / T445Y / K447Q / S602A quadruple mutant (McCS-M7) with improved activity compared with McCS-M2 or McCS-S602A. The M641L / I650V / S602A triple mutant (McCS-M8) with improved 602A activity was further superimposed on the mutation sites of McCS-M7 and McCS-M8, and the optimal D444N / T445Y / K447Q / S602A / M641L / I650V six mutant McCS-M10 was iterated. The GlcA transferase catalytic activity was increased to 21 times that of the wild-type McCS. After further superimposing McCS-M4 on the mutation site of McCS-M10, the effect did not increase but decreased, so McCS-M10 was finally determined to be the optimal mutant in this round.

[0155] 4. The fourth round of mutation of the chondroitin synthase mutant McCS-M1

[0156] Then, the optimal combination McCS-M10 iterated in point 3 of this example was combined with the site McCS-R480A with the greatest expression gain.

[0157] Combined with mutation site R480A alone to form

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

[0159] Combined with the active gain combination mutation M3 (M1-I478L / R480A / Q484N) where R480A is located, 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, the fourth round of mutation amplification was performed according to 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, according to the heterologous expression and purification method described in Example 1, point 1, chondroitin synthase mutants McCS-M12 and McCS-M13 were prepared, respectively.

[0162] Table 7. Primers for the fourth round of mutation

[0163]

[0164] 5. The soluble expression levels of wild-type chondroitin synthase 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 Fig.14 shown.

[0165] Depend on Fig.14 It can be seen that the soluble expression level of the chondroitin synthase mutant McCS-M12 was the highest, 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), so the chondroitin synthase mutant McCS-M12 was determined to be the optimal mutant.

[0166] The purified chondroitin synthase mutant McCS-M12 was identified by SDS-PAGE. Figure 1 shown.

[0167] Depend on Figure 1 It can be seen that a clear band with a size of about 78.9 KDa was presented 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: Analysis of Enzyme Properties of Chondroitin Synthase Mutant McCS-M12

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

[0170] The enzymatic reactions of different concentrations of UDP-GalNAc were carried out in parallel to measure the kinetic parameters of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12. The specific method is as follows:

[0171] When the concentration of the acceptor substrate GlcA-pNP / GalNAc-GlcA-pNP was 0.6mmol / L and the concentration of the donor substrate (UDP-GalNA or UDP-GlcA) varied from 0.1 to 0.5mmol / L, the curve relationship between the donor substrate concentration in each group of reactions and the amount of substrate converted by the unit amount of enzyme per unit time within a certain reaction time was determined. The curve was fitted with the Michaelis-Menten equation to calculate the kinetic constant of McCS. The kinetic curves of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12 are shown in Fig.15 shown.

[0172] The kinetic parameters of chondroitin synthase KfoC and chondroitin synthase tMcCS were determined again using the same method. The determination results 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 values ​​of kinetic constants of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12

[0174]

[0175] From Table 8 and Fig.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 was significantly improved. The GalNAc-T catalytic efficiency of the chondroitin synthase mutant McCS-M12 was increased by 8.8 times, and the GlcA-T catalytic efficiency was increased by 27 times (GalNAc-T: 8.8 times, GlcA-T: 27 times).

[0176] 2. The enzymatic 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 Example 1, point 1. The results are as follows: Fig.16 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. The unit of enzyme activity is the number of micromoles of substrate transferred per milligram of protein per minute.

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

[0179] 3. Determine the total enzyme activity of chondroitin synthase McCS, chondroitin synthase mutant tMcCS, chondroitin synthase mutant McCS-M12 and chondroitin synthase KfoC. The results are as follows: Fig.17 shown.

[0180] The specific reaction system is the same as in Example 1, the reaction time is 1 hour, and the reaction temperature is 20° C. The total enzyme activity is calculated by multiplying the enzyme activity by the protein expression level in one liter of culture system, which is the total number of micromoles of substrate that can be transferred by the purified protein per liter of culture system.

[0181] Depend on Fig.17 It can be seen that since 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; therefore, the total GalNAc-T enzyme 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); the total GlcA-T enzyme 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).

[0182] 4. The substrate specificity of chondroitin synthase McCS and chondroitin synthase mutant McCS-M12 was compared according to the method described in Example 2. The results are as follows: Fig.18 shown.

[0183] Depend on Fig.18 It can be seen that the recognition ability of the chondroitin synthase mutant McCS-M12 for the donor substrate has been further improved. Under the same conditions, the reaction degree of the chondroitin synthase mutant McCS-M12 to each recognizable substrate is higher than that of the wild type, and it recognizes UDP-Gal.

[0184] 5. Thermal stability test of chondroitin synthase and its mutants

[0185] The thermal stability test of chondroitin synthase McCS, chondroitin synthase mutant tMcCS and chondroitin synthase mutant McCS-M12 was carried out as follows: chondroitin synthase McCS, chondroitin synthase mutant tMcCS and chondroitin synthase mutant McCS-M12 were pre-incubated at 37°C for different time periods (0, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 hours), and then the GalNAc transferase activity reaction was carried out according to the method described in Example 1, point 1. The conversion rate of GlcA-pNP to GalNAc-GlcA-pNP was determined by HPLC. The stability half-life (T 1 / 2) were analyzed and calculated using GraphPad Prism 8 software. Three parallel experiments were performed in all groups. Fig.19 shown.

[0186] Depend on Fig.19 It can be seen that the chondroitin synthase mutant McCS-M12 still maintained more than 50% of its activity after being incubated at 37°C for 12 hours, while the wild-type chondroitin synthase McCS basically lost half of its activity within less than 2 hours of incubation 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 McCS, characterized in that The amino acid sequence 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 tMcCS, characterized in that: The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; The chondroitin synthase mutant tMcCS is obtained by deleting 263 amino acids at the C-terminus of the chondroitin synthase McCS.

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

4. A recombinant vector, characterized in that: The recombinant vector is constructed by inserting the nucleotide sequence of the chondroitin synthase McCS according to claim 1, the nucleotide sequence of the chondroitin synthase mutant tMcCS according to claim 2, or the nucleotide sequence of the chondroitin synthase mutant McCS-M12 according to claim 3 into a plasmid vector; More preferably, the plasmid vector is pET28a(+).

5. A recombinant cell, characterized in that The recombinant cell is obtained by transforming the recombinant vector according to claim 4 into a host cell; Further preferably, the host cell is Escherichia coli BL21 (DE3).

6. A method for preparing chondroitin synthase McCS, chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12 by fermentation, characterized in that: The method comprises culturing the host cell according to claim 5, and isolating and obtaining chondroitin synthase McCS, chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12.

7. The preparation method according to claim 6, characterized in that: The specific steps of the preparation method are: culturing the recombinant host cells containing the coding genes of the enzyme and the mutant in LB liquid culture medium until the OD600 is 0.6-0.8, adding IPTG to induce expression for 16-18 hours; collecting the bacteria, ultrasonically crushing, centrifuging and filtering, and purifying through a His tag to obtain chondroitin synthase McCS, chondroitin synthase mutant tMcCS or chondroitin synthase mutant McCS-M12.

8. Use of the chondroitin synthase McCS according to claim 1 in synthesizing chondroitin oligosaccharides or chondroitin polysaccharides.

9. Use of the chondroitin synthase mutant tMcCS according to claim 2 in synthesizing chondroitin oligosaccharides or chondroitin polysaccharides.

10. Use of the chondroitin synthase mutant McCS-M12 according to claim 3 in synthesizing chondroitin oligosaccharides or chondroitin polysaccharides.

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