Hyaluronate lyase and its use in degrading hyaluronic acid

Abstract

This invention discloses a hyaluronic acid lyase and its application in the degradation of hyaluronic acid. Specifically, this invention provides a hyaluronic acid lyase derived from *Enterobacter asburiae*, whose encoding gene was cloned and recombinantly expressed in an *E. coli* system, achieving an enzyme activity as high as 36715 U / mL. Enzymatic characterization experiments show that this enzyme can degrade hyaluronic acid within a pH range of 6.5-9.0, exhibiting high substrate specificity for hyaluronic acid. It can rapidly degrade high molecular weight hyaluronic acid into low molecular weight hyaluronic acid or oligosaccharides, demonstrating high degradation efficiency. The final degradation product is an unsaturated hyaluronic acid disaccharide. The hyaluronic acid lyase provided by this invention can efficiently produce unsaturated hyaluronic acid disaccharides, laying the foundation for further research and evaluation of the activity of hyaluronic acid oligosaccharides.

Description

Technical Field

[0001] This invention relates to a hyaluronic acid lysin and its application in the degradation of hyaluronic acid, belonging to the field of enzyme engineering technology. Background Technology

[0002] Hyaluronic acid (HA), commonly known as hyaluronic acid, is a large-molecule acidic mucopolysaccharide composed of repeating disaccharide units, D-glucuronic acid and N-acetylglucosamine, linked by alternating β-(1,3) and β-(1,4) glycosidic bonds. Its hydrophilicity, viscoelasticity, and biocompatibility have led to its widespread application in medicine, cosmetics, food, and other fields.

[0003] Recent studies have revealed that hyaluronic acid (HA) with relatively small molecular weights, especially o-HAs (oligosaccharides with a relative molecular weight of less than 10kJ), possesses important biological functions. For example, o-HAs promote angiogenesis, wound healing, and inhibit multidrug resistance in tumor cells, showing potential applications in pharmaceuticals, cosmetics, and health products. Oligosaccharides HA (o-HAs) and HA fragments (≤2×10⁻⁶) are important components of hyaluronic acid. 4 Both hyaluronic acid (o-HAs) and hyaluronic acid (HAs) exhibit anti-inflammatory and pro-inflammatory properties, depending on cell type and disease. Some studies have shown that o-HAs can reduce Toll-like receptor (TLR)-mediated inflammation, inhibit HA-CD44 activation of kinases, and slow tumor growth. However, o-HAs have also been found to promote inflammation in synovial fibroblasts, stimulate cell adhesion, and enhance angiogenesis. Unsaturated hyaluronic acid disaccharides exhibit unique anti-inflammatory activity. Therefore, the preparation of hyaluronic acid with specific molecular weights is of great significance.

[0004] Hyaluronidase (HAase) degrades hyaluronic acid into o-HAs, making it the most important method for preparing o-HAs. Research on hyaluronidase has gradually become a hot topic. Hyaluronidase is widely distributed in natural organisms, found in mammals, invertebrates (crustaceans, leeches, and insects), pathogenic fungi (Candida, Streptomyces), bacteria, and bacteriophages. Currently, various microorganisms have been found to produce hyaluronidase, mostly concentrated in Arthrobacter, Pasteurella, Streptococcus zooepidemicus, and Bacillus. All known microbial hyaluronidases are lyases, capable of degrading the β-(1,4) glycosidic bonds of hyaluronic acid to form oligosaccharides containing unsaturated double bonds. Compared to animal-derived enzymes, microbial enzymes have advantages such as high yield, high activity, diverse properties, and ease of heterologous recombination expression, making them the main research focus for industrial enzyme preparations.

[0005] Currently, most commercially available hyaluronidases are extracted from animal tissues, which are costly and relatively complex in technology. Therefore, it is necessary to clarify the characteristics of microbially produced hyaluronidases and understand the properties of microbially derived hyaluronidases as soon as possible, so as to obtain more safe and high-quality hyaluronidases. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a hyaluronic acid lyase derived from Enterobacter asburiae. This enzyme, recombinantly expressed in E. coli, exhibits an activity as high as 36715 U / mL, demonstrates high selectivity and excellent catalytic performance for the substrate hyaluronic acid, and excels in degrading hyaluronic acid to generate oligosaccharides such as hyaluronic acid disaccharide. Furthermore, it surpasses existing reported hyaluronic acid lyases in terms of degradation rate.

[0007] The first object of the present invention is to provide a hyaluronic acid lysin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] A second object of the present invention is to provide a gene encoding the hyaluronic acid lyase.

[0009] Furthermore, the sequence of the gene is shown in SEQ ID NO.2.

[0010] A third objective of this invention is to provide a recombinant expression vector carrying the gene.

[0011] A fourth object of the present invention is to provide an expression cassette containing the said gene.

[0012] Furthermore, the expression frame includes elements necessary for expressing the gene and / or elements that assist in expression, such as promoters, terminators, signal peptides, RBS, etc.

[0013] A fifth objective of the present invention is to provide a recombinant bacterium expressing the hyaluronic acid lysin.

[0014] Furthermore, the host bacteria of the recombinant bacteria include Escherichia coli.

[0015] A sixth object of the present invention is to provide a method for producing hyaluronic acid lyase, comprising the step of fermentation production using the recombinant bacteria.

[0016] Furthermore, during the fermentation process, the pH is preferably 7.0-8.0, and the temperature is preferably 20-40℃.

[0017] Furthermore, in the fermentation process, the seed culture medium or fermentation medium is LB medium. LB medium includes: 8-12 g / L tryptone, 3-7 g / L yeast extract, and 8-12 g / L sodium chloride.

[0018] A seventh object of the present invention is to provide the application of the hyaluronic acid lyase in the degradation of hyaluronic acid.

[0019] Furthermore, the hyaluronic acid is high molecular weight hyaluronic acid and / or medium molecular weight hyaluronic acid, such as 1000-2000kDa, more preferably 1000-1500kDa.

[0020] Furthermore, the application involves degrading high molecular weight hyaluronic acid or medium molecular weight hyaluronic acid into low molecular weight hyaluronic acid or oligomeric hyaluronic acid within a short period of time (e.g., 30 min, 40 min, 50 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h or any duration longer than 6 h), preferably with an average molecular weight of less than 20 kDa, less than 10 kDa, less than 5 kDa, less than 1 kDa or even less than 500 Da.

[0021] Furthermore, the conditions for degrading hyaluronic acid are: reaction temperature 25-45℃, reaction pH 6.5-9.0, and hyaluronic acid concentration 1-20g / L.

[0022] An eighth object of the present invention is to provide a method for producing low molecular weight hyaluronic acid or oligomeric hyaluronic acid, comprising the steps of using high molecular weight hyaluronic acid or medium molecular weight hyaluronic acid as a substrate and the hyaluronic acid lyase as a catalyst for preparation.

[0023] Furthermore, the reaction time is at least 1 hour. More preferably, at least 2 hours.

[0024] Furthermore, the oligomeric hyaluronic acid includes, but is not limited to, hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, and hyaluronic acid dodecasaccharide.

[0025] The beneficial effects of this invention are:

[0026] This invention provides a hyaluronic acid lyase and its application in the degradation of hyaluronic acid. The hyaluronic acid lyase is derived from *Enterobacter asburiae*. This invention achieved recombinant expression of its encoding gene in an *E. coli* system, with an enzyme activity of 36715 U / mL and a protein molecular weight of 87.88 kDa. Enzymatic characterization experiments show that this enzyme can degrade hyaluronic acid within a pH range of 6.5-9.0, with an optimal pH of 7.0 and an optimal reaction temperature of 40℃. More importantly, this enzyme exhibits high substrate specificity for hyaluronic acid and high degradation efficiency; 100 U / mL of the enzyme can reduce hyaluronic acid with an average molecular weight of 1360 kDa to below 20 kDa in 1 hour, with the final degradation product being an unsaturated hyaluronic acid disaccharide. The hyaluronic acid lyase provided by this invention can efficiently produce unsaturated hyaluronic acid disaccharides. Attached Figure Description

[0027] Figure 1 Electrophoresis images of PCR-amplified hyaluronic acid lyase-encoding genes and E. coli BL21 / pET22b(+)-HylEP0006 colony PCR verification nucleic acid. (a) Lanes 1-5 show the amplified hyaluronic acid lyase-encoding gene; lane M: 10,000 bp DNA marker. (b) Electrophoresis images of E. coli BL21 / pET22b(+)-HylEP0006 colony PCR verification nucleic acid. Lanes 1-5 show the gene fragment carrying the target gene obtained from recombinant bacterial colony PCR amplification; lane M: 5,000 bp DNA marker.

[0028] Figure 2 This is an SDS-PAGE image of purified hyaluronic acid lyase HylEP0006. Lanes: Hyl; HylEP0006; Lane M: Protein standard marker.

[0029] Figure 3 The optimal reaction temperature (a) and optimal reaction pH (b) for HylEP0006 are given.

[0030] Figure 4 The enzymatic hydrolysis curves of HylEP0006 with different concentrations of substrates are plotted as (a) with the double reciprocal of the enzyme kinetic parameters (b).

[0031] Figure 5The HPLC chromatograms show the degradation of hyaluronic acid (2 g / L) by 100 U / mL HylEP0006 at different times: 1 h (a), 2 h (b), and 6 h (c).

[0032] Figure 6 The graph shows the concentration changes of each component in the degradation of hyaluronic acid (2g / L) by 100U / mL HylEP0006 at different times (1h, 2h, 6h).

[0033] Figure 7 MS chromatogram (a) showing the degradation of hyaluronic acid (2 g / L) by 100 U / mL HylEP0006 for 6 h, and thin-layer chromatography analysis (b). Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] The methods and materials involved in this invention are as follows:

[0036] (1) Method for determining the activity of hyaluronic acid lysin

[0037] Preparation of 2g / L hyaluronic acid substrate: Dissolve 2g of hyaluronic acid in 1L of 50mM phosphate buffer to prepare a 2g / L hyaluronic acid substrate solution.

[0038] Assay method: The content of reduced ends produced by hyaluronic acid lyase degradation of hyaluronic acid was determined according to the 3,5-dinitrosohydrate (DNS) colorimetric method. Enzyme solution and hyaluronic acid solution were used as experimental groups, while inactivated enzyme solution and hyaluronic acid solution served as control groups. The reaction was carried out at 37℃ for 15 min, DNS reagent was added, and the reaction was terminated by boiling in a water bath for 10 min. After cooling, the absorbance was measured at 540 nm using a microplate reader. Glucose solutions of different concentrations were prepared to establish a glucose standard curve.

[0039] Enzyme activity calculation method: The enzyme activity unit (1U) is defined as the amount of enzyme required to produce 1 μg of glucose reducing equivalent per minute under the above experimental conditions. Each measurement is repeated 3 times. This is used as the standard method for determining the activity of hyaluronic acid lyase.

[0040] (2) Culture medium

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0042] (3)Strain

[0043] In the early stages, an Enterobacter asburiae strain that grows using hyaluronic acid as a single carbon source was obtained by screening in river water, and hyaluronic acid lyase was amplified from this strain.

[0044] (4) Sequence

[0045] HylEP0006 amino acid sequence (SEQ ID NO.1):

[0046] *

[0047] The nucleotide sequence of HylEP0006 (SEQ ID NO.2):

[0048]

[0049] (5) Hyaluronic acid

[0050] The hyaluronic acid involved in this invention includes:

[0051] High molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight higher than 2000 kDa;

[0052] Medium molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 50 kDa and 2000 kDa;

[0053] Low molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 10 kDa and 50 kDa.

[0054] Oligomeric hyaluronic acid refers to hyaluronic acid molecules with a molecular weight of less than 10 kDa, that is, less than 50 monosaccharide structures and a degree of polymerization of less than 25.

[0055] In addition, the average molecular weight of the hyaluronic acid raw material involved in this invention is 1360 kDa.

[0056] Example 1: Recombinant Expression and Purification of Hyaluronic Acid Lyase

[0057] A polysaccharide lyase 8 family gene from the genome of Enterobacterales was obtained from the NCBI database, and primers HylEP0006-IF ​​were designed.

[0058] (5'-aattaattcggatccgaattcATGAAAAAGACAAATTTAGCAT-3'), HylEP0006-1R(5'-aattaattcggatccgaattcGCACAGATCGCTACC-3'). PCR amplification was performed using the genome as a template, and the amplification products were recovered. Figure 1 (a) Simultaneously, the pET22b(+) plasmid was linearized using reverse PCR primers and recovered. The PCR amplification product and linearized plasmid were ligated using a one-step homologous recombination cloning kit and transformed into E. coli BL21(DE3) for plating and culture. Single colonies were collected for colony PCR identification. Figure 1 (b) OD 600 When the α=0.4, expression was induced with 0.02 mM IPTG and fermented in shake flasks at 20°C for 24 h. The cells were collected by centrifugation, resuspended in pH 7.0 phosphate buffer, sonicated for 15 min, and the supernatant was collected by centrifugation for enzyme activity assay and SDS-PAGE electrophoresis analysis.

[0059] The results showed that the hyaluronic acid lyase HylEP0006 gene was 2379 bp long, encoding 792 amino acids (amino acid sequence see SEQ ID NO.1, gene sequence see SEQ ID NO.2). Comparison with other hyaluronic acid lyases in the NCBI database revealed that HylEP0006 had the highest similarity (80.77%) to the hyaluronic acid lyase from *Citrobacter freundii* (GenBank ID: WEA82373.1). The recombinant *E. coli* BL21 / pET22b(+)-HylEP0006 showed an enzyme activity of 36715 U / mL. The recombinant protein was purified using nickel affinity chromatography to obtain electrophoretic grade HylEP0006 recombinant enzyme. SDS-PAGE results showed that the protein size of HylEP0006 was approximately 90 kDa, close to the theoretical molecular weight of 87 kDa, consistent with expectations. Figure 2 ).

[0060] Example 2: Determination of the enzymatic properties of hyaluronic acid lyase

[0061] To investigate the degradation activity of hyaluronic acid by hyaluronic acid lyase at different temperatures, the enzyme reaction temperature was varied, and the activity of hyaluronic acid lyase was measured at various temperatures to obtain the optimal reaction temperature. The temperature gradient was set at 25℃, 30℃, 32.5℃, 35℃, 37.5℃, 40℃, 42.5℃, 45℃, and 50℃. The enzyme activity at pH 7.0 and 40℃ (35539 U / mL) was taken as 100% relative enzyme activity.

[0062] The optimal reaction pH was determined by changing the pH of the substrate (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0) and measuring enzyme activity. The enzyme activity at pH 7.0 and 40℃ was taken as 100% relative enzyme activity.

[0063] To characterize the effect of metal ions on enzyme activity, solutions containing (Al) were prepared with final metal ion concentrations of 1 and 10 mmol / L. 3+ Mg 2+ Cu 2+ Zn 2+ Fe 3+ Ba 2+ Li + K + Ca 2+ The enzyme solution was incubated in the incubation system at room temperature for 1 hour, and the enzyme activity was detected using the reaction without the addition of metal ions as a control.

[0064] The results show that the optimal reaction temperature for the hyaluronic acid lysin HylEP0006 in this invention is 40℃, and it retains more than 80% of its relative enzyme activity in the 30-45℃ range. Figure 3 a). The optimal pH for HylEP0006 is 7.0 ( Figure 3 b). Different metal ions Al were investigated. 3+ Mg 2+ Na + Zn 2+ Fe 3+ Ba 2+ Li + K + Ca 2+ The effect on enzyme activity was compared with that of enzyme solution without any added metal ions. As shown in Table 1, when Ca... 2+ At a concentration of 1 mmol / L, it has a promoting effect on hyaluronic acid lysin, while Al 3+ Na + Zn 2+ Fe 3+ and Cu 2+ This significantly inhibits enzyme activity; increasing the concentration to 10 mmol / L, Mg 2+ Ca 2+ Both metal ions have a promoting effect on hyaluronic acid lyase, and the promoting effect becomes more and more obvious as the concentration of the two metal ions increases, while the other metal ions all show an inhibitory trend on the activity of hyaluronic acid lyase.

[0065] Table 1. Effects of metal ions on hyaluronic acid lyase HylEP0006

[0066]

[0067] Note: ND indicates that enzyme activity was not detected.

[0068] Example 3: Determination of substrate specificity and kinetic parameters of hyaluronic acid lyase

[0069] To investigate the substrate specificity of hyaluronic acid lyase, solutions of 2 g / L hyaluronic acid, chondroitin sulfate, sodium heparin, sodium alginate, and chitosan were prepared using pH 7.0 phosphate buffer and used to determine the enzyme activity of HylEP0006, which showed 100% degradation activity for hyaluronic acid. To investigate the kinetic parameters of its degradation of hyaluronic acid, a series of hyaluronic acid solutions with concentrations ranging from 0.02 to 0.16 mg / mL were prepared using pH 7.0 phosphate buffer and mixed with the enzyme solution. Enzymatic kinetic curves were plotted by real-time monitoring of the absorbance of the reaction solution at 232 nm, and the Km and Vmax values ​​were determined by double reciprocal plotting.

[0070] The results showed that HylEP0006 exhibited high substrate specificity for hyaluronic acid, but only 6.33% of the relative enzyme activity for chondroitin sulfate (Table 1). By double reciprocal plotting, the maximum reaction rate of the hyaluronic acid lyase HylEP0006 in catalyzing hyaluronic acid was V0. max =1.075A 232 / min, Michael constant K m =1.636 mg / mL ( Figure 4 a,b).

[0071] Table 2. Substrate specificity of hyaluronic acid lyase

[0072]

[0073] Example 4: Characterization of the degradation characteristics of hyaluronic acid by hyaluronic acid lyase

[0074] To verify the degradation characteristics of hyaluronic acid by the hyaluronic acid lyase HylEP0006, a 2 g / L hyaluronic acid solution was prepared using pH 7.0 phosphate buffer, and the enzyme solution was added to a concentration of 100 U / mL in the reaction system. The reaction was carried out under constant temperature of 37℃ with stirring, and samples were taken at 1, 2, and 6 h. The oligosaccharide distribution was detected by HPLC, the average relative molecular weight of the degradation products was detected by GPC gel permeation chromatography, and the final degradation products were detected by ESI-MS and TLC thin-layer chromatography.

[0075] HPLC results showed that during 1 hour of degradation, hyaluronic acid disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were generated in the product, with the disaccharide being the most abundant and accumulating continuously. During 2 hours, the contents of hexasaccharides, octasaccharides, and decasaccharides gradually decreased, while the contents of disaccharides and tetrasaccharides increased. However, at 6 hours, only the disaccharide accumulated. Figure 5 6). GPC results showed that 100 U / mL HylEP0006 could rapidly reduce the average molecular weight of hyaluronic acid from 1360 kDa to below 20 kDa within 1 h (Table 3). Analysis of the final product of hyaluronic acid degradation by HylEP0006 revealed a mass-to-charge ratio (m / z) of 378 for the main peak observed in the mass spectrum, consistent with the molecular weight of the unsaturated disaccharide. Combined with TLC results, the final product was an unsaturated hyaluronic acid disaccharide with an N-acetylglucosamine reduction terminus (…). Figure 7 a,b).

[0076] Table 3. Viscosity and average molecular weight of hyaluronic acid at different enzymatic hydrolysis times.

[0077]

[0078] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hyaluronic acid lysin, characterized in that, The amino acid sequence of the hyaluronic acid lyase is shown in SEQ ID NO.

1.

2. The gene encoding the hyaluronic acid lyase of claim 1.

3. A recombinant expression vector carrying the gene of claim 2.

4. An expression cassette containing the gene of claim 2.

5. A recombinant bacterium expressing the hyaluronic acid lysin of claim 1.

6. A method for producing hyaluronic acid lysin, characterized in that, The step includes fermentation production using the recombinant bacteria described in claim 5.

7. The application of the hyaluronic acid lyase according to claim 1 in the degradation of hyaluronic acid.

8. The application according to claim 7, characterized in that, The application includes using the hyaluronic acid lyase to degrade high molecular weight hyaluronic acid or medium molecular weight hyaluronic acid into low molecular weight hyaluronic acid or oligomeric hyaluronic acid, wherein high molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight higher than 2000 kDa, medium molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 50 kDa and 2000 kDa, and low molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 10 kDa and 50 kDa.

9. A method for producing low molecular weight hyaluronic acid or oligomeric hyaluronic acid, characterized in that, The method includes the steps of preparing hyaluronic acid using high molecular weight hyaluronic acid or medium molecular weight hyaluronic acid as a substrate and the hyaluronic acid lyase described in claim 1 as a catalyst, wherein high molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight higher than 2000 kDa, medium molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 50 kDa and 2000 kDa, and low molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight between 10 kDa and 50 kDa.

10. The method according to claim 9, characterized in that, The oligomeric hyaluronic acid includes hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, or hyaluronic acid dodecasaccharide.

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