Hyaluronate lyase expressed extracellularly, recombinant bacteria and application thereof

Abstract

The application discloses a hyaluronate lyase expressed extracellularly, and a recombinant bacterium and application thereof. The application provides a hyaluronate lyase derived from a streptococcus bacteriophage, a coding gene of the hyaluronate lyase is cloned, and the recombinant expression is realized in a bacillus subtilis system; after a signal peptide is replaced, the extracellular enzyme activity is 1.86*10 4 U / mL, and the protein molecular weight is 39.5 kDa. Enzymatic property characterization experiments show that the hyaluronate lyase is an endohydrolase, can exert a hyaluronic acid degradation effect in a pH range of 5-8, has an optimal reaction pH of 6, an optimal reaction temperature of 40 DEG C, and various metal ions have a promoting effect on the hyaluronate lyase. The hyaluronate lyase has good thermal stability and pH stability. The application utilizes an optimized culture medium to carry out enzyme production fermentation in a 20-L fermentation tank, and the highest activity of the hyaluronate lyase produced extracellularly by the recombinant bacterium can reach 1.07*10 5 U / mL at 40 h, which is the first report on the extracellular expression of the hyaluronate lyase in the literature so far.

Description

Technical Field

[0001] This invention relates to an extracellular secretory expression of hyaluronic acid lyase, its recombinant strain, and its applications, belonging to the field of biotechnology. Background Technology

[0002] Hyaluronic acid is a glycosaminoglycan composed of repeating glucuronic acid and N-acetylglucosamine disaccharide units. Its hydrophilicity, viscoelasticity, and biocompatibility have garnered widespread attention and led to its application in medicine, cosmetics, and food. Hyaluronic acid lyase (HA lyase) can break its β-1,4 glycosidic bonds through β-elimination, degrading it into smaller molecule hyaluronic acid, which has wide applications in hyaluronic acid processing, pharmaceuticals, and the fermentation industry.

[0003] In vivo and in vitro experiments have demonstrated that the bioactivity of hyaluronic acid is closely related to its chain length and molecular weight. Low molecular weight hyaluronic acid generally refers to hyaluronic acid with a relative molecular mass between 10-50 kDa, while hyaluronic acid fragments with a relative molecular mass less than 10 kDa are usually classified as hyaluronic acid oligosaccharides. Unlike high molecular weight hyaluronic acid, low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides possess unique bioactivities, such as promoting angiogenesis, antioxidation, and anti-inflammation, thus having potential application value in the pharmaceutical field. Furthermore, with in-depth research on hyaluronic acid oligosaccharides, experiments have revealed that hyaluronic acid oligosaccharides containing different numbers of disaccharide units have different biological functions; for example, unsaturated hyaluronic acid disaccharides exhibit unique anti-inflammatory activity. Therefore, the preparation of hyaluronic acid with specific molecular weights is of great significance.

[0004] Currently, the main methods for degrading hyaluronic acid include physical degradation, chemical degradation, and enzymatic degradation. Physical treatment is insufficient to degrade hyaluronic acid below 10 kDa; chemical methods can prepare hyaluronic acid oligosaccharides, but their drawbacks include the need for stringent reaction conditions and the potential introduction of unwanted side reactions, such as the destruction of monosaccharide residues in the sugar chain. In contrast, enzymatic degradation of hyaluronic acid is more environmentally friendly, with rapid reactions and milder conditions.

[0005] The expression hosts of microbial-derived hyaluronic acid lyases are almost exclusively *Escherichia coli*, and rarely found in *Bacillus subtilis*. Conventional expression in *E. coli* systems faces several challenges, such as low expression levels, inclusion body formation, and difficulty in extracellular secretion of the hyaluronic acid lyase. Therefore, this invention aims to improve the extracellular secretion capacity of hyaluronic acid lyase by expressing it in *Bacillus subtilis*, thereby reducing extraction costs, increasing production efficiency, improving product quality, and enhancing the environmental friendliness of the process. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a hyaluronic acid lyase mutant that can be efficiently expressed in Bacillus subtilis, exhibiting a significant increase in extracellular enzyme activity compared to the original mutant. Furthermore, this invention constructs a recombinant strain based on this mutant and develops a method for the efficient production of the hyaluronic acid lyase mutant, laying the foundation for the development of hyaluronic acid-rich foods.

[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 objective of the present invention is to provide a polynucleotide encoding the hyaluronic acid lyase.

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

[0010] A third objective of this invention is to provide an expression vector carrying the polynucleotide.

[0011] Furthermore, the expression vector uses plasmid pP43NMK as the vector.

[0012] A fourth object of the present invention is to provide a recombinant cell expressing the hyaluronic acid lysin.

[0013] Furthermore, the host of the recombinant cells can be plant cells, animal cells, microorganisms (such as bacteria and fungi), etc.

[0014] Preferably, Bacillus subtilis is used as the host.

[0015] Furthermore, when constructing recombinant bacteria using Bacillus subtilis as the host, a signal peptide is used to regulate the extracellular secretion expression of the hyaluronic acid lyase. The signal peptide is selected from one of the following: nprE signal peptide (existing signal peptide), dacB signal peptide, amyE signal peptide, nprB signal peptide, bsnA signal peptide, phoB signal peptide, bpr signal peptide, abnA signal peptide, apre signal peptide, ywbN signal peptide, and wapa signal peptide.

[0016] Furthermore, the Bacillus subtilis is Bacillus subtilis WB600.

[0017] A fifth objective of this invention is to provide a method for producing hyaluronic acid lyase, using the recombinant bacteria for fermentation production.

[0018] Furthermore, the fermentation is either batch fermentation or fed-batch fermentation.

[0019] Furthermore, the batch fermentation includes the following steps: inoculating the recombinant bacteria into the fermentation medium, and maintaining the pH at 5.5-8.0 and the rotation speed at 300-700 rpm during the fermentation process;

[0020] The fed-batch fermentation includes the following steps: inoculating the recombinant bacteria into the fermentation medium, maintaining the pH at 5.5-8.0 during fermentation, and adding the fed-batch medium at a rate of 8-12 mL / h when dissolved oxygen begins to rise.

[0021] Furthermore, the fermentation medium includes 5-70 g / L glycerol, with soybean meal hydrolysate and yeast powder as nitrogen sources, the total nitrogen source concentration being 40-100 g / L, and the mass ratio of soybean meal hydrolysate to yeast powder being 1-4:1.

[0022] Furthermore, the fermentation medium also includes 10-30 mM KH2PO4 and 50-100 mM K2HPO4, with a pH of 5.5-8.0.

[0023] Furthermore, the supplemental culture medium comprises 300-350 g / L glycerol, 170-210 g / L soybean meal hydrolysate, 40-90 g / L yeast extract, 60-110 mM K2HPO4, and 340-380 mM KH2PO4.

[0024] Furthermore, the fermentation temperature is 25-37℃, and the pH is 5.5-8.0.

[0025] A sixth object of the present invention is to provide the use of the hyaluronic acid lysin, polynucleotide, expression vector or recombinant cell in the degradation of hyaluronic acid.

[0026] Furthermore, the application involves degrading high molecular weight hyaluronic acid into low molecular weight hyaluronic acid or oligosaccharides.

[0027] Furthermore, the hyaluronic acid is a high molecular weight hyaluronic acid with a molecular weight of 1000-1500 kDa.

[0028] Furthermore, the conditions for degrading hyaluronic acid are: reaction temperature 35-45℃, reaction pH 5.5-8, stirring speed 200-240rpm, and hyaluronic acid concentration 0.5-2g / L.

[0029] The beneficial effects of this invention are:

[0030] This invention modifies the hyaluronic acid lyase derived from streptococcal bacteriophage, then clones its encoding gene and achieves recombinant expression in a Bacillus subtilis system. After signal peptide replacement, the extracellular enzyme activity is 1.86 × 10⁻⁶. 4The protein has a concentration of U / mL and a molecular weight of 39.5 kDa. Enzymatic characterization experiments showed that it is an endonuclease capable of degrading hyaluronic acid within a pH range of 5-8, with an optimal reaction pH of 6.0 and an optimal reaction temperature of 40℃. Metal ions have a promoting effect on the enzyme, which exhibits good thermal and pH stability. The fermentation and on-board scale-up process based on recombinant bacteria producing hyaluronic acid lysin was investigated. Using an optimized culture medium, the highest extracellular activity of hyaluronic acid lysin produced by the recombinant bacteria reached 1.07 × 10⁻⁶ H⁻¹ in a 20-L fermenter after 40 h. 5 U / mL. Attached Figure Description

[0031] Figure 1 Electrophoretic patterns of nucleic acid encoded by PCR amplification of the hyaluronic acid lyase gene and the linearized vector pP43NMK. (a) Lanes 1-2 show the linearized vector pP43NMK obtained by reverse PCR amplification; Lane M: 10,000 bp DNA Marker. (b) Lane 1 shows the amplified hyaluronic acid lyase encoded gene; Lane M: 10,000 bp DNA Marker.

[0032] Figure 2 This is an electrophoresis image of the nucleic acid used to verify the successful construction of the pP43NMK-HylP plasmid by colony PCR. Lane 1 represents the gene fragment carrying the target gene obtained from the PCR amplification of the recombinant bacterial colony; Lane M: 10,000 bp DNA Marker.

[0033] Figure 3 SDS-PAGE images of recombinant B. subtilis WB600 / pP43NMK-HylP and purified hyaluronic acid lyase HylP.

[0034] Figure 4 The results of shake-flask fermentation of recombinant bacteria with a replacement signal peptide recombinant plasmid.

[0035] Figure 5 Plot the double reciprocals of the optimal reaction temperature (a), temperature stability (b), optimal reaction pH (c), pH stability (d), metal ion (e), and enzyme kinetic parameters of HylP (f).

[0036] Figure 6 The image shows the LC-MS chromatogram of the degradation of hyaluronic acid (0.5 g / L) by 2000 U / mL HylP for 6 h.

[0037] Figure 7 This diagram illustrates the optimization effect of shake-flask culture medium on recombinant bacteria. The optimization conditions are: carbon source type (a), glycerol concentration (b), nitrogen source type (c), nitrogen source ratio (d), nitrogen source concentration (e), temperature (f), and pH (g).

[0038] Figure 8 The results are from a 20-L fermenter scale-up experiment of the recombinant bacteria. (a) shows the batch fermentation strategy with only initial pH controlled, and (b) shows the DO-stat feeding strategy. Detailed Implementation

[0039] 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.

[0040] The materials and methods involved in the following embodiments are as follows:

[0041] Methods for determining hyaluronic acid lysin activity

[0042] Preparation of 2 g / L hyaluronic acid substrate: Dissolve 2 g of hyaluronic acid in 1 L of 50 mM citrate buffer to prepare a 2 g / L hyaluronic acid substrate solution.

[0043] 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.

[0044] 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.

[0045] Culture medium:

[0046] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0047] TB medium: 5 g / L glycerol, 12 g / L tryptone, 24 g / L yeast extract, 17 mM KH2PO4, 72 mM K2HPO4.

[0048] Optimized TB culture medium: 65 g / L glycerol, 39 g / L soybean meal hydrolysate, 13 g / L yeast extract, 17 mM KH2PO4, 72 mM K2HPO4, pH 6.0.

[0049] Soybean meal hydrolysate was purchased from Tianjin Lifalong Chemical Technology Co., Ltd., and yeast powder was purchased from OXOID.

[0050] Example 1: Recombinant expression and purification of hyaluronic acid lyase gene

[0051] A family 16 polysaccharide lyase gene derived from streptococcal phage was obtained from a previously preserved enzyme library in the laboratory. Using this gene as a template, amplification primers hylP-IF (5'-GTACCATTATAG GTA AGAGAG GAATGT ACACAT GATGAC TGAAAATAT TCC TTT ACG TGT CCAGT-3') and hylP-IR (5'-TCATTAATG ATG ATG ATG ATGATG CTT CTT CAG GAT TAATTT CTT CAACTC CGC-3') were designed. PCR amplification was performed using the genome as a template, and the amplification products were recovered. Figure 1 Simultaneously, the pP43NMK 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, transformed into E. coli BL21(DE3), plated, and single colonies were inoculated into 10 mL LB shake flasks and cultured overnight. Plasmids were extracted, transferred to WB600 competent cells, and cultured at 37°C and 220 rpm for 2 h on a shaker. After centrifugation, the colonies were plated and single colonies were used for colony PCR identification. Figure 2 The hyaluronic acid lyase expression vector pP43NMK-HylP was transferred into B. subtilis WB600 and fermented in shake flasks for 24 h. The supernatant was collected by centrifugation and analyzed by enzyme activity assay and SDS-PAGE electrophoresis.

[0052] The results showed that the hyaluronic acid lyase HylP gene is 1134 bp long (SEQ ID NO.2) and encodes 378 amino acids (SEQ ID NO.1). The extracellular enzyme activity of the recombinant strain B. subtilis WB600 / pP43NMK-HylP reached 1.0 × 10⁻⁶. 4 U / mL. The recombinant protein was purified using nickel ion affinity chromatography to obtain electrophoretic grade HylP recombinase. SDS-PAGE results showed that the protein size of HylP was approximately 39.5 kDa, close to the theoretical molecular weight of 40 kDa, which is consistent with expectations. Figure 3 ).

[0053] SEQ ID NO.1

[0054] MTENIPLRVQFKRMSADEWARSDVILLEGEIGFETDTGFAKFGDGQNTFSKLKYLTGPKGPKGDTGLQGKTGGTGPRGPAGKPGTTDYDQLQNKPDLGAFAQKEETNSKITKLESSKADKSAVYSKAESKIELDKKLSLTGGIVTGQLQFKPNKSGIKPSSSVGGAINIDMSKSEGAAMVMYTNKDTTDGPLMILRSDKDTFDQSAQFVDYSGKTNAVNIVMRQPSAPNFSSALNITSANEGGSAMQIRGVEKVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFFSILPIYDSGGYLEKVYQTAKS

[0055] SEQ ID NO.2

[0056] ATGACTGAAAATATTCCTTTACGTGTCCAGTTCAAACGCATGTCAGCGGACGAGTGGGCACGTTCTGACGTGATTCTTTTAGAGGGGGAGATCGGATTCGAAACTGACACCGGCTTTGCAAAATTCGGGGACGGTCAGAATACTTTTAGTAAATTGAAATATCTGACCGGACCCAAAGGTCCCAAGGGAGACACTGGGTTACAGGGCAAAACAGGTGGAACGGGTCCGCGTGGTCCAGCTGGTAAGCCTGGCACTACGGATTACGATCAGCTGCAGAACAAGCCCGACCTTGGTGCGTTCGCGCAGAAAGAGGAGACAAACTCCAAGATTACGAAACTGGAATCGAGCAAAGCCGATAAAAGTGCAGTATACTCGAAGGCCGAGAGTAAGATTGAACTGGATAAGAAGTTAAGTTTGACTGGCGGTATCGTGACCGGGCAACTTCAGTTCAAGCCTAACAAGTCCGGTATCAAGCCATCTAGTAGCGTTGGCGGCGCTATTAACATTGATATGTCAAAGAGTGAAGGTGCTGCAATGGTCATGTATACTAATAAAGACACGACGGATGGTCCGTTGATGATCCTTCGCAGCGACAAGGATACTTTCGACCAGTCGGCGCAATTCGTAGACTACTCCGGCAAAACAAACGCAGTTAATATTGTCATGCGTCAGCCTTCCGCACCCAATTTCTCGTCGGCATTGAATATCACTTCAGCAAACGAAGGAGGAAGTGCTATGCAAATCCGTGGAGTTGAGAAGGTGAATGGACCAATAATAATGACTAGAGAAGAAAGAATGAAGATTGTTCATGAAATTAAGGAACGAATATTGGATAAATATGGGGATGATGTTAAGGCTATTGGTGTTTATGGCTCTCTTGGTCGTCAGACTGATGGGCCCTATTCGGATATTGAGATGATGTGTGTCATGTCAACAGAGGAAGCAGAGTTCAGCCATGAATGGACAACCGGTGAGTGGAAGGTGGAAGTGAATTTTGATAGCGAAGA GATTCTACTAGATTATGCATCTCAGGTGGAATCAGATTGGCCGCTTACACATGGTCAATTTTTCTCTATTTTGCCGATTTATGATTCAGGTGGATACTTAGAGAAAGTGTATCAAACTGCTAAATCG

[0057] Example 2: Optimization of recombinant expression elements of hyaluronic acid lyase

[0058] To improve the secretion efficiency of hyaluronic acid lyase HylP in Bacillus subtilis, the effect of signal peptide replacement on enzyme production efficiency was investigated based on the recombinant plasmid pP43NMK-HylP. Ten recombinant plasmids with signal peptide replacements were constructed by replacing the original signal peptide nprE in the plasmid: pP43-dacB-HylP, pP43-amyE-HylP, pP43-nprB-HylP, pP43-bsnA-HylP, pP43-phoB-HylP, pP43-bpr-HylP, pP43-abnA-HylP, pP43-apre-HylP, pP43-ywbN-HylP, and pP43-wapa-HylP. The recombinant plasmids were transformed into the BL21(DE3) host, and the extracted plasmids were then transformed into the WB600 host and fermented in TB medium in shake flasks for 24 h. Enzyme activity was then measured.

[0059] The results showed that among the 10 signal peptide replacement strains, the recombinant strain WB600 / pP43-abnA-HylP exhibited higher enzyme production efficiency than the original strain, with an extracellular enzyme activity reaching 1.86 × 10⁻⁶. 4 U / mL, the extracellular enzyme activity of the control strain increased by 86% ( Figure 4 The sequence of abnA is shown in SEQ ID NO.3-4.

[0060] SEQ ID NO.3:

[0061] MKKKKTWKRFLHFSSSAALAAGLIFTSAAPAEA

[0062] SEQ ID NO.4:

[0063] TTGAAAAAGAAAAAACATGGAAACGCTTCTTACACTTTTCGAGTGCAGCTCTGGCTGCAGGTTGATATTCACTTCTGCTGCTCCCGCAGAGGCA

[0064] Example 3: Determination of the enzymatic properties of hyaluronic acid lyase

[0065] To investigate the degradation activity of hyaluronic acid lyase on hyaluronic acid at different temperatures, the temperature in the enzyme activity standard assay was changed to 25℃, 30℃, 37℃, 40℃, 45℃, 50℃, and 55℃ to determine the optimal temperature for enzyme activity measurement. The enzyme solution was incubated at 40℃ and 50℃ for 2 hours, and the initial enzyme activity at each temperature was taken as 100% to obtain temperature stability curves. Hyaluronic acid substrates were prepared using citrate buffers of different pH values ​​for enzyme activity measurement to obtain the optimal pH value. The enzyme solution was placed in buffers of different pH values ​​and incubated at room temperature for 2 hours, and samples were taken to measure residual enzyme activity. The initial highest enzyme activity at each pH was taken as 100% to obtain pH stability curves. To characterize the effect of metal ions on enzyme activity, solutions containing Li3-containing metal ions with a final concentration of 10 mmol / L were prepared. + K + Mg 2+ Ca 2+ Zn 2+ Ni 2+ Mn 2+ Al 3+ Na + Cd 2+ Ba 2+ and Co 2+ The enzyme solution was incubated at room temperature for 1 hour using the incubation system, and the enzyme activity was detected using the reaction without the addition of metal ions as a control.

[0066] The results show that the optimal reaction temperature for the hyaluronic acid lysin HylP in this invention is 40℃, and the relative enzyme activity is over 80% in the 35-45℃ range. Figure 5 a) and the enzyme activity is stable after incubation at 40℃ for 2 hours. Figure 5 b). HylP has an optimal pH of 6 and exhibits over 80% relative enzyme activity in the pH range of 5.5-8. Figure 5 c). Incubation for 2 hours in a buffer solution with pH 9-12 resulted in a rapid decrease in HylP enzyme activity; however, incubation in a buffer solution with pH 4-8 showed relative stability and minimal change in residual enzyme activity. Figure 5 d). 10 mmol / L Al 3+ Ba 2+ Ions have varying degrees of inhibitory effects on HylP enzyme activity, while Ca... 2+ Zn 2+Ni 2+ K + Li + Mg 2+ Mn 2+ Na + Cd 2+ and Co 2+ All ions promoted the enzyme activity of HylP, with 10 mmol / L Mg... 2+ Co 2+ After ion treatment, the relative enzyme activity of HylP increased to approximately 140%. Figure 5 e).

[0067] Example 4: Determination of substrate specificity and kinetic parameters of hyaluronic acid lyase

[0068] To investigate the kinetic parameters of its degradation of hyaluronic acid, a series of hyaluronic acid solutions with different concentrations of 0.2-3 mg / mL were prepared using pH 5.5 citrate buffer and mixed with the enzyme solution. Enzymatic hydrolysis kinetic curves were plotted by real-time detection of the absorbance of the reaction solution at 232 nm, and the Km and Vmax values ​​were obtained by double reciprocal plotting.

[0069] The results showed that, through double reciprocal plotting, the Km value of HylP was calculated to be 0.9 mg / mL, and the Vmax value was 1.76 A. 232 / min( Figure 5 f).

[0070] Example 5: Characterization of the degradation characteristics of hyaluronic acid by hyaluronic acid lyase

[0071] To verify the degradation characteristics of hyaluronic acid by the hyaluronic acid lyase HylP, a 2 g / L hyaluronic acid solution was prepared with water, and enzyme solution was added to a concentration of 50 U / mL in the reaction system, with enzyme added every 1 hour. The reaction was carried out at 37°C with stirring, and samples were taken at 1, 2, 3, 4, 5, and 6 hours. The average relative molecular weight of the degradation products was determined by GPC gel permeation chromatography. Alternatively, a 0.5 g / L hyaluronic acid solution was prepared with water, and enzyme solution was added to a concentration of 2000 U / mL in the reaction system. The reaction was carried out at 37°C with stirring for 6 hours, and samples were taken. The final degradation products were detected by LC-MS.

[0072] GPC results showed that 50 U / mL HylP could reduce the average molecular weight of hyaluronic acid from 1300 kDa to below 69.5 kDa in 6 hours (Table 1). Analysis of the final product of HylP degradation of hyaluronic acid revealed a relative molecular mass of approximately 377. Combined with TLC results, the final product was an unsaturated hyaluronic acid disaccharide (HylP). Figure 6 ).

[0073] Table 1. Average molecular weight of hyaluronic acid at different enzymatic hydrolysis times.

[0074]

[0075] Example 6: Optimization of culture medium for recombinant hyaluronic acid lysin bacteria

[0076] To further enhance the hyaluronic acid lyase production capacity of the recombinant bacteria, the fermentation medium was optimized at the shake-flask level. Based on TB medium, seven culture conditions were optimized in sequence: carbon source type, glycerol concentration, nitrogen source type, ratio of soybean meal hydrolysate to yeast extract, nitrogen source concentration, initial pH, and temperature. After overnight culture in 10 mL LB medium, the recombinant bacteria were inoculated into different culture media formulations at a 2% inoculum size. After 24 hours of culture, the fermentation broth was collected to determine enzyme activity and cell concentration.

[0077] The results showed that the recombinant strain produced the best enzyme when the glycerol concentration was 65 g / L, the ratio of soybean meal hydrolysate to yeast powder was 3:1, the nitrogen source concentration was 52 g / L (the most economical and with little difference in enzyme activity), the initial pH was 6, and the fermentation temperature was 30℃. Figure 7 (a, b, c, d, e, f, g). Compared to TB medium, the recombinant bacteria produced 2 times more enzymes in the optimized medium, reaching 3.8 × 10⁻⁶. 4 U / mL.

[0078] Example 7: Scale-up of recombinant hyaluronic acid lysin bacteria in a 20-L fermenter

[0079] Based on the results of culture medium optimization, two methods were used to conduct batch fermentation of the recombinant bacteria in a 20-L fermenter: Method 1 controlled the pH at 6.0 and the fermentation speed at 500 rpm throughout the fermentation process; Method 2 controlled the pH of the fermentation broth at 6.0 during the fermentation process. During fermentation, when dissolved oxygen began to rise, feed medium (glycerol 325 g / L, soybean meal hydrolysate 195 g / L, yeast extract 65 g / L, 85 mM K2HPO4, 360 mM KH2PO4) was added at a rate of 10 mL / h.

[0080] The results showed that, under method one, the recombinant bacteria reached peak enzyme production at 24 hours, with an extracellular enzyme activity of 8.2 × 10⁻⁶. 4 U / mL Figure 8 a) Under Method 2, the enzyme activity of the recombinant bacteria was significantly increased to 1.07 × 10⁻⁶ using fed-batch culture medium. 5 U / mL Figure 8 b) Its extracellular enzyme activity is the highest level reported in the literature for microbial hyaluronic acid lyases (under the same enzyme activity definition).

[0081] 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. A polynucleotide encoding the hyaluronic acid lysin of claim 1.

3. An expression vector carrying the polynucleotide of claim 2.

4. Recombinant cells expressing the hyaluronic acid lysin of claim 1.

5. A recombinant Bacillus subtilis, characterized in that, The recombinant Bacillus subtilis heterologously expresses the hyaluronic acid lysin of claim 1, and the extracellular secretion expression of the hyaluronic acid lysin is regulated by a signal peptide, wherein the signal peptide is the abnA signal peptide, and the gene sequence of the abnA signal peptide is shown in SEQ ID NO.

4.

6. A method for producing hyaluronic acid lyase, characterized in that, The recombinant Bacillus subtilis as described in claim 5 is used for fermentation production.

7. The method according to claim 6, characterized in that, The fermentation is either batch fermentation or fed-batch fermentation. The batch fermentation includes the following steps: inoculating the recombinant Bacillus subtilis into the fermentation medium, and maintaining the pH at 5.5-8.0 and the rotation speed at 300-700 rpm during the fermentation process; The fed-batch fermentation includes the following steps: inoculating the recombinant Bacillus subtilis into the fermentation medium, maintaining the pH at 5.5-8.0 during fermentation, and adding the fed-batch medium at a rate of 8-12 mL / h when dissolved oxygen begins to rise.

8. The method according to claim 7, characterized in that, The fermentation medium comprises: 5-70 g / L glycerol, with soybean meal hydrolysate and yeast powder as nitrogen sources, the total nitrogen source concentration being 40-100 g / L, and the mass ratio of soybean meal hydrolysate to yeast powder being 1-4:

1.

9. The use of the hyaluronic acid lysin of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the recombinant cell of claim 4, or the recombinant Bacillus subtilis of claim 5 in the degradation of hyaluronic acid.

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