Specific hyaluronate lyase and its encoding gene and use

Abstract

The present application relates to a specific hyaluronate lyase and its coding gene and application, and belongs to the technical field of biology and enzyme engineering. The specific hyaluronate lyase sHAase is an amino acid sequence as shown in SEQ ID NO. 1, or an amino acid sequence with homology of > 90% to the amino acid sequence shown in SEQ ID NO. 1, or an amino acid sequence formed by modifying, substituting, deleting or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO. 1, and still having hyaluronic acid degradation activity. The present application also provides the coding gene, preparation method and use of the specific hyaluronate lyase sHAase. The hyaluronate lyase has very high substrate selectivity for hyaluronic acid, does not act on other glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, heparin or heparan sulfate, and can cleave hyaluronic acid into degradation products mainly composed of unsaturated disaccharides, which is conducive to detection and separation.

Description

Technical Field

[0001] This invention relates to a specific hyaluronic acid lyase, its encoding gene, and its applications, belonging to the fields of biological and enzyme engineering technology. More specifically, it relates to the gene sequence of a specific hyaluronic acid lyase, a recombinant expression vector, and a recombinant genetically engineered strain containing the vector, as well as the applications of the said hyaluronic acid lyase in the fields of glycosaminoglycan quality control, hyaluronic acid oligosaccharide preparation, food, cosmetics, medicine, and scientific research. Background Technology

[0002] Glycosaminoglycans (GAGs) are a class of linear, negatively charged polysaccharide biomolecules. Based on the composition of their repeating disaccharide units and their sulfation characteristics, they are generally divided into four families: chondroitin sulfate (CS) / dermatan sulfate (DS), heparin / heparan sulfate (HS), keratan sulfate (KS), and hyaluronic acid (HA).

[0003] Among the glycosaminoglycans mentioned above, chondroitin sulfate has been widely used globally as a dietary supplement and functional ingredient due to its potential benefits in joint health-related applications. Furthermore, chondroitin sulfate, heparin, and other GAGs are important active ingredients in various pharmaceutical preparations, and high-purity analytical-grade GAGs are frequently used as standards in biomedical and life science research. Currently, the raw materials for the industrial production of GAGs mainly come from the cartilage or connective tissue of terrestrial animals such as cattle, pigs, and chickens, as well as some marine organisms. The sources of these raw materials are complex, and co-extraction of tissues is common.

[0004] Due to the significant differences in raw material sources and the complexity of extraction and purification processes, GAGs products face severe quality control challenges during production. Existing research indicates that various glycosaminoglycan impurities may coexist in animal-derived chondroitin sulfate products, which directly affects product safety, efficacy, and batch consistency.

[0005] Impurities are a prominent issue in the preparation of pharmaceutical-grade and analytical-grade chondroitin sulfate. Hyaluronic acid, due to its co-extraction with cartilage and connective tissue, is one of the most common impurities in chondroitin sulfate preparations. Hyaluronic acid is composed of alternating D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), and its main chain structure shares some similarities with chondroitin sulfate. This structural similarity makes it difficult to effectively distinguish and completely separate the two using conventional physical or chemical separation methods. Therefore, detecting and removing hyaluronic acid impurities from chondroitin sulfate products is crucial for verifying product purity and authenticity, especially in pharmaceutical formulations and research reagent applications where extremely high purity is required.

[0006] Currently, methods for the analysis and identification of glycosaminoglycans mainly include spectroscopic analysis, chromatographic analysis, and nuclear magnetic resonance analysis. However, these methods often lack sufficient specificity in distinguishing hyaluronic acid from chondroitin sulfate matrix, or rely on complex and costly precision instruments, which hinders their widespread application in industrial production and routine quality control. In contrast, enzyme-based treatment methods, due to their high substrate specificity, mild reaction conditions, and strong controllability, show significant advantages in the detection and removal of hyaluronic acid.

[0007] Selective enzymatic degradation can break down hyaluronic acid into smaller oligosaccharide fragments, significantly simplifying subsequent separation and removal processes. When combined with conventional separation techniques such as ethanol precipitation, gel filtration chromatography, or ultrafiltration, the removal efficiency of hyaluronic acid impurities in chondroitin sulfate products can be further improved.

[0008] However, existing hyaluronidases still have significant limitations in substrate specificity. Studies have shown that many human and animal-derived hyaluronidases, while degrading hyaluronic acid, also degrade related glycosaminoglycan substrates such as chondroitin sulfate, making it difficult to achieve specific treatment of hyaluronic acid. Furthermore, while some microbial hyaluronidases exhibit relatively high hyaluronic acid specificity, their final degradation products are usually tetrasaccharides, hexasaccharides, or oligosaccharides with larger molecular weights, rather than the smallest disaccharide units.

[0009] For example, studies have reported that hyaluronidase derived from Streptomyces can degrade hyaluronic acid into oligosaccharide products such as unsaturated tetrasaccharides and hexasaccharides; hyaluronidase derived from leeches also exhibits high selectivity for hyaluronic acid, but its main degradation products are still oligosaccharides rather than disaccharides. Due to the heterogeneity of the degradation end products, existing technologies not only increase the complexity of quantitative analysis of hyaluronic acid, but also limit the possibility of achieving complete removal of hyaluronic acid through conventional separation methods.

[0010] Therefore, current technologies still lack an enzyme tool that possesses both high substrate specificity for hyaluronic acid and the ability to uniformly and completely degrade hyaluronic acid into its smallest structural unit—disaccharides. Such an enzyme tool is crucial for the accurate quantitative detection of trace amounts of hyaluronic acid impurities in glycosaminoglycan products and for their efficient removal through conventional processes. It is a key prerequisite for ensuring quality control of pharmaceutical-grade and analytical-grade glycosaminoglycan products. Summary of the Invention

[0011] To address the problems of insufficient substrate specificity, uneven degradation products, and difficulty in meeting the high-purity quality control and related pharmaceutical applications requirements of existing hyaluronic acid lyase technologies, this invention aims to provide a specific hyaluronic acid lyase, its encoding gene, and its applications. This hyaluronic acid lyase has a novel source, high substrate specificity, and can uniformly degrade hyaluronic acid into disaccharides. It can be applied in glycosaminoglycan quality control, hyaluronic acid oligosaccharide preparation, food, cosmetics, pharmaceuticals, and scientific research.

[0012] The technical solution of this invention is:

[0013] A specific hyaluronic acid lysin sHAase, wherein the specific hyaluronic acid lysin sHAase is any one of the amino acid sequences shown in 1), 2), or 3):

[0014] 1) The amino acid sequence as shown in SEQ ID NO.1;

[0015] 2) An amino acid sequence that has ≥90% homology with the amino acid sequence shown in SEQ ID NO.1;

[0016] 3) An amino acid sequence formed by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.1, and still having hyaluronic acid degradation activity.

[0017] According to a preferred embodiment of the present invention, the specific hyaluronic acid lyase sHAase is an endonuclease capable of selectively cleaving hyaluronic acid and exhibits optimal activity at pH 6.0 and 50°C.

[0018] A gene encoding a specific hyaluronic acid lysin sHAase, wherein the nucleotide sequence of the gene is any one of the gene encoding shown in (1), (2), or (3):

[0019] (1) The nucleotide sequence as shown in SEQ ID NO.2;

[0020] (2) The nucleotide sequence encoding the amino acid sequence of the above-mentioned specific hyaluronic acid lysin sHAase;

[0021] (3) A nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0022] According to a preferred embodiment of the present invention, the gene encoding the specific hyaluronic acid lysin sHAase is derived from the mouse intestinal metagenomics.

[0023] A recombinant expression vector into which the gene encoding the aforementioned specific hyaluronic acid lysin sHAase is inserted.

[0024] According to a preferred embodiment of the present invention, the expression vector is selected from Escherichia coli expression vectors, yeast expression vectors, Bacillus subtilis expression vectors, lactic acid bacteria expression vectors, Streptomyces expression vectors, bacteriophage vectors, filamentous fungal expression vectors, plant expression vectors, insect expression vectors, or mammalian cell expression vectors.

[0025] A recombinant bacterium or transgenic cell line in which the encoding gene for the aforementioned specific hyaluronic acid lysin sHAase is inserted into the host bacterium or cell line.

[0026] According to a preferred embodiment of the present invention, the host bacteria or cell line is selected from Escherichia coli host cells, yeast host cells, Bacillus subtilis host cells, lactic acid bacteria host cells, actinomycete host cells, filamentous fungal host cells, insect cells, or mammalian cells.

[0027] The present invention also provides a method for preparing the above-mentioned specific hyaluronic acid lysin sHAase, comprising: cloning the gene encoding hyaluronic acid lysin sHAase into an expression vector to obtain a recombinant expression vector; and then introducing the recombinant expression vector into a host cell for expression, thereby obtaining the recombinant hyaluronic acid lysin sHAase.

[0028] The application of the aforementioned specific hyaluronic acid lysin sHAase in the preparation of oligomeric hyaluronic acid salts.

[0029] The application of the aforementioned specific hyaluronic acid lysin sHAase in the detection, removal, or quantitative identification of hyaluronic acid impurities in glycosaminoglycan products.

[0030] The application of the aforementioned specific hyaluronic acid lysin sHAase in the preparation of food, medical aesthetic repair products, or drugs.

[0031] According to a preferred embodiment of the present invention, the drug uses a specific hyaluronic acid lysin sHAase as a drug diffusion factor to enhance the diffusion and absorption of the drug in tissues by degrading hyaluronic acid in the extracellular matrix; or it regulates the content of hyaluronic acid in the microenvironment to promote drug penetration and assist in the treatment of anti-tumor and anti-fibrotic related diseases.

[0032] Beneficial effects:

[0033] 1. The hyaluronic acid lyase sHAase provided in this invention is derived from the mouse intestine and belongs to the endopeptidase type, capable of selectively cleaving hyaluronic acid. Compared with existing technologies, this hyaluronic acid lyase exhibits extremely high substrate selectivity for hyaluronic acid and essentially does not act on other glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, heparin, or heparan sulfate. It can cleave hyaluronic acid into degradation products mainly composed of unsaturated disaccharides, which is beneficial for detection and separation.

[0034] 2. By utilizing the substrate selectivity of the hyaluronic acid lysin sHAase provided by this invention, and combining it with gel filtration chromatography or conventional experimental methods, hyaluronic acid impurities in glycosaminoglycan products such as chondroitin sulfate and heparin can be effectively detected or quantitatively identified, and hyaluronic acid impurities in glycosaminoglycan products such as chondroitin sulfate and heparin can be removed.

[0035] 3. The hyaluronic acid lysin (sHAase) provided by this invention can be used in the preparation of food, medical aesthetic repair tools, or pharmaceuticals. For example, sHAase can be used as a drug diffusion factor to improve drug diffusion and absorption in tissues by degrading hyaluronic acid in the extracellular matrix; or it can be used to treat adverse reactions related to hyaluronic acid fillers in the field of medical aesthetic repair; or it can be used as an adjunct therapy for anti-tumor treatment by regulating the content of hyaluronic acid in the tumor microenvironment to promote drug penetration; or it can be used as an adjunct therapy for anti-fibrosis-related diseases. Attached Figure Description

[0036] Figure 1 This is a three-dimensional protein structural model of hyaluronic acid lysin sHAase.

[0037] Figure 2 Polyacrylamide gel electrophoresis pattern showing the expression and purification of hyaluronic acid lysin sHAase;

[0038] In the figure, lane 1 represents the protein molecular weight standard, with bands ranging from 180 kD, 130 kD, 100 kD, 70 kD, 55 kD, 40 kD, 35 kD, 25 kD, 15 kD, and 10 kD from top to bottom; lane 2 represents the control strain culture before cell wall disruption, with a loading volume of 10 µL; lane 3 represents the recombinant strain culture after cell wall disruption, with a loading volume of 10 µL; lane 4 represents the supernatant of the recombinant strain after cell wall disruption, with a loading volume of 10 µL; and lane 5 represents the protein purified by nickel column chromatography, with a loading volume of 10 µL.

[0039] Figure 3 The curve shows the effect of temperature on the activity of hyaluronic acid lysin sHAase.

[0040] Figure 4The curve shows the effect of pH on the activity of hyaluronic acid lysin sHAase.

[0041] Figure 5 The curve shows the effect of temperature on the stability of hyaluronic acid lysin sHAase.

[0042] Figure 6 The bar graph shows the effects of metal ions and chemical reagents on the activity of hyaluronic acid lysin sHAase.

[0043] Figure 7 High-performance liquid chromatography (HPLC) analysis of degradation products of different substrates by hyaluronic acid lysin sHAase;

[0044] In the figure, A is the HPLC of HA degradation products, B is the HPLC of CS-A degradation products, C is the HPLC of CS-C degradation products, D is the HPLC of CS-D degradation products, E is the HPLC of CS-E degradation products, and F is the HPLC of DS degradation products; where 1 represents unsaturated hyaluronic acid disaccharide.

[0045] Figure 8 HPLC analysis of unsaturated chondroitin tetrasaccharide and unsaturated hyaluronic acid tetrasaccharide by hyaluronic acid lysase sHAase;

[0046] In the figure, A represents the degradation results of unsaturated chondroitin tetrasaccharide; B represents the degradation results of unsaturated hyaluronic acid tetrasaccharide; among them, 1 represents unsaturated hyaluronic acid disaccharide; 2 represents unsaturated hyaluronic acid tetrasaccharide; and 3 represents unsaturated chondroitin (Ch) tetrasaccharide.

[0047] Figure 9 HPLC analysis of chondroitin after degradation and purification by hyaluronic acid lysin sHAase and positive control CSase ABC;

[0048] In the diagram, 1 represents unsaturated chondroitin disaccharide. Detailed Implementation

[0049] The following embodiments are provided to fully disclose some common techniques for implementing the present invention, and not to limit the scope of application of the invention. The inventors have made every effort to ensure the accuracy of the parameters (e.g., quantity, temperature, etc.) in the embodiments; however, some experimental errors and deviations should also be taken into account. Unless otherwise stated, molecular weight in the present invention refers to average molecular weight, and temperature refers to degrees Celsius.

[0050] Kunming mice (KM mice) were fed a 1% CS-A solution for an extended period, and their feces were collected as samples.

[0051] Example 1: Preparation of fecal genome from Kunming mice

[0052] Total genomic DNA was extracted from feces of Kunming mice using the EZNA® Soil DNA Kit (Omega Bio-tek, Norcross, GA, US).

[0053] The specific method is as follows: First, add 500mg of magnetic beads, 0.5g of Kunming mouse (KM mouse) fecal sample, and 1mL of SLX-MLus Buffer to a 2mL centrifuge tube, and vortex at 45Hz for 250s; add 100µL of DS Buffer, and mix by inversion; incubate at 70℃ for 10min, then at 95℃ for 2min; centrifuge at 13000rpm for 5min at room temperature; transfer 800µL of supernatant to a new 2mL centrifuge tube, add 270µL of P2 Buffer and 100µL of HTR Reagent; mix by inversion, and incubate at -20℃ for 5~10min; centrifuge at 13000rpm for 5min at room temperature; transfer the supernatant to a new 2mL centrifuge tube, add an equal volume of XP5 Buffer and 40µL of magnetic beads, and mix by inversion for 8min; use a magnetic rack to absorb the residue, discard the residue, remove the tube, and add 500µL of XP5. Buffer, mix well; magnetic rack adsorption, discard residual liquid, remove tube, add 600µL of poly-β-hydroxybutyrate (PHB), mix well; magnetic rack adsorption, discard residual liquid, remove tube, add 600µL of SPW Wash Buffer, mix well; magnetic rack adsorption, discard residual liquid, remove tube, add 600µL of SPW Wash Buffer, mix well; magnetic rack adsorption, discard residual liquid, centrifuge at 13000rpm for 10s at room temperature; magnetic rack adsorption, discard residual liquid with pipette, let stand at room temperature for 8min; add 100µL of Elution Buffer, mix well, let stand for 5min; magnetic rack adsorption, transfer supernatant to a new 1.5mL centrifuge tube to obtain total DNA from Kunming mouse feces. The concentration and purity of the extracted DNA were determined using a TBS-380 and NanoDrop2000 centrifuge, respectively.

[0054] Example 2: Prediction and Sequence Analysis of the Hyaluronic Acid Lysase sHAase Gene

[0055] The total DNA from the feces of Kunming mice obtained in Example 1 was fragmented using a Covaris M220, with each fragment being approximately 400 bp. Next, a PE library was constructed using the NEXTFLEX™ Rapid DNA-Seq Kit, and finally, bridge PCR and sequencing were performed using NovaSeqReagent Kits / HiSeq X Reagent Kits.

[0056] After sequencing, the raw sequencing data was first quality controlled using FastP (https: / / github.com / OpenGene / fastp). This involved splitting the raw sequences, quality trimming, and removing contaminants to obtain high-quality control data, ensuring the accuracy of subsequent analyses. Then, Megahit (https: / / github.com / voutcn / megahit) software was used to assemble sequences at different sequencing depths. MetaGene (http: / / metagene.cb.ku-tokyo.ac.jp / ) was used to predict the ORF of contigs in the assembly results, selecting genes with a nucleic acid length greater than or equal to 400 bp and translating them into amino acid sequences. The corresponding tool for the CAZy database, hmmscan (http: / / hmmer.janelia.org / search / hmmscan), was used to align the amino acid sequences of the non-redundant gene set with the CAZy database (alignment parameters were set with an expected e-value of 1e-5) to obtain the carbohydrate active enzyme annotation information corresponding to the genes, denoted as sHAase. Based on the CAZy annotation, it is speculated that this sHAase is chondroitin sulfate AC enzyme (CSase AC), which can selectively degrade chondroitin sulfate and hyaluronic acid.

[0057] NCBI analysis showed that the coding region of the sHAase gene is 2049 bp long, and its nucleotide sequence is shown in SEQ ID NO.1. The GC content is 57.8%, encoding 682 amino acids. The theoretical molecular weight of the sHAase protein is 76.10 kDa, and the pI is 6.30. Further online analysis using BLAST software showed that hyaluronic acid lyase sHAase has the highest similarity (17.92%) to hyaluronic acid lyase (AHB61202.1) from Bacillus sp. A50, and therefore it was named hyaluronic acid lyase sHAase.

[0058] BLAST software was used to analyze the conserved domains of hyaluronic acid lyase (sHAase). The results showed that amino acids 347-583 of sHAase belong to the polysaccharide lyase 8 (PL8) family. Homology modeling of the sHAase protein structure was then performed using the SWISS-MODEL homology modeling server (http: / / swissmodel.expasy.org), resulting in the final 3D protein structure model of sHAase, as shown below. Figure 1 As shown.

[0059] Example 3: Heterologous expression of hyaluronic acid lyase sHAase

[0060] 1. Using the total DNA from the feces of Kunming mice obtained in Example 1 as a template, PCR amplification was performed to obtain the target gene hyaluronic acid lyase sHAase.

[0061] The primers are as follows:

[0062] sHAase-F: 5'-ggagatatacatatgACACTCGAAGATGTCCGAAG-3';

[0063] sHAase-R: 5'-gtggtggtgctcgagAAACTGTACTTTTACACTTG-3'.

[0064] PrimeSTAR™ HSDNA polymerase was purchased from Takara Bio Inc. The PCR reaction system was operated according to the product instructions provided by the company. PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 60℃ annealing for 30 s, 72℃ extension for 3 min, 35 cycles; 72℃ extension for 10 min, 4℃ stabilization for 15 min.

[0065] The PCR product (hyaluronic acid lyase sHAase of the target gene) was ligated to the pET-30a expression vector, which had been double-digested with NdeI and XhoI, using the homologous recombinase Exnase II in a 37°C water bath for 30 minutes. The ligation product was transformed into Escherichia coli DH5α strain and plated onto Luria-Bertani solid medium containing 50 µg / mL kanamycin. After incubation at 37°C for 12–14 h, single clones were picked and cultured in Luria-Bertani liquid medium containing 50 µg / mL kanamycin at 37°C and 200 rpm for 12–14 h. The plasmid was then extracted. The plasmid was verified by bacterial PCR using forward and reverse primers. The results showed that the amplified product was of the correct size, preliminarily proving that the constructed recombinant plasmid was correct. 20 µL of the recombinant plasmid was sent to Sangon Biotech for sequencing.

[0066] Sequencing results showed that the hyaluronic acid lyase gene sHAase (as shown in SEQ ID NO.1) fragment was successfully inserted between the restriction sites NdeI and XhoI of the pET-30a expression vector. The insertion direction was correct, and no base mutations, deletions, or additions occurred. Therefore, it was further confirmed that the constructed recombinant plasmid was correct, and the recombinant expression vector was named pET30a-sHAase.

[0067] The ClonExpress II One Step Cloning kit was purchased from Novizan Biotechnology, the restriction endonucleases NdeI, XhoI, and PrimeSTAR™ HS DNA polymerase were purchased from Takara Bio, and the pET-30a vector was purchased from Novagen Biotechnology. The enzyme-substrate reaction system, reaction temperature, and reaction time all followed the instructions for use.

[0068] 2. The recombinant plasmid pET30a-sHAase was transformed into *E. coli* BL21(DE3) (purchased from Novagen, USA) to obtain recombinant bacteria. The recombinant bacteria were then cultured according to the company's instructions, and the expression of the recombinant hyaluronic acid lysin pET30a-sHAase was induced. The target protein was purified using a Ni Sepharose 6 Fast Flow (GE) gel, with *E. coli* BL21(DE3) as a control. The following were obtained: pre-cell wall disruption culture of the control strain, post-cell wall disruption culture of the recombinant bacteria, supernatant of the recombinant bacteria after cell wall disruption, and protein purified by nickel column chromatography.

[0069] Finally, polyacrylamide gel electrophoresis was used to detect the bacterial culture of the control strain before cell disruption, the bacterial culture of the recombinant strain after cell disruption, the supernatant of the recombinant strain after cell disruption, and the protein purified by nickel column chromatography. The results are as follows: Figure 2 As shown.

[0070] Depend on Figure 2 It can be seen that the purified hyaluronic acid lyase sHAase appears as a single band on the electrophoresis gel, and the position matches the predicted molecular weight, with a purity of 95%.

[0071] Example 4: Analysis of the basic enzymatic properties of hyaluronic acid lyase sHAase

[0072] 1. The effect of temperature on enzyme activity

[0073] First, 3 mg / mL HA, 150 mM pH 7.0 NaH2PO4-Na2HPO4 buffer, hyaluronic acid lysin (sHAase) enzyme solution, and deionized water were mixed in a volume ratio of 10:10:3:7. The mixture was then reacted at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 20 min. After the reaction, enzyme activity was determined by UV spectrophotometry. Optimal enzyme activity was defined as 100% relative activity. The results are shown below. Figure 3 As shown.

[0074] Depend on Figure 3It is known that hyaluronic acid lyase sHAase reaches its maximum activity against HA at 50℃, indicating that the optimal temperature for hyaluronic acid lyase sHAase to work against HA is 50℃.

[0075] The method for determining enzyme activity using ultraviolet light, referencing existing technologies, uses inactivated enzymes as a negative control. It employs a spectrophotometer to measure the 232nm light absorption of the reaction product to determine the product yield and thus the enzyme activity.

[0076] 2. The effect of pH on enzyme activity

[0077] Mix 3 mg / mL HA, 150 mM buffer, hyaluronic acid lysin (sHAase) enzyme solution, and deionized water in a ratio of 10:10:3:7 (volume ratio). The reaction buffer consisted of 150 mM NaAc-HAc (pH=5.0-6.0), 150 mM NaH2PO4-Na2HPO4 (pH=6.0-8.0), and 150 mM Tris-HCl (pH=7.0-10.0). React at the optimal temperature for 20 minutes. After the reaction, determine the enzyme activity using UV spectrophotometry. Optimal enzyme activity was defined as 100% relative activity. The results are shown below. Figure 4 As shown.

[0078] Depend on Figure 4 It can be seen that the hyaluronic acid lysin sHAase exhibits the highest enzyme activity for HA in NaH2PO4-Na2HPO4 (pH=6.0) buffer.

[0079] 3. The effect of temperature on enzyme stability

[0080] Hyaluronic acid lysin sHAase, after heat treatment at different temperatures (0℃~70℃) for 1, 2, 4, 8, 12, and 24 h, was mixed with 3 mg / mL HA. Residual enzyme activity was measured at the optimal temperature and pH. The enzyme activity of the untreated enzyme solution was defined as 100% relative activity. The test results are as follows: Figure 5 As shown.

[0081] Depend on Figure 5 It is known that the enzyme activity of hyaluronic acid lyase (sHAase) is very stable at temperatures ranging from 0 to 40°C, retaining extremely high enzyme activity (>100%) even after incubation for 24 hours at the corresponding temperatures. When the enzyme solution is incubated at 50°C for 8 hours, the enzyme activity of sHAase remains at 95%, but after incubation at the same temperature for 12 hours, the enzyme activity is 75.94%. Therefore, subsequent reactions with this enzyme can be carried out at 40°C.

[0082] 4. The effect of metal ions on enzyme activity

[0083] A mixture of 3 mg / mL HA, 150 mM pH 6.0 NaH2PO4-Na2HPO4 buffer, hyaluronic acid lysin HAase enzyme solution, and deionized water was prepared in a volume ratio of 20:20:6:11. Different metal ions were then added to the reaction system, with a final concentration of 5 mM. The reaction was carried out under optimal conditions for 20 minutes. After the reaction, the residual enzyme activity was measured by UV spectrophotometry. The enzyme activity without added metal ions was defined as 100% relative activity. The results are shown below. Figure 6 As shown.

[0084] Depend on Figure 6 It can be seen that Ag + Cu 2+ Co 2+ Ni 2+ Hg 2+ Pb 2+ Mn 2+ Zn 2+ Fe 3+ Cr 3+ EDTA and β-mercaptoethanol all inhibited the enzyme activity of sHAase, while several metal ions such as K+ inhibited it. + Li + Na + and Mg 2+ It improved the enzyme's activity in degrading HA.

[0085] Example 5: Determination of the enzyme activity of hyaluronic acid lyase sHAase

[0086] Mix 3 mg / mL glycosaminoglycan substrate, 150 mM pH 6.0 NaH2PO4-Na2HPO4 buffer, sHAase enzyme solution, and deionized water in a certain proportion and react under optimal conditions for 1-10 min. Add an equal amount of inactivated enzyme to the negative control. After the reaction, determine the enzyme activity by the aforementioned ultraviolet spectrophotometric method.

[0087] The glycosaminoglycan substrate is CS-A, CS-C, CS-E, HA, or DS.

[0088] The reaction system consisted of: 50 μl of 3×NaH2PO4-Na2HPO4 (150 mM, pH=6.0), 50 μl of glycosaminoglycan substrate (3 mg / ml), 3 μl of sHAase (15 μg / μl), and 7 μl of ddH2O.

[0089] The results showed that the hyaluronic acid lyase sHAase did not degrade CS and DS, but only specifically degraded HA, and the enzyme activity was 5.23 U / mg.

[0090] Example 6: HPLC analysis of degradation products of different substrates by hyaluronic acid lyase (sHAase)

[0091] A mixture of 1 mg / mL substrate (CS-A, CS-C, CS-E, DS, HA, or Ch), 150 mM NaH2PO4-Na2HPO4 buffer (pH=6.0), hyaluronic acid lysin sHAase enzyme solution, and deionized water was prepared in a 5:5:1:4 (volume ratio). The mixture was reacted under optimal conditions until complete degradation. The degradation products were analyzed by HPLC, and the results are shown below. Figure 7 As shown.

[0092] HPLC analysis conditions: gel column was Superdex peptide 10 / 300 GL (GE), mobile phase was 0.2M ammonium bicarbonate, flow rate was 0.4 mL / min, and detection condition was UV 232 nm.

[0093] Depend on Figure 7 It is known that after hyaluronic acid lyase (sHAase) degrades HA, the degradation products are only unsaturated disaccharides, indicating that sHAase can completely degrade HA into disaccharides. However, weak peak signals also appeared at the non-sulfated disaccharide positions during the degradation of Ch, CS-A, CS-C, CS-E, and DS, which need to be further confirmed by mass spectrometry and other methods.

[0094] Example 7: Substrate specificity analysis of hyaluronic acid lyase sHAase

[0095] 1. To further identify the structure of the non-sulfated disaccharide described in Example 6, unsaturated hyaluronic acid tetrasaccharide and unsaturated chondroitin tetrasaccharide were degraded by hyaluronic acid lyase sHAase under optimal conditions for 12 h. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 min and then placed on ice to cool. After centrifugation at 12,000 rpm for 10 min, 25 μL of the supernatant was taken as a sample for 2-AB labeling.

[0096] After labeling, the sample was dried using a centrifuge and 0.5 μg of the sample was analyzed by anion exchange HPLC on a YMC Pack PA-G column (YMC-Pack, Kyoto, Japan). The obtained disaccharides were identified by comparing their elution positions with those of standardized CS and HA unsaturated disaccharides. The results are as follows: Figure 8 As shown.

[0097] The reaction system consisted of: 10 μl of 3×NaH2PO4-Na2HPO4 (150 mM, pH=6.0), 1 μl of HA tetrasaccharide / Ch tetrasaccharide (1 mg / mL), 3 μl of sHAase (1 μg / μl), and 16 μl of ddH2O.

[0098] Anion exchange chromatography (HPLC) analysis conditions: column type YMC-Pack PA-G (YMC-Pack, Kyoto, Japan); mobile phase concentration of NaH2PO4 increased from 16 mM to 460 mM within 60 min; flow rate was 1 mL / min; detection time was 60 min; detector was a fluorescence detector; detection wavelength was 330 nm for excitation and 420 nm for emission.

[0099] 2. In order to further eliminate the interference of trace HA impurities in commercial chondroitin products on specific analysis and to confirm that the hyaluronic acid lysin sHAase of this invention has no degradation activity on chondroitin polymers, this experiment purified and re-enzymatically hydrolyzed the chondroitin substrate for verification.

[0100] The specific method is as follows: First, commercial chondroitin was used as a sample, and hyaluronic acid lyase (sHAase) was used to completely degrade it under optimal conditions to remove contaminated HA. After the reaction, the reaction was terminated by boiling water bath and the supernatant was collected by centrifugation. 5% (v / v) sodium acetate and 3 volumes of ice-cold anhydrous ethanol were added to the supernatant, and precipitation was carried out at -20℃. The precipitate was collected by centrifugation, washed with ethanol, dried, and redissolved to obtain purified chondroitin with HA impurities removed. The purified chondroitin was then mixed with hyaluronic acid lyase (sHAase) and the positive control enzyme CSaseABC, respectively, and reacted under their respective optimal conditions. The products were analyzed by gel filtration chromatography, and the results are as follows. Figure 9 As shown.

[0101] The sHAase reaction system consisted of: 10 μl of 3×NaH2PO4-Na2HPO4 (150 mM, pH=6.0), 1 μl of purified chondroitin (3 mg / mL), 3 μl of sHAase (1 μg / μl), and 16 μl of ddH2O.

[0102] The CSase ABC reaction system consisted of: 10 μl of 3×NaH2PO4-Na2HPO4 (150 mM, pH=6.0), 1 μl of purified chondroitin (3 mg / mL), 3 μl of CSase ABC (1 μg / μl), and 16 μl of ddH2O.

[0103] Anion exchange chromatography (HPLC) analysis conditions: Superdex peptide 10 / 300 GL (GE) column, 0.2 M ammonium bicarbonate mobile phase, flow rate 0.4 mL / min, detection time 60 min, UV detector, detection wavelength 232 nm.

[0104] Depend on Figure 8 and Figure 9 It can be seen that in the tetrasaccharide degradation experiment, the hyaluronic acid lyase sHAase of the present invention can completely degrade HA tetrasaccharide into disaccharide, but cannot degrade chondroitin tetrasaccharide ΔO-O. In the polymer degradation experiment, the positive control CSaseABC can completely degrade purified chondroitin into disaccharide, while the chromatogram of purified chondroitin treated with hyaluronic acid lyase sHAase is consistent with the blank control, and no degradation products are detected.

[0105] Combined with the results of Example 6, it can be seen that the trace disaccharide peaks generated during the degradation of various commercial CS substrates are confirmed to be caused by the degradation of HA impurities mixed in the CS products. The reason for the appearance of this peak is likely due to the high similarity of physicochemical properties between HA and Ch, and the limitations of currently used separation and purification techniques, which result in a small amount of HA mixed in various CS products. This phenomenon is common.

[0106] The above results fully demonstrate that the hyaluronic acid lysin sHAase of the present invention has absolute substrate specificity for HA, no degradation activity for chondroitin, and is a specific hyaluronic acid lysin.

[0107] Example 8: Evaluation of the effectiveness of hyaluronic acid lyase (sHAase) in detecting trace HA impurities in CS-A

[0108] To evaluate the effectiveness, sensitivity, and accuracy of the hyaluronic acid lyase sHAase combined with gel filtration chromatography described in this invention for detecting micro HA in CS-A, a spiked recovery experiment was conducted.

[0109] The specific method was as follows: 1 μg of HA standard was added to 9, 99, and 999 μg of high-purity CS-A, respectively, to prepare mixed experimental samples with final mass ratios of 10%, 1%, and 0.1% w / w, denoted as 10% HA-spiked CS-A, 1% HA-spiked CS-A, and 0.1% HA-spiked CS-A. Simultaneously, a positive control (containing only 1 μg of HA standard) and a negative matrix control (containing only high-purity CS-A) were prepared in parallel, denoted as Positive Control (HA) and CS-A. All samples were incubated with hyaluronic acid lyase (sHAase) at 40°C to specifically degrade HA. After the reaction was complete, the reaction was terminated by boiling for 10 minutes, followed by cooling with ice water for 10 minutes. After centrifugation, the supernatant was collected, and the degradation products were separated and quantified using a Superdex Peptide 10 / 300 GL column (GE Healthcare). HA was quantified by integrating the peak areas of the characteristic disaccharide degradation products of HA. Finally, the accuracy and recovery rate of the method were evaluated by comparing the measured HA amount with the known initial concentration, and the results are shown in Table 1.

[0110] Table 1. Quantitative analysis and recovery rate of HA spiked in CS-A samples

[0111]

[0112] Note that the negative matrix control (containing only high-purity CS-A) contains no HA and is not listed in Table 1.

[0113] Table 1 shows that the positive control (pure HA) exhibited a clear characteristic disaccharide peak after enzymatic digestion; the negative matrix control (pure CS-A) showed no detected peak at the corresponding retention time, confirming the high specificity of sHAase under these experimental conditions; the corresponding disaccharide peaks were detected in HA spiked samples at 10%, 1%, and 0.1% (w / w). Further quantitative analysis of the results revealed that the recoveries of HA reached 99.11% and 98.66% at high and medium spiking levels (1%), respectively. Even at a trace level of 0.1% (w / w), this method successfully quantified HA with a recovery rate of 96.12%.

[0114] Example 9: Application of hyaluronic acid lyase sHAase in GAGs quality control

[0115] To determine whether HA was present as an impurity in various GAGs produced by different companies, 100 μg of GAGs were completely degraded using 0.5 μg of hyaluronic acid lyase (sHAase) under optimal reaction conditions. The GAGs were then analyzed by gel permeation chromatography on a Superdex Peptide 10 / 300 GL column (GE Healthcare) at a wavelength of 232 nm. The HA content in the GAGs was quantified by integrating peak areas, and the results are shown in Table 2.

[0116] The GAGs products involved in this embodiment were purchased from T Co., Ltd., G Co., Ltd., Sigma-Aldrich, M Co., Ltd., and B Co., Ltd., respectively.

[0117] Table 2. Quantitative analysis of HA contamination in commercial GAGs products using sHAase enzymatic detection method.

[0118]

[0119] Table 2 shows that HA impurities were present in all GAGs from multiple companies. Heparin, DS, and CS-E produced by companies T Co., Ltd. and G Co., Ltd. exhibited high purity. In contrast, CS produced by companies M Co., Ltd. and B Co., Ltd. contained relatively high levels of HA. Furthermore, significant amounts of HA were detected in bovine CS-A and porcine small intestinal mucosa-derived heparin from Sigma. This indicates that the hyaluronic acid lysin sHAase provided in this invention can not only detect HA in various GAGs but also completely convert HA into disaccharides. Combined with methods such as ethanol precipitation, gel filtration chromatography, or ultrafiltration, HA disaccharides can be effectively removed, thereby achieving the purification of GAGs.

[0120] Example 10: Uses of hyaluronic acid lyase sHAase

[0121] This invention demonstrates through the above experiments that sHAase, a hyaluronic acid lysin derived from the mouse intestine, can selectively cleave hyaluronic acid, exhibiting extremely high substrate selectivity for hyaluronic acid and essentially not acting on other glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, heparin, or heparan sulfate. It can cleave hyaluronic acid into degradation products primarily composed of unsaturated disaccharides, thus enabling its application in the following fields:

[0122] 1. Drug delivery: Utilizing the specificity of hyaluronic acid lyase sHAase, it can be used as a drug diffusion factor and combined with macromolecular drugs (such as monoclonal antibodies) to significantly increase the diffusion area of ​​subcutaneously injected drugs.

[0123] 2. Medical Aesthetics: Utilizing the highly efficient degradation effect of hyaluronic acid lyase sHAase, it is prepared into a repair injection to dissolve excess hyaluronic acid fillers injected into the face. Because it does not degrade its own CS / DS, its safety is significantly better than existing testicular hyaluronidase.

[0124] 3. Clinical treatment: In tumor models, adjuvant administration of hyaluronic acid lyase sHAase can reduce tumor interstitial pressure; in fibrosis models, it can soften scar tissue.

Claims

1. A specific hyaluronic acid lysin sHAase, characterized in that, The specific hyaluronic acid lysin sHAase is any one of the amino acid sequences shown in 1), 2), or 3). 1) The amino acid sequence as shown in SEQ ID NO.1; 2) An amino acid sequence that has ≥90% homology with the amino acid sequence shown in SEQ ID NO.1; 3) An amino acid sequence formed by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.1, and still having hyaluronic acid degradation activity.

2. A gene encoding the specific hyaluronic acid lysin sHAase as described in claim 1, characterized in that, The nucleotide sequence of the coding gene is any one of the coding genes shown in (1), (2), or (3): (1) The nucleotide sequence as shown in SEQ ID NO.2; (2) The nucleotide sequence encoding the amino acid sequence of the specific hyaluronic acid lysin sHAase; (3) A nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The gene encoding the specific hyaluronic acid lyase sHAase described in claim 2 was inserted into the expression vector.

4. A recombinant bacterium or transgenic cell line, characterized in that, The encoding gene for the specific hyaluronic acid lyase sHAase described in claim 2 was inserted into the host bacteria or cell line.

5. A method for preparing the specific hyaluronic acid lysin sHAase according to claim 1, characterized in that, include: The gene encoding hyaluronic acid lyase sHAase was cloned into an expression vector to obtain a recombinant expression vector; then the recombinant expression vector was introduced into host cells for expression, thereby obtaining recombinant hyaluronic acid lyase sHAase.

6. The use of the specific hyaluronic acid lysin sHAase described in claim 1 in the preparation of oligomeric hyaluronic acid salts.

7. The use of the specific hyaluronic acid lysin sHAase described in claim 1 in the detection, removal or quantitative identification of hyaluronic acid impurities in glycosaminoglycan products.

8. The use of the specific hyaluronic acid lysin sHAase described in claim 1 in the preparation of food, medical aesthetic repair products, or pharmaceuticals.

9. The application as described in claim 8, characterized in that, The drug uses a specific hyaluronic acid lysin sHAase as a drug diffusion factor to enhance drug diffusion and absorption in tissues by degrading hyaluronic acid in the extracellular matrix; or it regulates the content of hyaluronic acid in the microenvironment to promote drug penetration and assist in the treatment of anti-tumor and anti-fibrotic related diseases.

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