High-enzyme-activity heat-resistant hyaluronidase mutant and application thereof

Abstract

The invention discloses a high-enzyme-activity heat-resistant hyaluronidase mutant and an application of the high-enzyme-activity heat-resistant hyaluronidase mutant. The hyaluronidase mutant is obtained by mutating the 218th amino acid of a wild type hyaluronidase amino acid sequence from asparagine to aspartic acid and mutating the 267th amino acid of the wild type hyaluronidase amino acid sequence from glutamine to arginine, and the amino acid sequence of the hyaluronidase mutant is as shown in SEQ ID NO. 1. According to the invention, the hyaluronidase mutant N218D / Q267R with improved enzyme activity and heat resistance is constructed, the specific enzyme activity of the hyaluronidase mutant N218D / Q267R is 2.09 times that of a wild type under the optimal reaction condition, and the hyaluronidase mutant N218D / Q267R has good heat resistance and good industrial application potential.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a high-enzyme-activity heat-resistant hyaluronidase mutant and its application, and more specifically to a hyaluronidase mutant derived from Bacillus sp.A50 and its encoding gene, as well as its application in the preparation of small-molecule hyaluronic acid and oligomeric hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a linear glycosaminoglycan formed by the condensation of disaccharide units composed of alternating D-glucuronic acid and N-acetylglucosamine. Its molecular formula is (C 14 H 21 NO 11 ) n , in various forms, widely exists in the extracellular matrix of various organisms, such as cockscombs, vitreous body, synovial fluid, cartilage, etc. Hyaluronic acid is a white powdery substance with strong water absorption. It can be used as a natural moisturizer. It forms a gel after dissolving in water and exhibits excellent rheological properties. The viscosity of the aqueous solution is linearly related to the average molecular weight. The larger the average molecular weight, the stronger the viscosity of the hydrogel. As a linear macromolecule, hyaluronic acid has a wide range of molecular weights. According to the size of the molecular weight, it can be divided into large molecular weight hyaluronic acid (>1000kDa), medium molecular weight hyaluronic acid (250kDa-1000kDa), small molecular weight hyaluronic acid (10-250kDa) and oligomeric hyaluronic acid (<10kDa). Depending on the molecular weight, hyaluronic acid can exhibit a variety of biological functions: large molecular hyaluronic acid has strong viscoelasticity and good mechanical properties, and is often used as a tissue filler to assist in repairing cartilage and restoring joints; medium molecular hyaluronic acid is moderate in size and can form a permeable membrane on the skin, playing a role in moisturizing and lubricating; small molecular hyaluronic acid and oligomeric hyaluronic acid can penetrate the skin barrier, inhibit inflammation, scavenge free radicals, delay aging, and promote the repair of damaged cells. In addition, hyaluronic acid is also widely used in food and daily necessities as a dietary supplement and cosmetic raw material.

[0003] With the increasing application of hyaluronic acid, the preparation method of hyaluronic acid has also become a hot topic of research. Among them, large molecular hyaluronic acid is mainly obtained by biological extraction and microbial fermentation, while small molecular hyaluronic acid and oligomeric hyaluronic acid are prepared by degrading large molecular hyaluronic acid by physical, chemical and enzymatic degradation. Compared with non-enzymatic degradation, enzymatic hydrolysis has the advantages of mild reaction conditions, high catalytic efficiency, controllable product molecular weight and low cost. It is the most suitable method for preparing small molecular hyaluronic acid and oligomeric hyaluronic acid. Hyaluronidase (HAase) is a class of glycosidases that can hydrolyze hyaluronic acid, and some of them also have substrate selectivity for glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and dermatan sulfate (DS). Hyaluronidase was first discovered in the testicles of mammals. Because it can promote the diffusion of substances such as vaccines, dyes and toxins, it is also called "diffusion factor". According to the different mechanisms of catalytic degradation, hyaluronidase can be divided into three categories: the first is hyaluronic acid 4-glycoside hydrolase (EC3.2.1.35) from mammals; the second is hyaluronic acid 3-glycoside hydrolase (EC3.2.1.36) from the salivary glands of leeches and hookworms; the third is hyaluronic acid lyase (EC4.2.2.1) from microorganisms. Although enzymatic hydrolysis is the optimal solution for preparing small molecule hyaluronic acid and oligomeric hyaluronic acid, animal-derived hyaluronidase has limited sources, low purity, and immunogenicity, and microbial-derived hyaluronidase has low enzymatic activity, narrow optimal reaction range, and poor stability, which limits its large-scale application in industrial production. Therefore, the key to achieving industrial production of small molecule hyaluronic acid and oligomeric hyaluronic acid is to obtain heat-resistant hyaluronidase with high enzyme activity using genetic engineering and enzyme engineering technology. Summary of the invention

[0004] The purpose of the present invention is to provide a hyaluronidase mutant derived from Bacillus sp. A50, a coding gene, a recombinant vector containing the mutant gene, and the use of the hyaluronidase mutant in catalytic preparation of small molecule hyaluronic acid and oligomeric hyaluronic acid.

[0005] The technical solution adopted by the present invention is:

[0006] A hyaluronidase mutant, whose amino acid sequence is shown in SEQ ID NO.1, has higher enzyme activity and better thermal stability than wild-type hyaluronidase. The hyaluronidase mutant is obtained by mutating the 218th amino acid from asparagine to aspartic acid and the 267th amino acid from glutamine to arginine from Bacillus sp.A50. The present invention uses error-prone PCR technology to carry out directed evolution of hyaluronidase, further improving the enzyme activity of hyaluronidase and improving its heat resistance.

[0007] The present invention also relates to the coding gene of the hyaluronidase mutant. Specifically, the nucleotide sequence of the coding gene is shown in SEQ ID NO.2.

[0008] The present invention also relates to a recombinant expression vector containing the coding gene. These recombinant expression vectors can be constructed by connecting the hyaluronidase mutant nucleotide sequence of the present invention to various vectors using conventional methods in the art. The vectors can be various conventional vectors in the art, such as various plasmids, phages or viral vectors.

[0009] Preferably, the recombinant expression vector is pET-28a.

[0010] The present invention also relates to an engineered bacterium containing the recombinant vector. As an application of a recombinant expression vector, a genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be any conventional host microorganism in the art, mainly satisfying that the recombinant expression vector can stably self-replicate and the hyaluronidase mutant gene of the present invention carried by the vector can be effectively expressed.

[0011] Preferably, the host microorganism is Escherichia coli BL21 (DE3).

[0012] The present invention also relates to a hyaluronidase mutant, a coding gene of the hyaluronidase mutant, and application of the engineering bacteria in catalytic preparation of hyaluronic acid.

[0013] When used as a biocatalyst for production, the hyaluronidase mutant of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, carrier-immobilized enzymes, cell fragments, etc.; the bacterial cell forms include living bacterial cells and dead bacterial cells.

[0014] Specifically, the application is: using the hyaluronidase mutant as a catalyst and macromolecular hyaluronic acid as a substrate, reacting at pH = 6.5 and 45° C. for 0.5-3 hours to obtain hyaluronic acid in the reaction solution.

[0015] Preferably, the hyaluronic acid is small molecule hyaluronic acid and oligomeric hyaluronic acid.

[0016] The molecular weight of the obtained hyaluronic acid is 3KDa to 9KDa.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that: the present invention constructs a hyaluronidase mutant N218D / Q267R with improved enzyme activity and heat resistance. Under the optimal reaction conditions, the specific enzyme activity of the hyaluronidase mutant N218D / Q267R reaches 2.17×10 6 U / mg, which is 2.09 times that of the wild type, and has good heat resistance. After reacting at the optimum temperature (45°C) for 3 hours, the relative enzyme activity is 82.3%, while the wild type has only 43.4% relative enzyme activity after reacting at the optimum temperature (40°C) for 3 hours. The thermal stability is significantly improved, and it has good potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the agarose gel electrophoresis result of error-prone PCR amplification of pET-28a-hly. Lane M: 5000bp DNA marker; Lane 1: error-prone PCR amplification plasmid pET-28a-hly product.

[0019] Figure 2 This is the SDS-PAGE electrophoresis of the purification of hyaluronidase and mutant N218D / Q267R. Lane M: 17-180KDa protein marker; Lane 1: hyaluronidase WT purification; Lane 2: hyaluronidase mutant N218D / Q267R purification.

[0020] Figure 3 It is the specific enzyme activity of hyaluronidase and mutants T105K, N218D / Q267R, and L587F.

[0021] Figure 4 The effect of temperature and time on the activity and stability of hyaluronidase and mutant N218D / Q267R.

[0022] Figure 5 The effect of pH on the activity and stability of hyaluronidase and mutant N218D / Q267R.

[0023] Figure 6 This is a diagram of the reaction process of HA degradation by hyaluronidase and mutant N218D / Q267R. DETAILED DESCRIPTION

[0024] Example 1

[0025] Construction of hyaluronidase-producing strain E. coli BL21(DE3) / pET-28a-hly

[0026] The hyaluronidase gene (hly) from Bacillus sp. A50 was synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed into the pET-28a plasmid, named pET-28a-hly, and the vector was transformed into the E. coli DH5α strain. The recombinant bacteria E. coli DH5α was inoculated into 5mL LB test tube culture medium and cultured at 37°C and 220rpm for 12h. After the culture was completed, the bacteria were collected by centrifugation at 12000rpm for 1min, and the plasmid was extracted with a plasmid extraction kit. The obtained plasmid was transferred into the expression host of E. coli BL21 (DE3) to obtain the hyaluronidase-producing strain E. coli BL21 (DE3) / pET-28a-hly, and a glycerol strain was prepared by adding sterile glycerol with a final concentration of 20% and stored at -80°C.

[0027] Example 2

[0028] Construction and screening of hyaluronidase mutant gene library

[0029] Using the pET-28a-hly plasmid obtained in Example 1 as a template, a hyaluronidase mutation library was constructed by error-prone PCR reaction. The error-prone PCR reaction system is shown in Table 1, and the reaction procedure is: 95°C 2min, followed by 35 cycles of 95°C 30s, 58°C 30s and 72°C 45s. This condition can achieve an average of 1-2 mutations per 1000bp base. After the PCR reaction, the amplified product was detected by 1% agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 3324bp. The initial plasmid template was digested with QuickCut Dpn I at 37°C for 1h, and the gene library fragment was purified and recovered with a gel recovery kit. The purified fragment obtained was connected to the pET-28a plasmid to obtain the recombinant plasmid pET-28a-hly-X. The recombinant plasmid pET-28a-hly-X was transformed into the E. coli BL21 (DE3) host, and then spread on the enrichment medium plate and cultured overnight. The enrichment medium formula is: peptone 10g / L, yeast extract 10g / L, K2PO4·3H2O2g / L, MgSO4·7H2O 0.2g / L, HA1g / L, 2.5% agar powder.

[0030] Upstream mutation primer: 5'-cg ggatcc aatgaatctactttacta-3'

[0031] Downstream mutation primer: 5'-cc ctcgagttattatctgattgagtt-3'

[0032] Note: The underlined lines are restriction enzyme cleavage sites.

[0033] Table 1 Error-prone PCR reaction system

[0034]

[0035] Add 500 μL of LB liquid culture medium containing 50 μg / mL kanamycin to each well of the 96-well plate, then pick a single colony with a sterile toothpick and place it in the corresponding well, select 2 wells to add a WT single colony as a positive control, select two wells to add phosphate buffer as a negative control, cover the plate, and culture at 37°C, 180 rpm for 6 h.

[0036] Take 50 μL of the cultured seed solution and add it to a new 96-well plate, add 900 ul of liquid LB medium containing 50 μg / mL kanamycin, cover the plate, shake and culture at 37°C, 280 rpm for 2 hours, add 10 μL of 0.02M IPTG and 100 μL of liquid LB medium to each well, cover the plate, shake and culture at 28°C, 180 rpm for 18 hours. Add 50 μL of cell lysis solution (50 mmol / L Tris-HCl (pH=8.0), 2 mM EDTA, 100 mM NaCl, 0.5% TritionX-100, 1 mg / mL lysozyme, 100 μg / mL PMSF) to the well, lyse at 25°C for 2 hours to obtain a crude enzyme solution.

[0037] Take 200 μL crude enzyme solution in a 96-well plate, add 200 μL 5g / L HA solution (pH=6.5), react at 45°C for 30 minutes, and terminate the reaction in a boiling water bath for 2 minutes. Then add 50 μL DNS reagent, heat in a microwave oven for 30 seconds on medium heat to develop color, and observe the color development results. Use an enzyme marker to detect the absorbance of the reaction solution at 540nm. All results were measured three times and the average value was taken. Some results are shown in Table 2.

[0038] Table 2 DNS method absorbance detection results (partial)

[0039]

[0040] According to the results of the DNS method, samples B5, E11, and G8 with higher absorbance than the positive control were labeled, and 50 μL of seed solution was added to 10 mL of LB liquid medium containing kanamycin. After 12 hours of shaking culture at 37°C and 180 rpm, the bacterial solution was taken and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the mutation sites of the three mutant strains were T105K, N218D / Q267R, and L587F. 200 μL of the corresponding seed solution was taken into a glycerol tube, and sterile glycerol with a final concentration of 20% was added to prepare the glycerol strain and stored at -80°C.

[0041] Example 3

[0042] Expression and purification of hyaluronidase and its mutants

[0043] The recombinant E. coli glycerol bacteria obtained in Example 1 and Example 2 were taken out and inoculated into 5 mL LB medium containing 50 μg / mL kanamycin, respectively, and cultured at 37°C, 220 rpm for 14 h. Then, they were inoculated into 50 mL LB medium containing 50 μg / mL kanamycin at an inoculum size of 1% (v / v), and cultured at 37°C, 220 rpm until OD 600 When the pH reaches 0.6-0.8, add IPTG with a final concentration of 0.2mM, and continue induction culture at 28℃ and 200rpm for 20h. After the induction, collect the bacterial solution, centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, collect the bacteria, rinse twice with PBS buffer and resuspend, place on ice water mixture for ultrasonic disruption, the working conditions of ultrasonic cell disruptor are: power 750W, working time 5s, interval time 10s, a total of 99 times. After the disruption is completed, centrifuge at 4℃ and 8000rpm for 10min, collect the supernatant as the crude enzyme solution, and filter with 0.45μm microporous filter membrane for use.

[0044] Use nickel ion affinity chromatography column for protein purification, rinse the column with deionized water at 4°C (about 10 column volumes), and then balance the column with low salt concentration buffer (500mmol / L NaCl, 50mM Tris, pH=7.0). When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer pumped into the column (about 5 column volumes of buffer), add the reserved membrane-passed crude enzyme solution to the column. First, rinse with a buffer containing a low concentration of imidazole (500mmol / L NaCl, 50mmol / L imidazole, 50mM Tris, pH=7.0) to remove impurities until baseline equilibrium, and then elute the target protein with an eluent containing a high concentration of imidazole (500mmol / L NaCl, 500mmol / L imidazole, 50mM Tris, pH=7.0). Collect the eluent of the absorption peak, which is the pure enzyme solution of the target protein, and perform SDS-PAGE protein gel electrophoresis to verify the band.

[0045] Example 4

[0046] Determination of hyaluronidase and mutant enzyme activities

[0047] Hyaluronidase from microorganisms can catalyze HA to form unsaturated disaccharide structures, resulting in maximum light absorption at 232nm. The enzyme activity can be determined by ultraviolet spectrophotometry to detect the light absorption of unsaturated disaccharides produced by HA degradation. The activity unit (U) of the enzyme is defined as: the amount of enzyme that catalyzes the formation of 1μmol of unsaturated disaccharides per minute under standard conditions. Specific enzyme activity definition (U / mg): the number of enzyme activity units per unit mass (mg) of enzyme protein under standard reaction conditions.

[0048] The enzymatic activity of hyaluronidase and mutants was determined under standard reaction conditions. The enzyme solution obtained in Example 3 was diluted to 10 μg / L with a phosphate buffer solution of pH=6.5. A certain amount of HA was dissolved in a phosphate buffer solution of pH=6.5 to prepare a substrate solution of 5 mg / mL. 9.9 mL of HA solution was drawn into a stoppered test tube, three tubes were taken as a group and parallel, incubated at the optimum temperature for 10 min, 100 μL of the diluted enzyme solution was taken and mixed, and the incubation reaction was continued for 30 min. After the reaction was completed, it was boiled and inactivated for 2 min. The enzyme solution inactivated in advance plus the HA substrate solution was used as a blank control, and the absorbance at 232 nm was measured. The protein quality was detected by the Coomassie Brilliant Blue-G250 method.

[0049] Results Figure 3 The specific enzyme activity of wild-type hyaluronidase is 1.04×10 6 U / mg, and the specific enzyme activity of mutant T105K was 1.42×10 6 U / mg, and the specific enzyme activity of mutant N218D / Q267R was 2.17×10 6 U / mg, and the specific enzyme activity of mutant L587F was 1.63×10 6 U / mg. The mutant N218D / Q267R had the highest specific enzyme activity, which was 2.09 times that of the wild type, and subsequent catalytic conditions were explored for it.

[0050] Example 5

[0051] Effects of temperature and time on the activity and stability of hyaluronidase and its mutant N218D / Q267R

[0052] A certain amount of HA was dissolved in a phosphate buffer solution at pH = 6.5 to prepare a 2 mg / mL substrate solution. 9.9 mL of HA solution was drawn into a stoppered test tube, and three tubes were taken as a group in parallel, and kept warm for 10 min at different temperatures (30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C). 100 μL of the enzyme solution obtained in Example 3 was added to each group, mixed, and the reaction was continued for 30 min. After the reaction, it was boiled and inactivated for 2 min. The enzyme solution inactivated in advance plus the HA substrate solution was used as a blank control, and the absorbance at 232 nm was measured. The highest enzyme activity was defined as 100% relative enzyme activity.

[0053] 100 μL of the enzyme solution obtained in Example 3 was kept at the optimal temperature (40°C for the wild type and 45°C for the mutant) for a period of time (15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min), and added to stoppered test tubes containing 2 mg / mL HA substrate solution, respectively. The temperature was kept for 30 min, and then boiled and inactivated for 2 min. The enzyme solution inactivated in advance plus HA substrate solution was used as a blank control, and the absorbance at 232 nm was measured. The enzyme activity at 0 min was defined as 100% relative enzyme activity.

[0054] Results Figure 4 The optimum temperature of wild-type hyaluronidase is 40°C, and the optimum temperature of mutant N218D / Q267R is 45°C. In terms of thermal stability, the relative enzyme activity of wild-type hyaluronidase is only 43.4% after 3 hours of incubation at the optimum temperature, while the relative enzyme activity of mutant N218D / Q267R is 82.3% after 3 hours of incubation at the optimum temperature, which still retains good enzyme activity.

[0055] Example 6

[0056] Effects of pH on the activity and stability of hyaluronidase and its mutant N218D / Q267R

[0057] A certain amount of HA was dissolved in a buffer system with different pH values ​​to prepare a 2 mg / mL substrate solution. The buffers used under different conditions were: citric acid-Na2HPO4 buffer (pH4.0-5.0) and NaH2PO4-Na2HPO4 buffer (pH5.0-8.5). 9.9 mL of HA solution was pipetted into a stoppered test tube, and three tubes were taken as a group and incubated at the optimum temperature for 10 min. 100 μL of the enzyme solution obtained in Example 3 was added to each group, mixed, and the reaction was continued for 30 min. After the reaction was completed, it was boiled and inactivated for 2 min. The enzyme solution inactivated in advance plus the HA substrate solution was used as a blank control. The absorbance at 232 nm was measured, and the highest enzyme activity was defined as 100% relative enzyme activity.

[0058] 100 μL of the enzyme solution obtained in Example 3 was diluted with different buffer systems (pH 4.0-8.5), kept at the optimal temperature for a period of time (1 h, 2 h), and added to stoppered test tubes containing 2 mg / mL HA substrate solution. The temperature was kept for 30 min, and then boiled and inactivated for 2 min. The enzyme solution inactivated in advance plus HA substrate solution was used as a blank control, and the absorbance at 232 nm was measured. The enzyme activity at 0 min was defined as 100% relative enzyme activity.

[0059] Results Figure 5 The optimum pH of wild-type hyaluronidase and mutant N218D / Q267R is 6.5, and both lose enzyme activity at pH = 4.0. In terms of stability, the wild-type and mutant N218D / Q267R have good stability in the range of 6.0-7.0, and the relative enzyme activities are 91.3% and 97.0% respectively after being incubated for 2 hours at pH 6.5.

[0060] Example 7

[0061] Application of hyaluronidase and mutant N218D / Q267R in the preparation of small molecule hyaluronic acid and oligomeric hyaluronic acid

[0062] Take 30g / L hyaluronic acid solution (pH=6.5) and keep it at the optimal temperature for 10min. Add 100:1 hyaluronidase and mutant to the substrate solution, respectively, with an enzyme concentration of 10μg / L, and continue to keep warm to start the enzymatic reaction. During the reaction, take out part of the reaction solution at intervals, heat and boil for 2min to terminate the reaction, and filter it with a 0.45μm filter membrane for later use. The enzymatic reaction time is controlled at 0-3h.

[0063] The molecular weight of hyaluronic acid in the reaction solution was determined by viscometer method. 3 volumes of ethanol were added to the enzymatically hydrolyzed reaction solution, and the mixture was thoroughly mixed and precipitated at 4°C for 1 hour. The precipitate was centrifuged at 4°C and 5000 rpm for 15 minutes. The precipitate was washed with an equal volume of ethanol saline solution (75% ethanol, 25% 0.2M NaCl) and resuspended, and centrifuged at 4°C and 8000 rpm for 15 minutes. The supernatant was discarded and the precipitate was dried to constant weight. The precipitate was dissolved with 0.2M NaCl solution and diluted to a certain concentration such that 1.3 <t n / t0<1.5, and t0>100s, the time interval between two outflows does not exceed 0.2s. Where t0 is the outflow time of the blank solvent, t n is the elution time of the sample solution.

[0064] The intrinsic viscosity [η] and the average molecular weight Mw of the sample were calculated according to the one-point method formula.

[0065]

[0066]

[0067] Where t0 is the elution time of the blank solvent, t1 is the elution time of the sample solution, and c is the concentration of the sample solution after dilution.

[0068] Results Figure 6 After adding wild-type hyaluronidase to the macromolecular hyaluronic acid (2.30MDa) solution, the molecular weight of hyaluronic acid dropped to 1.23MDa after 30s, and after 30min of reaction, the molecular weight of hyaluronic acid dropped to 108KDa, reaching the size of small-molecule hyaluronic acid, but the reaction speed slowed down after 45min, and oligomeric hyaluronic acid with a molecular weight of <10KDa could be obtained after 1.5h of reaction, and the molecular weight of hyaluronic acid obtained after 3h of reaction was 7100Da. After the mutant N218D / Q267R was added to the solution, the molecular weight of hyaluronic acid dropped to 0.91MDa after 30s, and a small-molecule hyaluronic acid with a size of 171KDa was obtained after 10min of reaction, and an oligomeric hyaluronic acid with a size of 9KDa could be obtained after 0.5h of reaction. The molecular weight of the hyaluronic acid finally obtained after 3h of reaction could reach as low as 3000Da, and the catalytic efficiency was greatly improved compared with the wild-type hyaluronidase, and it could degrade and obtain oligomeric hyaluronic acid with a smaller molecular weight.

[0069] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A hyaluronidase mutant, characterized in that: The hyaluronidase mutant is obtained by mutating the 218th amino acid of the wild-type hyaluronidase amino acid sequence from asparagine to aspartic acid, and the 267th amino acid from glutamine to arginine. The amino acid sequence of the hyaluronidase mutant is shown in SEQ ID NO.

1.

2. A hyaluronidase mutant according to claim 1, characterized in that: The wild-type hyaluronidase amino acid sequence is Bacillus ( Bacillus sp. A50) amino acid sequence.

3. The gene encoding the hyaluronidase mutant according to claim 1.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

5. A recombinant expression vector containing the gene encoding the hyaluronidase mutant according to claim 3.

6. The recombinant expression vector according to claim 5, characterized in that The vector is a pET series expression vector.

7. An engineered bacterium containing the recombinant expression vector as claimed in claim 5.

8. Use of the hyaluronidase mutant according to claim 1, the encoding gene of the hyaluronidase mutant according to claim 3, or the engineered bacteria according to claim 7 in the preparation of small molecule hyaluronic acid or oligomeric hyaluronic acid.

9. The use according to claim 8, characterized in that The hyaluronidase mutant is used as a catalyst and macromolecular hyaluronic acid is used as a substrate. The reaction is carried out at pH=6.5 and 45° C. for 0.5-3 hours to obtain hyaluronic acid in the reaction solution.

10. The use according to claim 9, characterized in that: The molecular weight of the obtained hyaluronic acid is 3Kda~9KDa.

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