Chondroitin 4-o-sulfate transferase with improved enzyme activity and stability and method for producing the same

Abstract

This invention discloses a chondroitin 4-O-sulfate transferase with enhanced enzyme activity and stability, and its production method, belonging to the field of biotechnology. This invention truncates and performs site-directed mutagenesis on mouse-derived chondroitin 4-O-sulfate transferase, increasing the enzyme activity to 6062.36 U / L, 2.4 times that of the original strain; and extending the half-life of the final mutant to 228 h at 37°C, 28.5 times that of the original strain. Further optimization of the culture medium reduces production costs while increasing the enzyme activity to 10791.08 U / L, 4.3 times that of the original strain, thus promoting the industrial production of non-animal-derived chondroitin sulfate.

Description

Technical Field

[0001] This invention relates to a chondroitin 4-O-sulfate transferase with improved enzyme activity and stability, and its production method, belonging to the field of biotechnology. Background Technology

[0002] Chondroitin sulfate (CS) is a heteropolysaccharide formed by alternating β-1,3 and β-1,4 glycosidic bonds between glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) and undergoing varying degrees of sulfonation modification. It belongs to the glycosaminoglycan family. Based on the sulfonation position of the disaccharide units, CS can be classified into five main types: CS-O [GlcA-GalNAc], CS-A [GlcA-GalNAc(4S)], CS-C [GlcA-GalNAc(6S)], CS-D [GlcA(2S)-GalNAc(6S)], and CS-E [GlcA-GalNAc(4S,6S)]. Different types of CS exhibit diverse biological functions.

[0003] Currently, chondroitin sulfate is still primarily obtained through animal tissue extraction. However, this method has many limitations, such as long breeding cycles, limited raw material supply, potential risks of cross-contamination with pathogens, and potential allergen residues. These factors restrict its widespread application in the food and pharmaceutical industries. Therefore, developing non-animal-derived CS (chondroitin sulfate) is of great significance. Mythocondro, as a non-animal-derived CS, has been approved by the U.S. Food and Drug Administration (FDA) for use as a food ingredient since 2017. Furthermore, studies have shown that microbial-derived CS has a potential protective effect in alleviating knee osteoarthritis.

[0004] Currently, chondroitin 4-O-sulfate transferase (C4ST) has been successfully produced in *Escherichia coli* and *Pichia pastoris* through genetic engineering. In the synthesis of CS, chondroitin 4-O-sulfate transferase (C4ST) is a key enzyme in the first step, converting chondroitin into chondroitin sulfate (CSA). However, the C4ST enzyme activity expressed in *Pichia pastoris* and *E. coli* remains low, and its stability is poor. This bottleneck significantly limits the industrial production of CS. Therefore, developing C4ST expression strains with high enzyme activity and high stability is of significant research value for improving CS production efficiency. Summary of the Invention

[0005] To address the aforementioned issues, this invention mutates chondroitin 4-O-sulfate transferase to obtain a mutant with high enzyme activity and high stability. Combined with culture medium optimization, this yields a chondroitin 4-O-sulfate transferase mutant with enhanced enzyme activity and stability, along with its production method. This achieves high-activity, low-cost production of the target product, providing a high-activity, high-stability, and low-cost chondroitin 4-O-sulfate transferase mutant and its production method for the industrial production of CS.

[0006] This invention provides a chondroitin 4-O-sulfate transferase mutant, which uses the chondroitin 4-O-sulfate transferase shown in SEQ ID NO.1 as the starting sequence, shortens amino acids 1 to 59, and has one or more of the following mutations:

[0007] (1) Mutate valine at position 85 to leucine;

[0008] (2) Mutate the glutamic acid at position 114 to aspartic acid;

[0009] (3) Mutate the glycine at position 297 to aspartic acid;

[0010] (4) Mutate the aspartic acid at position 312 to tyrosine;

[0011] (5) Mutate valine at position 333 to leucine.

[0012] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the first to 59 amino acids truncated, and the valine at position 85 mutated to leucine, and the glycine at position 297 mutated to aspartic acid.

[0013] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the first to 59 amino acids truncated, and the glutamic acid at position 114 mutated to aspartic acid, and the glycine at position 297 mutated to aspartic acid.

[0014] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the first to 59 amino acids truncated, and the glycine at position 297 mutated to aspartic acid, and the aspartic acid at position 312 mutated to tyrosine.

[0015] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the first to fifth amino acids truncated, and the glutamic acid at position 114 mutated to aspartic acid, the glycine at position 297 mutated to aspartic acid, and the aspartic acid at position 312 mutated to tyrosine.

[0016] In one embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO.4.

[0017] A second objective of this invention is to provide a gene encoding the chondroitin 4-O-sulfate transferase mutant.

[0018] The present invention also provides a recombinant expression vector carrying the gene.

[0019] In one embodiment, the vector includes, but is not limited to, the plasmid pPIC9K.

[0020] The present invention also provides mutants of the chondroitin 4-O-sulfate transferase or microbial cells containing the gene.

[0021] In some embodiments, the microbial cells are Pichia pastoris GS115.

[0022] In one embodiment, the mutant is expressed using Pichia pastoris GS115 as the host and plasmid pPIC9K as the expression vector.

[0023] The present invention also provides a method for improving the activity and stability of chondroitin 4-O-sulfate transferase, wherein the method involves truncating the amino acid sequence of chondroitin 4-O-sulfate transferase and mutating valine at position 85, glutamic acid at position 114, glycine at position 297, aspartic acid at position 312, and / or valine at position 333.

[0024] In one embodiment, the parent of the chondroitin 4-O-sulfate transferase has the amino acid sequence shown in SEQ ID NO.1.

[0025] In one embodiment, a solubilizing tag SUMOPro3 is also fused to the N-terminus of the chondroitin 4-O-sulfate transferase mutant; the amino acid sequence of the solubilizing tag SUMOPro3 is shown in SEQ ID NO.6.

[0026] In one embodiment, the expression of the chondroitin 4-O-sulfate transferase mutant is promoted by the fusion signal peptide OST1-α; the amino acid sequence of the fusion signal peptide OST1-α is shown in SEQ ID NO.5.

[0027] In one embodiment, the method further includes fermenting the recombinant Pichia pastoris in a fermentation medium containing glycerol, cottonseed peptone, yeast extract, phosphate, and biotin.

[0028] In one embodiment, the fermentation medium contains: 10 g / L glycerol, 20 g / L cottonseed peptone, 10 g / L yeast extract, 100 mM phosphate, and 1.0 × 10⁻⁶ ppm. -4g / L biotin.

[0029] The present invention also provides a method for producing chondroitin 4-O-sulfate transferase, characterized in that the recombinant microbial cells are cultured in a fermentation medium to prepare the chondroitin 4-O-sulfate transferase mutant.

[0030] In one embodiment, the fermentation medium uses glycerol as a carbon source and cottonseed peptone as a nitrogen source.

[0031] In one embodiment, the fermentation medium contains: 10 g / L glycerol, 20 g / L cottonseed peptone, 10 g / L yeast extract, 100 mM phosphate, and 1.0 × 10⁻⁶ ppm. -4 g / L biotin.

[0032] The present invention also provides the use of the chondroitin 4-O-sulfate transferase, or the recombinant microbial cell, or the recombinant Pichia pastoris in the preparation of chondroitin sulfate or chondroitin sulfate-containing products.

[0033] Beneficial effects:

[0034] (1) The present invention firstly increased the enzyme activity to 6062.36 U / L by site-directed mutagenesis of mouse chondroitin 4-O-sulfate transferase, which is 2.4 times that of the original strain; the mutation not only increased the enzyme activity but also improved the enzyme stability, extending the half-life of the mutant at 37°C to 228 h, which is 28.5 times that of the original strain.

[0035] (2) By optimizing the culture medium, the enzyme activity of the recombinant bacteria expressing the mutant after fermentation was increased to 10791.08 U / L, which is 4.3 times that of the starting strain. The production cost was also reduced, which promoted the industrial production of non-animal chondroitin sulfate. Attached Figure Description

[0036] Figure 1 This is an assay of the C4ST enzyme activity resulting from a single-point mutation in Example 1.

[0037] Figure 2 The activity of C4ST enzyme with combined mutations in Example 2 was measured.

[0038] Figure 3 Stability determination of C4ST enzyme in the superior mutant prepared in Example 3.

[0039] Figure 4 The results show the optimization of the carbon source for the fermentation medium in Example 4.

[0040] Figure 5 The results show the optimization of the main nitrogen source in the fermentation medium in Example 5.

[0041] Figure 6The results show the optimized biotin concentration in the fermentation medium in Example 5.

[0042] Figure 7 The results of YNB component analysis in Example 5 are shown.

[0043] Figure 8 The results show the optimization of the fermentation medium YNB in ​​Example 5. Detailed Implementation

[0044] The materials and methods involved in the embodiments of the present invention are as follows:

[0045] Pichia pastoris GS115 is used to construct recombinant microbial cells.

[0046] YPD medium (g / L): yeast extract 10, peptone 20, glucose 20.

[0047] BMGY medium (g / L): yeast extract 10 g, peptone 20 g, 100 mM K₂HPO₄-KH₂PO₄ buffer (pH 6.0), biotin 4.0 × 10⁻⁶ g / L -4 Yeast-free amino nitrogen source (YNB) 13.4, glycerol 10.

[0048] BMMY medium (g / L): yeast extract 10, peptone 20, 100mM K2HPO4-KH2PO4 buffer (pH 6.0); biotin 4.0×10 -4 Yeast without amino nitrogen source (YNB) 13.4%; Methanol 1% (v / v).

[0049] C4ST growth medium (g / L): yeast extract 10 g, cottonseed peptone 20 g, 100 mM K₂HPO₄-KH₂PO₄ buffer (pH 6.0), biotin 1.0 × 10⁻⁶ g / L -4 Glycerin 10.

[0050] C4ST induction medium (g / L): yeast extract 10 g, cottonseed peptone 20 g, 100 mM K₂HPO₄-KH₂PO₄ buffer (pH 6.0), biotin 1.0 × 10⁻⁶ g / L -4 Methanol 1% (v / v).

[0051] Methods for determining chondroitin 4-O-sulfatase:

[0052] C4ST enzyme activity was determined by measuring the absorbance of PNPs formed during the reaction at 400 nm. The reaction system consisted of 1.5 mL of PNPS, 0.5 mM PAP, 2 mg / mL AST IV, 20% (v / v) glycerol, 2 mg / mL chondroitin, and an appropriate amount of C4ST supernatant. The reaction was carried out at 37 °C for 2 h. The reaction was terminated by boiling in a water bath for 10 min. After centrifugation at 10000 × g for 10 min, the supernatant was discarded, and the absorbance at 400 nm was measured. The C4ST enzyme activity unit is defined as the amount of enzyme required to produce 1 μM PNP per hour under specific reaction conditions (37 °C).

[0053] Example 1: Preparation method of chondroitin 4-O-sulfatyltransferase site-directed mutant

[0054] Using standard PCR procedures, the primers used are shown in Table 1. Amino acids 1-59 were truncated from the sequence shown in SEQ ID NO.1, and a lysis-promoting tag SUMOPro3 was fused to the N-terminus of the sequence. The signal peptide was replaced with the fusion signal peptide OST1-α. The sequence encoding the OST1-α signal peptide shown in SEQ ID NO.7, the C4ST gene sequence shown in SEQ ID NO.2, and the SUMOPro3 sequence linked at the 5' end (shown in SEQ ID NO.8) were synthesized to construct the recombinant plasmid pPIC9K-OST1-α-SUMOPro3-C4ST. Using the recombinant plasmid pPIC9K-OST1-α-SUMOPro3-C4ST as a template, the following mutations were performed: S72L, T74M, T74W, V85L, T86W, N92W, N92M, M95Y, E114D, L134V, S139T, T163F, S178I, E238P, E253Y, Q291W, S296I, G297D, D312Y, V333L, and N347S. Unless otherwise specified, amino acid sites are described based on the positions of amino acids in the sequence shown in SEQ ID NO.1. Taking G297D as an example, using the starting plasmid pPIC9K-C4ST as a template, circular PCR was performed using primers G297D-F / G297D-R. This was then transformed into E. coli JM109 competent cells. After successful sequencing, the recombinant plasmid pPIC9K-C4ST-G297D was obtained. The successfully sequenced plasmid was linearized, recovered by column chromatography, and introduced into Pichia pastoris GS115 using electroporation. The resulting recombinant strain was inoculated into YPD medium and cultured at 30°C for 16-20 h. A 10% inoculum was transferred to 50 mL of BMGY medium and cultured in a 250 mL Erlenmeyer flask at 30°C for 24 h. The cells were collected, centrifuged at 5000 rpm for 5 min, resuspended three times with 0.9% sodium chloride, and centrifuged again to obtain the cells. The cells were then transferred to 50 mL of BMMY medium and induced with 1% methanol for 96 h. After cultivation, the fermentation broth was centrifuged at 4℃ and 6000 rpm for 10 min, and the supernatant was collected to obtain the crude extracellular enzyme. Using the truncated C4ST△59 as a control (WT), enzyme activity was measured at 37℃, and the results are as follows. Figure 1 As shown, the enzyme activities of mutants expressing V85L, E114D, G297D, D312Y and V333L were higher than those of the original strain (2508.3 U / L), with mutant G297D showing the most significant effect, with an enzyme activity of 3262.23 U / L.

[0055] Table 1 Primer sequences for introducing mutations

[0056]

[0057]

[0058] Example 2: Preparation method of double mutant of chondroitin 4-O-sulfatyltransferase

[0059] The PCR procedure was the same as in Example 1. Using the plasmid pPIC9K-C4ST-G297D containing the mutant G297D constructed in Example 1 as a template, double mutants with other site mutations were constructed based on G297D. Using the corresponding primers in Table 1, mutations of V85L, E114D, D312Y, and V333L were introduced to obtain corresponding double mutant plasmids such as pPIC9K-C4ST-G297D / V85L, pPIC9K-C4ST-G297D / E114D, pPIC9K-C4ST-G297D / D312Y, and pPIC9K-C4ST-G297D / V333L. These were then transformed and shake-flask fermented according to the procedure in Example 1, and the enzyme activity was detected after fermentation.

[0060] like Figure 2 As shown, the enzyme activity of the double mutant G297D / E114D can reach 5259.69 U / L, which is 2.1 times that of the original strain.

[0061] Example 3: Preparation method of chondroitin 4-O-sulfatyltransferase triple mutant

[0062] Using the recombinant plasmid pPIC9K-C4ST-G297D / E114D containing the mutant G297D / E114D constructed in Example 2 as a template, triple mutations of V85L and D312Y were introduced using primer pairs V85L-F / V85L-R and D312Y-F / D312Y-R, respectively. Transformation and shake-flask fermentation were performed according to the procedure in Example 1, and enzyme activity was detected after fermentation.

[0063] like Figure 2 As shown, the enzyme activity of the mutant G297D / E114D / D312Y can reach 6062.36 U / L, which is 2.4 times that of the original strain.

[0064] Example 4: Stability analysis of chondroitin 4-O-sulfatyltransferase and mutants

[0065] The extracellular crude enzymes prepared in Examples 1-3 were passed through a membrane and then the recombinant protein was purified using a Ni column. Buffer A: 20 mM PB buffer, 500 mM NaCl, pH 7.4; Buffer B: 20 ​​mM PB buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4. First, the pre-packed HisTrap HP column (GE Healthcare) was equilibrated with buffer A. Then, the sample was loaded and passed through the membrane. The column was washed with buffer A, followed by gradient elution at 10% of buffer B to remove impurities. The target protein was eluted at 40% of buffer B. Then, molecular sieve purification was performed using a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare). The gel chromatography column was equilibrated with buffer C (20 mM PB buffer, 150 mM NaCl, pH 7.4). After loading the sample, elution was performed with buffer C. The elution process was monitored at 280 nm, and the target sample was collected. The results showed (…). Figure 3 The half-life of the mutant protein G297D / E114D / D312Y at 37°C was extended from 8 hours to 228 hours compared to the parent strain, which is 28.5 times that of the original strain. The half-lives of mutant proteins G297D / E114D and G297D at 37°C were also increased, being 22.5 times and 4.5 times that of the original strain, respectively.

[0066] Example 5: Optimizing the culture medium to improve enzyme production capacity

[0067] To further improve the expression level of chondroitin 4-O-sulfate transferase and reduce production costs, single-factor optimization of the culture medium was performed based on BMGY / BMMY:

[0068] (1) Optimization of carbon source: Keeping other conditions of BMGY unchanged, fermentation culture was carried out using glucose, glycerol, sorbitol, mannitol and sucrose at a concentration of 10 g / L, respectively, and the growth status and enzyme activity of the strain were measured.

[0069] (2) Optimization of main nitrogen sources: Keeping other conditions of BMGY and BMMY unchanged, the types of peptones were adjusted to 20 g / L urea, fish peptone, cottonseed peptone, plant peptone, beef peptone, FP108 yeast peptone, FP103 yeast peptone, FP318 trypsin peptone, FP400 soybean peptone, and FP220 wheat peptone to explore the effects of different nitrogen sources on the growth status and enzyme activity of the strains;

[0070] (3) Biotin concentration optimization: Keeping other conditions of BMGY and BMMY unchanged, adjust the biotin concentration to 8×10 -4 4×10 -4 2×10-4 The study investigated the effects of different concentrations of biotin on the growth and enzyme activity of the strain.

[0071] (4) YNB optimization: Keeping other conditions of BMGY and BMMY unchanged, the YNB components were gradually reduced to 13.4 g / L core pentasalt (containing 10 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.2 g / L sodium chloride and 0.2 g / L calcium chloride), 10 g / L ammonium sulfate, 5 g / L ammonium sulfate and no YNB added, to explore the effect of YNB on the growth status and enzyme activity of the strain.

[0072] Enzyme activity assays of the fermentation supernatant showed that using 10 g / L glycerol, 20 g / L cottonseed peptone, 10 g / L yeast extract, 100 mM phosphate, and 1.0 × 10⁻⁶ enzymes was optimal. -4 When biotin is used as a component of the culture medium for fermentation, the C4ST enzyme activity in the supernatant can reach 10791.08 U / L, which is 1.78 times that of BMGY / BMMY.

[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A chondroitin 4-O-sulfotransferase mutant characterized in that, Starting with the chondroitin 4-O-sulfatyltransferase sequence shown in SEQ ID NO.1, amino acids 1 to 59 were truncated, and one or more of the following mutations were incorporated: (1) Mutate valine at position 85 to leucine; (2) Mutate the glutamic acid at position 114 to aspartic acid; (3) Mutate the glycine at position 297 to aspartic acid; (4) Mutate the aspartic acid at position 312 to tyrosine; (5) Mutate valine at position 333 to leucine.

2. The chondroitin 4-O-sulfatase mutant according to claim 1, characterized in that, Based on the sequence shown in SEQ ID NO.1, amino acids 1 to 59 were truncated, and glutamic acid at position 114 was mutated to aspartic acid, glycine at position 297 was mutated to aspartic acid, and aspartic acid at position 312 was mutated to tyrosine.

3. A gene encoding the chondroitin 4-O-sulfate transferase mutant of claim 1 or 2.

4. Microbial cells expressing the chondroitin 4-O-sulfate transferase mutant of claim 1 or 2 or containing the gene of claim 3.

5. A recombinant Pichia pastoris, characterized in that, Using Pichia pastoris GS115 as the host and plasmid pPIC9K as the expression vector, the chondroitin 4-O-sulfate transferase mutant as described in claim 1 or 2 was expressed.

6. A method for improving the activity and / or stability of chondroitin 4-O-sulfate transferase, characterized in that, The parental amino acid sequence of chondroitin 4-O-sulfate transferase shown in SEQ ID NO.1 was truncated, and mutations were made in valine at position 85, glutamic acid at position 114, glycine at position 297, aspartic acid at position 312, and / or valine at position 333.

7. The method according to claim 6, characterized in that, The solubilizing tag SUMOPro3 is also fused to the N-terminus; the amino acid sequence of the solubilizing tag SUMOPro3 is shown in SEQ ID NO.

6.

8. A method for preparing chondroitin 4-O-sulfate transferase, characterized in that, The recombinant microbial cells of claim 4 or the recombinant Pichia pastoris of claim 5 are cultured in a fermentation medium.

9. The method according to claim 8, characterized in that, The fermentation medium contains glycerol, cottonseed peptone, yeast extract, phosphate, and biotin.

10. The use of the chondroitin 4-O-sulfate transferase of claim 1 or 2, or the recombinant microbial cell of claim 4, or the recombinant Pichia pastoris of claim 5, or the method of any one of claims 6 to 9 in the preparation of chondroitin sulfate or chondroitin sulfate-containing products.

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