High-activity and thermal-stability PL29 family hyaluronate lyase derived from bacteroides and application of high-activity and thermal-stability PL29 family hyaluronate lyase

Abstract

The invention discloses high-activity and thermal-stability PL29 family hyaluronic acid lyase derived from bacteroides and application of the PL29 family hyaluronic acid lyase, and belongs to the technical field of microorganisms. The invention provides the hyaluronic acid lyase BfiHAase derived from Bacteroides pinnatae, the hyaluronic acid lyase BfiHAase has good enzymatic activity and thermal stability, the optimal reaction pH value of the BfiHAase is 6.08, the optimal reaction temperature is 40 DEG C, the hyaluronic acid lyase BfiHAase has high enzyme activity, and the highest enzyme activity under the optimal condition can reach 454.9893 U / mg and exceeds that of most hyaluronic acid lyase. Meanwhile, the enzyme has good thermal stability, can keep more than 90% of activity after being stored at 4 DEG C for 6 hours, and can still keep more than 70% of activity after being stored for one day, which indicates that the enzyme has great industrial application potential.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biotechnology, specifically to a highly active, thermostable PL29 family hyaluronic acid lyase derived from Bacteroides and its applications. Background Technology

[0002] Hyaluronic acid (HA) is a high-value linear glycosaminoglycan widely found in vertebrate connective tissues, vitreous humor, and synovial fluid. It is composed of repeating D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) disaccharide units linked by β-1,3 and β-1,4 glycosidic bonds. Due to its unique water-retention, viscoelasticity, lubricity, and biocompatibility, HA has extremely important applications in medicine (such as ophthalmic surgery, orthopedic injections, and drug delivery), cosmetics (moisturizers, anti-wrinkle agents), and food and health products.

[0003] According to the CAZy database (Carbohydrate-Active enZymes database), enzymes with hyaluronic acid cleavage activity are mainly distributed in the PL8, PL16, PL21, PL29, and PL35 families. Enzymes from different families exhibit significant differences in structure, substrate specificity (preferentiality towards glycosaminoglycans such as HA and chondroitin sulfate), mode of action, and optimal reaction conditions. The known PL29 enzyme has insufficient thermostability at physiological temperatures (37°C) or higher, resulting in a short active half-life, requiring frequent replenishment or large doses in practical applications.

[0004] Currently, commercially available hyaluronic acid lyases are mainly extracted from animal tissues, which suffers from limited resources, low specific activity, and poor stability. In contrast, microbially derived hyaluronic acid lyases exhibit higher activity and stronger stability, showing great application potential in the preparation of low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides. Therefore, many research institutions are dedicated to studying the preparation of hyaluronic acid lyases via microbial fermentation. Various microorganisms have been found to produce hyaluronic acid lyases, including... Streptococcus , Staphylococcus , Clostridium , Propionibacterium , Peptostreptococcus as well as Streptomyces However, to date, there are few reports characterizing hyaluronic acid lysins in Bacteroides. Microbial hyaluronic acid lysins are not limited by source, are easy to extract, have high purity, and are not prone to causing immune reactions. Therefore, the search for and mining of thermostable original hyaluronic acid lysins from strains is still of great significance. Summary of the Invention

[0005] Through in-depth research and creative effort, the applicant of this invention addresses the shortcomings and deficiencies of existing technologies by providing a hyaluronic acid lyase, its gene, and its applications. This invention also constructs a recombinant expression vector for the hyaluronic acid lyase gene and a host cell for expressing the hyaluronic acid lyase. The hyaluronic acid lyase protein sequence provided by this invention differs from known sequences and exhibits different enzyme activities compared to similar sequences. Under the same measurement conditions, the enzyme activity in the fermentation broth is higher than that of most hyaluronic acid lyases reported in current literature and patents, and it maintains high activity while being stored at low temperatures for a longer period. This enzyme exhibits good temperature stability and high product purity, offering unique advantages in the preparation of low-molecular-weight hyaluronic acid in pharmaceutical and biochemical applications.

[0006] Technical issues: The technical problem to be solved by this invention is to provide a highly active hyaluronic acid lyase with good thermal stability and its application, update the strain source information of the PL29 family of polysaccharide lyases, and discover more hyaluronic acid lyases from different strains.

[0007] Technical solution: To solve the above-mentioned technical problems, the present invention provides a hyaluronic acid lysin, wherein the amino acid sequence of the hyaluronic acid lysin is as shown in any one of SEQ ID NO.1~5.

[0008] In one embodiment of the present invention, the amino acid sequence of the hyaluronic acid lysin BfiHAase is shown in SEQ ID NO. 1; the BfiHAase is derived from... B. finegoldii Bacteroides filamentosa FNMHLBE3K7.

[0009] In one embodiment of the present invention, the amino acid sequence of the hyaluronic acid lysin BnoHAase is shown in SEQ ID NO. 2; the BnoHAase is derived from... B. nordii Nodibronema FTJS11K9.

[0010] In one embodiment of the present invention, the amino acid sequence of hyaluronic acid lysin BclHAase is shown in SEQ ID NO. 3; the hyaluronic acid lysin BclHAase is derived from... B. clarus Bacteroides clausti FFJLY22K22.

[0011] In one embodiment of the present invention, the amino acid sequence of the hyaluronic acid lysin BceHAase is shown in SEQ ID NO. 4; the BceHAase is derived from... B. cellulosilyticus FSDTAELIBHI5.

[0012] In one embodiment of the present invention, the amino acid sequence of the hyaluronic acid lysin BxyHAase is shown in SEQ ID NO. 5, and the BxyHAase is derived from... B. xylanisolvens Xylanobacterium FBJ60K5.

[0013] The present invention also provides a gene encoding the hyaluronic acid lyase, the nucleotide sequence of which is shown in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 or SEQ ID NO.10.

[0014] In one embodiment of the present invention, the gene sequence encoding hyaluronic acid lysin BfiHAase is shown in SEQ ID NO. 6.

[0015] In one embodiment of the present invention, the gene sequence encoding the hyaluronic acid lysin BnoHAase is shown in SEQ ID NO.7.

[0016] In one embodiment of the present invention, the gene sequence encoding hyaluronic acid lyase BclHAase is shown in SEQ ID NO. 8.

[0017] In one embodiment of the present invention, the gene sequence encoding the hyaluronic acid lysin BceHAase is shown in SEQ ID NO. 9.

[0018] In one embodiment of the present invention, the gene sequence encoding hyaluronic acid lyase BxyHAase is shown in SEQ ID NO. 10.

[0019] The present invention also provides a recombinant vector carrying the gene for the above-mentioned hyaluronic acid lysin.

[0020] The present invention also provides a gene carrying the above-mentioned hyaluronic acid lyase, or a recombinant cell carrying the above-mentioned recombinant vector.

[0021] In one embodiment of the present invention, the recombinant cells are bacteria.

[0022] The present invention also provides a recombinant Escherichia coli that expresses the gene encoding hyaluronic acid lyase described above.

[0023] In one embodiment of the present invention, pE-SUMO is used as the expression vector.

[0024] In one embodiment, the recombinant Escherichia coli genetically engineered bacteria uses Escherichia coli BL21(DE3) as the expression host.

[0025] The present invention also provides a method for constructing the above-mentioned recombinant Escherichia coli, the method being: (1) PCR amplification of the gene fragment of hyaluronic acid lyase shown in any of SEQ ID NO.6~10 from the strain.

[0026] (2) Using pE-sumo as the expression vector, the amplified hyaluronic acid lyase gene was ligated into the expression vector to construct a recombinant expression vector. The above recombinant expression plasmid was transformed into E.coli BL21(DE3), and positive transformants were screened and sequenced for verification.

[0027] This invention provides a method for producing the above-mentioned hyaluronic acid lyase. The method involves adding the above-mentioned recombinant Escherichia coli to a seed culture medium and culturing it at 37°C and 220 rpm until the OD600 is between 0.6 and 0.8 to obtain a seed solution. The seed solution is then cultured at 22°C and isopropyl-β-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5 mM for induced expression for 8-10 h at a rotation speed of 160 rpm.

[0028] The present invention also provides a method for cleaving hyaluronic acid, characterized in that the method comprises adding the above-mentioned hyaluronic acid cleaving enzyme, or the above-mentioned recombinant cells, or the above-mentioned recombinant Escherichia coli to a reaction system containing hyaluronic acid for reaction.

[0029] In one embodiment of the present invention, the reaction temperature is 30–40°C.

[0030] In one embodiment of the present invention, the final concentrations of each substance in the reaction system are 50 mmol / L sodium acetate, 5 mmol / L calcium acetate, 5 mg / mL hyaluronic acid, and pH 7.4.

[0031] The present invention also provides the use of the above-mentioned hyaluronic acid lysin, or the above-mentioned recombinant cells, or the above-mentioned recombinant Escherichia coli in the preparation of products containing low molecular weight hyaluronic acid.

[0032] Beneficial effects: 1. This invention provides five hyaluronic acid lysins, including those derived from... Bacteroides finegoldii BfiHAase, a hyaluronic acid lysin derived from Bacteroides filamentosa FNMHLBE3K7, is a hyaluronic acid lysin derived from... B. nordii BnoHAase from Nodibacterium FTJS11K9, derived from B. clarus BclHAase from Bacteroides FFJLY22K22, derived from B. cellulosilyticus BceHAase from Bacteroides fibrobacterium FSDTAELIBHI5, derived from B. xylanisolvensThe BxyHAase of *Bacillus xylanosporus* FBJ60K5, a hyaluronic acid lyase, exhibits good thermal stability. Good thermal stability of hyaluronic acid lyases is significant for improving operational stability in catalytic processes, enabling batch reuse, and reducing production costs. Furthermore, this hyaluronic acid lyase demonstrates good enzyme activity; under optimal conditions after purification, its specific activity can reach 198.4 U / mg ~ 446.5538 U / mg.

[0033] 2. The method of this invention utilizes bioinformatics technology to deeply mine the CAZymes gene of human intestinal microorganisms. Based on the method of this invention, a novel high-performance hyaluronic acid lysin can be rapidly obtained, and it also provides feasible ideas for the discovery of other novel enzymes.

[0034] 3. This invention provides a hyaluronic acid lysin with good thermal stability. Since high molecular weight hyaluronic acid has very low solubility, for the production of low molecular weight hyaluronic acid, a hyaluronic acid lysin with better heat resistance can appropriately increase the reaction temperature and shorten the entire production cycle. In addition, hyaluronic acid lysins are expensive, and a thermally stable hyaluronic acid lysin can last longer throughout the entire production process, reducing the amount of enzyme used and saving production costs. Attached Figure Description Figure 1 Phylogenetic analysis of five enzymes. (Red indicates the five enzymes we identified, blue indicates the two enzymes from the PL29 family that have been identified, and the others are predicted enzymes from various Bacteroidetes genera within the PL29 family.) Figure 2 Gel images of protein expression and purification: Figure A shows BfiHAase, Figure B shows BnoHAase, Figure C shows BclHAase, Figure D shows BceHAase, and Figure E shows BxyHAase. Lane M is the protein marker, lane 1 is the whole bacterial culture for protein expression, lane 2 is the supernatant after sonication lysis of protein expression, lane 3 is the precipitate after sonication lysis of protein expression, lane 4 is the flow-through buffer for protein purification, lanes 5 and 6 are elution buffers with low concentrations of imidazole (20 mM and 50 mM), respectively, and lane 7 is elution buffer with high concentrations of imidazole (150 mM).

[0035] Figure 3 The effect of temperature on enzyme activity. The optimal temperature corresponding to the enzyme activity of each enzyme was set to 100%. Figures A to E show the activity changes of BceHAase, BxyHAase, BclHAase, BnoHAase, and BfiHAase in the temperature range of 25℃ to 50℃, respectively.

[0036] Figure 4 Thermostability of enzymes. Changes in the activities of five enzymes after storage at 4℃ (A) and 40℃ (B) for 24 hours, respectively.

[0037] Figure 5 The effect of pH on enzyme activity. Figures A through E show the activity changes of BceHAase, BxyHAase, BclHAase, BnoHAase, and BfiHAase in different buffer solutions at different pH levels.

[0038] Figure 6 Enzyme kinetic curves and parameters. Figures A to E show the reaction rate curves and reaction parameters of BceHAase, BxyHAase, BnoHAase, BclHAase, and BfiHAase in the HA concentration range of 0.1-5 mg / mL.

[0039] Figure 7 Molecular docking verification diagrams. Figures A to E show the docking results of BceHAase, BxyHAase, BfiHAase, BclHAase, and BnoHAase with HA, respectively. Detailed Implementation The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] The PCR primers used in the following examples were synthesized by Shanghai Sangon Biotech Co., Ltd., and sequencing was performed by Shanghai Sangon Biotech Co., Ltd. DNA and protein markers were purchased from Thermo Fisher Scientific. Plasmid extraction kits and PCR product gel extraction kits were purchased from Novizan Biotech Co., Ltd. Nickel columns and desalting columns used for purification were purchased from Jiaxing Qianchun Biotech Co., Ltd. Hyaluronic acid (molecular weight 1.5-2.5 million Da) was purchased from Shanghai Maclean Biotech Co., Ltd.

[0041] The culture media involved in the following examples are as follows: LB liquid medium: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L.

[0042] LB solid medium: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar powder 15 g / L.

[0043] The detection methods involved in the following embodiments are as follows: The method for determining hyaluronic acid lysin activity is as follows: Enzyme activity was determined using the 232 nm ultraviolet absorption method with hyaluronic acid as the substrate to measure the activity of hyaluronic acid lyase. Under this method, enzyme activity was defined as the amount of enzyme required to produce 1 μmol Δ4,5-unsaturated uronic acid per minute within the given environment, which is 1 U. The reaction system was as follows: 800 μL of substrate buffer (a mixture of 50 mmol / L sodium acetate, 5 mmol / L calcium acetate, and 5 mmol / L hyaluronic acid) was added to a 1.5 mL centrifuge tube; 200 μL of diluted, purified, and desalted enzyme solution was added; the reaction was allowed to proceed for 30 min; the reaction was terminated by boiling for 5 min; the mixture was centrifuged at 12000 rpm for 5 min, and the absorbance at 232 nm was measured in the supernatant. Specific enzyme activity was calculated using the following formula: .

[0044] In the formula: A is the specific activity of the enzyme, in U / mg; ΔA232 is the absorbance change at 232 nm; ε is the molar absorptivity of the unsaturated double bond, which is 5500 L / (mol) (cm); b is the width of the cuvette; t is the enzyme reaction time; Vtotal is the total volume of the enzyme reaction system; Venzyme is the volume of the enzyme; cenzyme: enzyme concentration, in mg / mL.

[0045] Example 1: Enzyme Sequence Analysis This invention is derived from Bacteroides filamentosa ( Bacteroides finegoldii A novel hyaluronic acid lyase, named BfiHAase, was discovered in FNMHLBE3K7, with its amino acid sequence shown in SEQ ID NO.1; from Nodicobacterium ( B. nordii A novel hyaluronic acid lyase, named BnoHAase, was discovered in FTJS11K9, with its amino acid sequence shown in SEQ ID NO.2; from Bacteroides clarus (… B. clarus A novel hyaluronic acid lyase, named BclHAase, was discovered in FFJLY22K22 and its amino acid sequence is shown in SEQ ID NO.3; from Bacteroides fibronectin ( B. cellulosilyticus A novel hyaluronic acid lyase, named BceHAase, was discovered in FSDTAELIBHI5, with the amino acid sequence shown in SEQ ID NO.4; from *Bacillus xylana* (… B. xylanisolvens A novel hyaluronic acid lyase, named BxyHAase, was discovered in FBJ60K5. Its amino acid sequence is shown in SEQ ID NO.5.

[0046] This study analyzed the physical properties of five identified hyaluronic acid lyases, including their molecular weight, isoelectric point, and signal peptide (Table 1). Simultaneously, the amino acid sequences of the screened hyaluronic acid lyases were analyzed. Phylogenetic analysis was performed using the maximum likelihood method with amino acid sequences from different members of the PL29 family selected from the CAZy database and the current five amino acid sequences, followed by multiple sequence alignment. Figure 1 As can be seen, the five amino acid sequences are distantly related to known PL29 family enzymes, and the amino acid sequences are significantly different, indicating that they have low similarity to currently reported sequences and have the potential to be developed into novel hyaluronic acid lyases.

[0047] Table 1. Analysis of the physical properties of hyaluronic acid lyase

[0048] Example 2: Obtaining the target gene for hyaluronic acid lyase According to the methods provided in *Molecular Cloning: A Laboratory Manual*, the culture was started after 36 hours of culture. B. finegoldii (Bacteroides frenulum FNMHLBE3K7) B. nordii (Nordibacterium FTJS11K9). B. clarus (Bacteroides clausti FFJLY22K22) B. cellulosilyticus (Bacteroides fibrinolyticus FSDTAELIBHI5) and B. xylanisolvens Total DNA was extracted from *Bacteroides xylanopsinus* FBJ60K5 and sent for whole-genome sequencing and gene function annotation. The CAZymes gene of *Bacteroides* was analyzed using bioinformatics techniques, and its classification was determined according to the CAZy family. B. finegoldii (Bacteroides frenulum FNMHLBE3K7) B. nordii (Nordibacterium FTJS11K9) B. clarus (Bacteroides clausti FFJLY22K22) B. cellulosilyticus (Bacteroides fibrinolyticus FSDTAELIBHI5) and B. xylanisolvens The hyaluronic acid lysin genes BfiHAase (nucleotide sequence SEQ ID NO. 6), BnoHAase (nucleotide sequence SEQ ID NO. 7), BclHAase (nucleotide sequence SEQ ID NO. 8), BceHAase (nucleotide sequence SEQ ID NO. 9), and BxyHAase (nucleotide sequence SEQ ID NO. 10) in *Bacteroides xylanae* FBJ60K5.

[0049] from B. finegoldii (Bacteroides frenulum FNMHLBE3K7) B. nordii(Nordibacterium FTJS11K9) B. clarus (Bacteroides clausti FFJLY22K22) B. cellulosilyticus (Bacteroides fibrinolyticus FSDTAELIBHI5) and B. xylanisolvens Genes of the polysaccharide lyase family PL29 were obtained from the whole genome of *Bacteroides xylanopsinus* FBJ60K5. Primers B.fi.Hyl, B.no.Hyl, B.cl.Hyl, B.ce.Hyl, and B.xy.Hyl were designed using these primers as templates for PCR amplification, and the amplification products were recovered. Simultaneously, the pE-sumo plasmid was linearized using inverse PCR primers pE-sumoF and pE-sumoR and recovered to obtain the gene sequences of the target gene and the linearized vector. The primers, components, and amplification conditions used are shown in Tables 2 and 3.

[0050] Table 2 Primer Sequences

[0051] Table 3 PCR reaction system

[0052] Add water to 50uL.

[0053] The PCR reaction conditions were as follows: 94℃, 5 min pre-denaturation; 95℃, 30 s denaturation; 55℃, 30 s annealing; 72℃, 2 min extension; 30 cycles of steps 2 to 4; 72℃, 5 min; 12℃. The target DNA fragment and linearized vector were obtained by purifying the target gene DNA using a gel purification kit provided by Vazyme.

[0054] Example 3: Construction of recombinant Escherichia coli Homologous recombination ligation of PCR amplification products and linearized vectors was performed using the Vazyme ClonExpress II One Step Cloning Kit. The recombinant products were transformed into E. coli DH5α, plated, and cultured. Positive clones were screened by plasmid extraction and PCR, and then identified by sequencing. Hyaluronic acid lyase expression vectors pE-sumo-BfiHAase, pE-sumo-BnoHAase, and pE... sumo BclHAase, pE sumo BceHAase and pE sumo BxyHAase was transformed into E. coli BL21(DE3) to obtain recombinant Escherichia coli.

[0055] Example 4: Expression, purification, and activity assay of hyaluronic acid lyase The specific steps are as follows: 1. Preparation of crude hyaluronic acid lysin solution The recombinant Escherichia coli prepared in Example 3 was inoculated into 5 mL LB liquid medium containing 50 μg / ml kanamycin sulfate resistance and cultured at 37°C and 220 rpm for 12 h to prepare seed solutions.

[0056] The prepared seed culture was inoculated into 100 mL LB fermentation medium (250 mL shake flask) at a 1% (v / v) inoculation rate and cultured at 37℃ and 220 rpm until the OD600 reached 0.6. At 0.8, IPTG with a final concentration of 0.5 mM was added, and the mixture was induced at 26℃ and 160 rpm for 9-10 h to prepare fermentation broths.

[0057] The prepared fermentation broth was centrifuged at 8000 r / min for 10 min at 4℃ to collect the cells. The cells were washed twice with sterile PBS buffer (pH 7.4), resuspended in 20 mL buffer, and sonicated to disrupt the cells. The supernatant was collected after centrifugation at 12000 r / min at 4℃ for 10 min and then subjected to SDS-PAGE. PAGE analysis. Electrophoresis results are as follows: Figure 2 As shown in the figure. The results indicate that all five enzymes were mainly expressed in soluble form.

[0058] 2. Preparation of pure hyaluronic acid lysin solution (1) Purification was performed using Ni-NTA Sepharose 6FF (His-Tag) affinity chromatography. The nickel column was equilibrated with 10 mL of equilibration buffer (20 mmol / L PB, pH 7.4) before loading the sample. Non-specific binding proteins were removed by washing with 10 mL of binding buffer (20 mmol / L PB, 500 mmol / L NaCl, 20 or 50 mmol / L imidazole). The recombinant target protein was eluted with 10 mL of elution buffer (20 mmol / L PB, 500 mmol / L NaCl, 150 mmol / L imidazole), and the eluent was collected as the purified recombinase. PD was used. 10. A pre-packed desalting column was used to desalt the purified enzyme. After equilibration with 25 mL of equilibration buffer (20 mmol / L PB, 500 mmol / L NaCl, pH 7.4), 2.5 mL of the sample was loaded, followed by elution with 3.5 mL of buffer. 2.5 mL of the sample was collected from the loading point. The 6 mL eluent is the desalted enzyme solution.

[0059] Using the above method, pure enzyme solutions containing BceHAase, BclHAase, BxyHAase, BnoHAase and BfiHAase were prepared respectively.

[0060] The purified enzyme was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) to assess the purification efficiency, and the target protein expression yield was determined using the Bradford method. The results are as follows: Figure 2 As shown, the effect of gel purification can be achieved. The protein concentration of the purified recombinant HAase was determined by the Bradford method. The protein concentrations of BceHAase, BclHAase, BxyHAase, BnoHAase and BfiHAase were 0.1747 mg / mL, 0.2189 mg / mL, 0.1814 mg / mL, 0.1239 mg / mL and 0.2012 mg / mL, respectively.

[0061] The enzyme activities of the above-mentioned purified enzymes were measured at 37℃ and pH 7.0. The results showed that the specific enzyme activities of BceHAase, BclHAase, BxyHAase, BnoHAase and BfiHAase were 236.6005 U / mg, 320.3768 U / mg, 198.4 U / mg, 317.9 U / mg and 446.5538 U / mg, respectively.

[0062] Example 5: Determination of the enzymatic properties of hyaluronic acid lyase 1. Effect of temperature on hyaluronic acid lyase activity Temperature can alter the rate of enzyme catalytic reactions and also lead to a decrease or inactivation of enzyme protein activity. This experiment will determine the optimal reaction temperatures for hyaluronic acid lyases BclHAase, BceHAase, BxyHAase, BfiHAase, and BnoHAase.

[0063] The specific experiment is as follows: A buffer system with a final concentration of 50 mmol / L sodium acetate, 5 mmol / L calcium acetate, 5 g / L hyaluronic acid, and pH 7.42 was prepared. 200 μL of the pure enzyme solution prepared in Example 3 was added to each 800 μL buffer system to obtain the final reaction system. The reaction system was placed at 25, 30, 35, 40, 45, and 50 °C. The change in A232 in the reaction solution was measured periodically (after 30 min of reaction). The enzyme activity value measured at the optimal temperature was taken as 100%, and the relative enzyme activity at other temperatures was calculated. The results are shown in Table 4 and... Figure 3 As shown.

[0064] Table 4. Relative enzyme activities of different enzymes at different temperatures

[0065] Experimental results showed that the optimal reaction temperature for all five enzymes was 40℃, and the activity of BfiHAase did not change significantly within the range of 30-45℃.

[0066] Furthermore, BclHAase, BceHAase, BxyHAase, BfiHAase, and BnoHAase were stored at 4℃ and 40℃ for 24 hours, respectively, to examine the temperature stability of each sample, and the relative enzyme activities were compared (the highest enzyme activity was 100%).

[0067] Experimental results (Table 5 and Figure 4 The results show that under storage conditions of 4℃, BfiHAase exhibits minimal activity change within 6 hours, retaining over 95% activity. After 6 hours of storage at 40℃, BfiHAase still retains over 90% activity. Regardless of whether it's 4℃ or 40℃, BfiHAase maintains over 75% activity after one day of storage. BceHAase maintains over 70% activity after 24 hours of storage at either 4℃ or 40℃. BnoHAase shows a higher activity retention rate at 4℃, retaining over 85% activity after 24 hours, but only retains 69.51% activity after 24 hours at 40℃. BxyHAase similarly only retains over 70% activity after 24 hours of storage at 4℃. BclHAase retains over 70% activity after 24 hours of storage at 40℃, but only retains 65.94% activity after storage at 4℃. The above results indicate that the hyaluronic acid lyase samples prepared in this study not only have high yield and high enzyme activity, but also stable properties, with the same optimal temperature and good temperature stability, showing promising development and application prospects in biomedicine and biochemical engineering.

[0068] Table 5. Thermal stability of different enzymes at 4℃

[0069] 2. Effect of pH on hyaluronic acid lyase activity Enzymatic reactions have their optimal pH range. Too high or too low a pH will affect the activity of the enzyme in catalytic reactions. This experiment will determine the optimal reaction pH for hyaluronic acid lyases BclHAase, BceHAase, BxyHAase, BfiHAase, and BnoHAase.

[0070] The specific experiment is as follows: A buffer system with a final concentration of 50 mmol / L sodium acetate, 5 mmol / L calcium acetate, and 5 g / L hyaluronic acid was prepared. 200 μL of the pure enzyme solution prepared in Example 3 was added to each 800 μL buffer system to obtain the final reaction system. The pH of the reaction system was adjusted to 3.0–10.0 according to the pH adaptability of different buffer solutions, and the reaction was carried out at 40°C. The changes in reaction solution A232 were measured periodically (after 30 min of reaction). The enzyme activity value measured at the optimal pH value was taken as 100%, and the relative enzyme activity at other pH values ​​was calculated. The results are shown in Table 6 and... Figure 5 As shown.

[0071] Table 6 Enzyme activities of different enzymes under different pH conditions

[0072] Experimental results showed that BclHAase and BnoHAase remained stable in phosphate buffer at pH 7.01-7.98, and maintained over 70% of their activity between pH 6.08-8.02. The optimal pH values ​​for these five enzymes differed slightly, but all fell within the range of 6.0-8.0. Compared to the other four enzymes, BfiHAase had a slightly more acidic optimal pH, possibly due to its higher content of acidic amino acids. It maintained over 50% activity even at pH close to 4, demonstrating its potential for industrial applications.

[0073] 3. Effect of substrate concentration on enzyme-catalyzed reaction rate Substrate concentration significantly affects the efficiency of enzymatic reactions. Under otherwise constant conditions, the effect of substrate concentration on reaction rate follows a rectangular hyperbolic relationship. At low substrate concentrations, the reaction rate is directly proportional to the substrate concentration, indicating a first-order reaction. As the substrate concentration increases, the reaction rate no longer increases proportionally, indicating a mixed-order reaction. When the substrate concentration reaches a certain level, the reaction rate no longer increases, reaching its maximum, indicating a zero-order reaction.

[0074] The specific experiment is as follows: A buffer system with a final concentration of 50 mmol / L sodium acetate and 5 mmol / L calcium acetate was prepared. Hyaluronic acid with different final concentrations (0.1, 0.5, 1, 2, 3, 4, 5 mg / ml) was added to each system. 200 μL of the pure enzyme solution prepared in Example 3 was added to each 800 μL buffer system to obtain the final reaction system. The reaction system was placed at the optimal temperature and pH conditions for each enzyme and reacted for 30 min. After the reaction, the catalytic constant of the enzyme was fitted using a Michaelis-Menten curve. Km Value and maximum reaction rate V The maximum value is shown in Table 7. Figure 6As shown. The results indicate that BfiHAase has the highest k cat / Km The value can reach 10.999 μM. -1 min -1 And its Km The relatively low value indicates that it has a strong substrate affinity and catalytic efficiency for HA.

[0075] Table 7 Catalytic kinetic parameters of different enzymes

[0076] Example 6: Molecular docking verification of hyaluronic acid lyase and hyaluronic acid substrate Molecular docking can predict how substrate molecules bind to the active site of an enzyme, recognizing key amino acid residues, hydrogen bonds, hydrophobic interactions, and more. This helps in understanding the catalytic mechanism and substrate specificity of enzymes. It can also simulate how reaction products are released from the active site.

[0077] This study used AlphaFold3 to perform homology modeling of the target protein, simulating the three-dimensional structure of the hyaluronic acid lyase target protein. Molecular docking between the hyaluronic acid lyase and hyaluronic acid was performed using AutoDock vina. Finally, PyMOL software was used for graphical visualization, and the results are shown below. Figure 7 As shown, the binding energy of BfiHAase can reach -5.8 kcal / mol, and the enzyme kinetic parameters indicate that BfiHAase has a strong binding ability to hyaluronic acid substrates.

[0078] Although the embodiments of this invention have been disclosed above, they are not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of this invention. Therefore, the scope of protection of this invention should be determined by the claims.

Claims

1. A hyaluronic acid lysin, characterized in that, The amino acid sequence of the hyaluronic acid lyase is shown in any one of SEQ ID NO. 1 to 5.

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

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

4. A recombinant microbial cell expressing the hyaluronic acid lyase of claim 1, or containing the recombinant vector of claim 3.

5. A recombinant Escherichia coli, characterized in that, The hyaluronic acid lyase described in claim 1 was expressed using pE-SUMO as an expression vector.

6. The recombinant Escherichia coli as described in claim 5, characterized in that, The expression host was Escherichia coli BL21(DE3).

7. A method for degrading hyaluronic acid, characterized in that, The hyaluronic acid lysin of claim 1, the recombinant microbial cell of claim 4, or the fermentation broth and / or lysis buffer of the recombinant Escherichia coli of claim 5 or 6 are added to a reaction system containing hyaluronic acid for reaction.

8. The method as described in claim 7, characterized in that, The reaction temperature is 30–40℃, and the reaction pH is 6–7.

9. The method as described in claim 8, characterized in that, The molecular weight of the hyaluronic acid is 1.5-2.5 million Da.

10. The use of the hyaluronic acid lyase according to claim 1 in the degradation of hyaluronic acid or the preparation of low molecular weight hyaluronic acid.

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