Method for efficiently separating and purifying hyaluronidase

Abstract

The invention provides a method for efficiently separating and purifying hyaluronidase, and belongs to the technical field of hyaluronidase preparation.According to the method for separating and purifying hyaluronidase, magnetic beads are added into thallus crushing liquid or fermentation supernate, purification is conducted in combination with certain incubation conditions and elution conditions, and high-purity hyaluronidase can be obtained; the prepared hyaluronidase is high in enzyme activity, the purification method is easy to operate and low in cost, expensive and precise instruments are not needed, and the method is suitable for purification systems of different scales.

Description

Technical Field

[0001] The invention relates to a method for efficiently separating and purifying hyaluronidase, and belongs to the technical field of hyaluronidase preparation. Background Art

[0002] Hyaluronic acid (HA) is a high-molecular-weight mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid disaccharide units repeatedly connected alternately. It is an important component of human skin, vitreous body, synovial fluid and cartilage tissue. Due to its high viscoelasticity and plasticity, strong water retention and permeability and good biocompatibility, it is widely used in food, cosmetics and medicine. The biological properties of HA are closely related to its chain length and relative molecular weight. HA with high molecular weight fraction (average molecular weight of 1 MDa-1.5 MDa) has good moisturizing properties, and HA with low molecular weight fraction (average molecular weight of 20 kDa-400 kDa) has anti-wrinkle effect because it can penetrate the skin barrier. Hyaluronic acid fragments with a relative molecular weight of less than 10 kDa are usually called hyaluronic acid oligosaccharides. With the deepening of research on hyaluronic acid oligosaccharides, researchers have found that hyaluronic acid oligosaccharides with different degrees of polymerization have different biological activities, such as unsaturated hyaluronic acid disaccharides have unique anti-inflammatory activity, and unsaturated hyaluronic acid tetrasaccharides have pro-angiogenic activity.

[0003] At present, the degradation methods of HA mainly include physical degradation, chemical degradation and enzymatic degradation. Among them, it is difficult to degrade HA into oligosaccharides by physical treatment. Although chemical methods can obtain oligosaccharide products, they require harsh reaction conditions and may introduce unnecessary side reactions. Hyaluronic acid is specifically degraded by hyaluronic acid lyase. The reaction conditions are mild, easy to control, and the product structure is uniform, which overcomes the shortcomings of physical and chemical methods. Hyaluronidase has gradually become the main tool for preparing hyaluronic acid oligosaccharides.

[0004] Hyaluronidase is an endogenous glycosidase that degrades the glycosidic bonds in hyaluronic acid polymers to produce hyaluronic acid of different molecular weights. In 1939, this enzyme was first discovered by Chain and Duthie in animal testicular extracts and bacterial filtrates: as a "diffusion factor", the enzyme can accelerate the diffusion of dyes and toxins injected into the body. Typical application scenarios of hyaluronidase include assisting drug diffusion, preparing hyaluronic acid of a specific molecular weight, etc. The preparation of hyaluronidase with good activity and high purity is of great significance for the large-scale preparation of high-purity hyaluronic acid oligosaccharides.

[0005] At present, hyaluronidase is mainly obtained by fermentation of genetically engineered bacteria to obtain recombinant hyaluronidase. In the prior art, the purification of hyaluronidase is mostly carried out by membrane filtration (CN 115287275 A), chromatography (CN 118086255A) and high-performance liquid phase (CN 114507655 A). Among them, the purification steps of the membrane filtration method are: the leech hyaluronidase is sequentially passed through the plate and frame, (low-pressure) ceramic membrane, (high-pressure) ceramic ultrafiltration membrane process and a certain concentration of buffered salt solution is added to reduce the bacterial and small molecule impurities in the hyaluronidase fermentation liquid. The method process is cumbersome, the enzyme activity yield is low, the impurity protein is not easy to remove, and the product purity is low; the purification method described in the chromatography method is: the recombinant Escherichia coli fermentation liquid is crushed to obtain a crushed liquid, the crushed liquid is centrifuged to collect the supernatant, the supernatant is filtered and impurities are removed, and the recombinant bacteria supernatant is obtained, and then the AKTA protein purification system is used, and the nickel column affinity chromatography is used for purification, and the elution peak is collected to obtain a hyaluronidase enzyme liquid. The method needs to be pre-treated by centrifugation and membrane filtration to prevent the obstruction of the chromatography column, and expensive instruments and equipment need to be used, and more energy is spent on the use and maintenance of the equipment, which is not suitable for the industrial continuous production of enzyme-cut small molecule hyaluronic acid; the purification method described in the high-performance liquid phase method is to use cation exchange gel chromatography and molecular sieve purification, and then prepare a method for preparing hyaluronidase using a preparative high-performance liquid chromatograph. The method also requires expensive and precise instruments, which is costly and is not suitable for industrial amplification production. At present, due to the limitations of the above-mentioned commonly used purification methods, the purification preparation of hyaluronidase has not yet been industrialized. Therefore, a purification method with simple steps, low cost, high purification degree and high enzyme activity recovery rate is needed to make up for the shortcomings of traditional hyaluronidase purification technology.

[0006] There have been relevant reports in the prior art on the separation and purification of proteins using magnetic beads, but the separation and purification of different proteins require different technical methods and the application effects are also different; although hyaluronidase is a protein, it has certain catalytic activity, and there are also many types of amino acid sequence compositions of hyaluronidase. There are many factors that affect the enzyme activity during the separation and purification of hyaluronidase, and the effects of different separation and purification methods on the activity of hyaluronidase cannot be predicted. There are no reports in the prior art on the separation and purification of hyaluronidase using magnetic beads. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for efficiently separating and purifying hyaluronidase.

[0008] In order to solve the problems of difficulty in separation and purification of recombinant hyaluronidase, complicated steps and high cost, the present invention provides a separation and purification method for recombinant hyaluronidase with strong operability and good repeatability, which can obtain high-purity and high-activity.

[0009] The technical solution of the present invention is as follows: A method for separating and purifying hyaluronidase comprises the following steps: (1) fermenting the recombinant engineered bacteria expressing hyaluronidase, collecting the bacterial cells after fermentation of the recombinant engineered bacteria expressing hyaluronidase intracellularly, and disrupting the cells to obtain a bacterial cell disruption solution; Or after fermentation of the recombinant engineered bacteria expressing extracellular hyaluronidase, the bacteria are removed and the fermentation supernatant is retained; (2) Purification of intracellularly expressed hyaluronidase: Add the equilibrated magnetic beads to the bacterial cell disruption solution obtained in step (1) for 4-8 o C, incubate at 80-100 rpm for 15-45 min. After the incubation, collect the magnetic beads and remove the supernatant. Or purification of extracellularly expressed hyaluronidase: add the equilibrated magnetic beads to the fermentation supernatant obtained in step (1) for 4-8 o C, incubate at 80-100 rpm for 15-45 min. After the incubation, collect the magnetic beads and remove the supernatant. (3) Resuspend the magnetic beads collected in step (2) using a washing buffer with a volume 5-10 times the solid volume of the magnetic beads, mix well, collect the magnetic beads again, remove the supernatant, and repeat the washing step 1-2 times; (4) Resuspend the magnetic beads in elution buffer 5-10 times the volume of the magnetic beads. o C, incubate at 80-100 rpm for 1-10 min, collect the magnetic beads, and recover the eluate containing hyaluronidase.

[0010] Preferably, according to the present invention, in step (1), the recombinant engineered bacteria expressing hyaluronidase is a recombinant Escherichia coli or a recombinant yeast.

[0011] Further preferably, in step (1), the recombinant Escherichia coli is a host bacterium, and the hyaluronidase encoding gene sequence is transferred into the Escherichia coli using a vector; The recombinant yeast uses Pichia pastoris as a host bacterium, and the hyaluronidase encoding gene sequence is transferred into Pichia pastoris using a vector.

[0012] Further preferably, the fermentation method of recombinant Escherichia coli comprises the following steps: The recombinant Escherichia coli was inoculated into LB liquid culture medium, cultured at 35-37°C and 200-220 rpm for 8-10 h to obtain a primary seed solution; the primary seed solution was transferred to a secondary seed culture medium at an inoculation rate of 2%-3% by volume, cultured at 35-37°C and 200-220 rpm for 8-10 h to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation medium at an inoculation rate of 10%-11% by volume to obtain the recombinant Escherichia coli by fermentation.

[0013] Further preferably, the fermentation conditions are: the initial fermentation parameters are set to a fermentation temperature of 35-37 o C, initial ventilation volume 1.0-1.2 L / min, fermentation to dissolved oxygen 50%-60%, adjust ventilation volume to 3.0-4.0 L / min according to dissolved oxygen, dissolved oxygen associated stirring 50%-60%, set upper limit speed 700 rpm and lower limit speed 150 rpm, control pH at 7.0-7.5 by automatic addition of ammonia water during fermentation, and ferment for 8-10 h in total.

[0014] Further preferably, LB liquid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, the final concentration of ampicillin is 0.05 mg / mL, and the solvent is water; Secondary seed medium: 10 g / L glycerol, 24 g / L peptone, 12 g / L yeast powder, 10 g / L NaCl, final concentration of ampicillin is 0.05 mg / mL, solvent is water; Fermentation medium: glycerol 20 g / L, peptone 24 g / L, yeast powder 12 g / L, NaCl 10 g / L, MgSO 4 ·H 2 O 1.25 g / L, the final concentration of ampicillin was 0.05 mg / mL, and the solvent was water.

[0015] Preferably according to the present invention, in step (1), the amino acid sequence of the hyaluronidase contains a His tag.

[0016] More preferably, the amino acid sequence of the hyaluronidase is SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3.

[0017] More preferably, the amino acid sequence of the hyaluronidase is SEQ ID NO.1, and is expressed by recombinant Escherichia coli; The amino acid sequence of the hyaluronidase is SEQ ID NO.2 or SEQ ID NO.3, and is expressed by recombinant yeast.

[0018] Preferably, according to the present invention, in step (1), after the fermentation of the recombinant engineered bacteria expressing intracellular hyaluronidase is completed, the fermentation broth is centrifuged at 4000-5000 rpm for 15-20 min to collect the bacterial sludge, and the bacterial sludge is resuspended in phosphate buffer at a solid-liquid mass volume ratio of 1: (10-15) g / mL, and the bacterial sludge is crushed 2-3 times with a homogenizer at a high pressure of 800-900 bar to prepare a bacterial cell crushing liquid.

[0019] More preferably, the phosphate buffer is 10 mM-30 mM, pH 6.0-9.0.

[0020] More preferably, the phosphate buffer is 10 mM-30 mM, pH 7.0-8.0.

[0021] More preferably, the phosphate buffer is 20 mM, pH 7.5.

[0022] According to the preferred embodiment of the present invention, in step (2), the magnetic beads are chelated Ni 2+ Cross-linked superparamagnetic agarose microspheres.

[0023] Preferably, according to the present invention, in step (2), the balancing method of magnetic beads comprises the following steps: Use the equilibration buffer to mix with the magnetic beads, collect the magnetic beads, remove the supernatant, then add the equilibration buffer to resuspend the magnetic beads, collect the magnetic beads, and remove the supernatant; The equilibration buffer includes: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 5 mM-15 mM imidazole, pH 7.0-8.0.

[0024] Further preferably, the equilibration buffer comprises: 20 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole, pH 7.5.

[0025] Preferably, according to the present invention, in step (2), before adding the magnetic beads, the content of the target protein in the sample is estimated to determine a sufficient amount of magnetic beads to be added.

[0026] Preferably according to the present invention, in step (2), the incubation time is 25-35 min.

[0027] Further preferably, in step (2), the incubation time is 30 min.

[0028] Preferably according to the present invention, in step (3), the washing buffer is: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 15 mM-25 mM imidazole, pH 7.0-8.0.

[0029] More preferably, the washing buffer is: 20 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 7.5.

[0030] Preferably according to the present invention, in step (4), the elution buffer comprises: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 200 mM-300 mM imidazole, pH 7.0-8.0.

[0031] Further preferably, the elution buffer is: 20 mM phosphate buffer, 300 mM sodium chloride, 250 mM imidazole, pH 7.5.

[0032] Preferably according to the present invention, in step (4), the incubation time is 3-7 min.

[0033] Further preferably, in step (4), the incubation time is 5 min.

[0034] Preferably, according to the present invention, in step (4), the elution step is repeated twice to recover the eluate containing hyaluronidase.

[0035] According to a preferred embodiment of the present invention, in step (4), after the elution is completed, the collected magnetic beads are resuspended in a regeneration buffer having a volume 5-10 times that of the solid volume of the magnetic beads, and then the magnetic beads are resuspended in a regeneration buffer having a volume 4-8 o C, incubate at 80-100 rpm for 10-15 min, collect the magnetic beads and discard the supernatant, repeat this step 1-2 times; wash the regenerated magnetic beads 1-3 times with distilled water 5-10 times the solid volume of the magnetic beads, add 25% ethanol solution 3-5 times the solid volume of the magnetic beads, and store at 4-8 o C conditions.

[0036] Further preferably, the regeneration buffer is 8 M urea.

[0037] Preferably according to the present invention, in step (2), step (3) or step (4), the method for collecting the magnetic beads is to use a magnetic rod to collect the magnetic beads.

[0038] The beneficial effects of the present invention include at least the following: The present invention has found that by combining magnetic beads with certain incubation conditions and elution conditions, a high-purity hyaluronidase can be obtained, and the enzyme activity of the obtained hyaluronidase is high, and the enzyme activity of the purified hyaluronidase A reaches 18.381 million U / mL, the enzyme activity of the hyaluronidase B reaches 5.682 million U / mL, and the enzyme activity of the hyaluronidase C reaches 7.436 million U / mL. The purification method provided by the present invention has obvious advantages in extracting and purifying hyaluronidase in the recombinant Escherichia coli cell lysate expressing hyaluronidase. Compared with hyaluronidase B and hyaluronidase C, the extraction and purification effect of hyaluronidase A expressed intracellularly is better.

[0039] The purification method provided by the present invention is simple to operate, low in cost, does not require expensive and sophisticated instruments, and is applicable to purification systems of different scales, and can be applied to small-scale system trial production in laboratories, large-scale system production in factories, and can also be applied to automated experiments and production, thereby improving work efficiency and saving labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the SDS-PAGE detection result of each component obtained by purifying hyaluronidase by magnetic beads. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0042] Unless otherwise specified, the experimental reagents used in the examples are common commercially available products; unless otherwise specified, the experimental operations involved in the examples are routine operations in the art.

[0043] Magnetic beads are chelated Ni 2+ Cross-linked superparamagnetic agarose microspheres were purchased from Nanjing Yuzhulong Biotechnology Co., Ltd.

[0044] Hyaluronidase A is a hyaluronidase derived from Citrobacter freundii that was successfully expressed in Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.1 (disclosed in patent document CN 116286764 A, application number is 202310217653.4); Hyaluronidase B is a hyaluronidase derived from brown spider venom that was successfully expressed in Pichia pastoris, and its amino acid sequence is shown in SEQ ID NO.2 (disclosed in patent document CN 118931881 A, application number is 202411352942.6); Hyaluronidase C is a hyaluronidase fusion protein derived from leech and mountain big-toothed ant that was successfully expressed in Pichia pastoris, and its amino acid sequence is shown in SEQ ID NO.3 (disclosed in patent document CN117887691 A, application number is 202311831261.3).

[0045] The hyaluronidase recombinant Escherichia coli expressing hyaluronidase A in the present invention is constructed according to the method disclosed in patent document CN116286764 A (application number 202310217653.4), specifically, the recombinant plasmid pET-grpET7-HylC constructed in Example 2 of the patent is transformed into a BL21 (DE3) host to obtain a recombinant bacterium.

[0046] The construction of the hyaluronidase recombinant Pichia pastoris expressing hyaluronidase B in the present invention is obtained according to the method disclosed in patent document CN 118931881 A (application number 202411352942.6), and the construction method is specifically referred to Example 6 of the patent.

[0047] The construction of the hyaluronidase recombinant Pichia pastoris expressing hyaluronidase C in the present invention is based on the patent document CN 117887691 A (application number 202311831261.3): the construction method specifically refers to Example 5 of the patent.

[0048] Example 1 High-density culture of recombinant Escherichia coli of hyaluronidase A in a 20 L fermenter A single colony of recombinant Escherichia coli on the plate was picked and inoculated into 20 mL LB liquid medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and the final concentration of ampicillin was 0.05 mg / mL), and cultured at 37 °C and 220 rpm for 8 h to obtain the first-level seed solution; the first-level seed solution was transferred to 750 mL second-level seed medium (10 g / L glycerol, 24 g / L peptone, 12 g / L yeast powder, 10 g / L NaCl, and the final concentration of ampicillin was 0.05 mg / mL) at an inoculum volume fraction of 2%, and cultured at 37 °C and 220 rpm for 8 h to obtain the second-level seed solution; the second-level seed culture solution was inoculated into the fermentation tank medium (20 g / L glycerol, 24 g / L peptone, 12 g / L yeast powder, 10 g / L NaCl, MgSO 4 ·H 2 O1.25 g / L, the final concentration of ampicillin was 0.05 mg / mL). The initial fermentation parameters were set as follows: fermentation temperature 37 o C, initial aeration rate of 1.0 L / min, fermentation to dissolved oxygen 50%-60%, adjust aeration rate to 3.0 L / min according to dissolved oxygen, dissolved oxygen associated stirring 50%-60%, upper limit speed 700 rpm, lower limit speed 150 rpm, pH controlled at 7.5 by automatic addition of ammonia water during fermentation, fermentation for 8 h to obtain fermentation liquid.

[0049] Cultivation of Hyaluronidase B or Hyaluronidase C Recombinant Pichia pastoris Fermentation Broth The obtained hyaluronidase recombinant Pichia pastoris was inoculated into 50 mL YPD liquid medium, and cultured overnight at 30 °C and 220 rpm to obtain the first-level seed solution. The solution was then transferred to a 100 mL YPD medium second-level seed shake flask at a volume ratio of 2% and cultured at 30 °C and 220 rpm until the OD 600The pH value was about 6.0, and the fermentation culture was carried out using BSM fermentation medium. The successfully activated secondary seeds were inoculated into the fermenter at a volume ratio of 10%. The initial culture temperature was 30 °C, the stirring speed was 300 rpm, the ventilation volume was 2.0 L / min, and the pH was adjusted to 5.5 using 50% ammonia water. The dissolved oxygen in the fermenter was maintained above 20% by adjusting the dissolved oxygen-associated stirring speed. After fermentation for 18-20 hours, when the glycerol in the BSM fermentation medium was consumed (detected using a biosensor analyzer), 50% (m / m) glycerol containing 1.2% (v / v) PTM1 was started to promote bacterial growth, and dissolved oxygen feedback was associated with stirring. When the DO value was greater than 20%, glycerol was added for 12 hours at a feed rate of 1.0 mL / min to maintain the dissolved oxygen at 20%-30%. When the dissolved oxygen rises rapidly again, the biosensor analyzer is used for detection. When the glycerol added is exhausted, methanol containing 1.2% (v / v) PTM1 is used for induction. The temperature is set at 25 °C and the speed is 900 rpm. Four hours before induction, the flow acceleration of the methanol feed solution is 0.06 mL / min to promote the adaptation of the bacteria to the carbon source to methanol. After 4 hours of induction, the feed rate is adjusted to 0.12 mL / min, and the feeding method is dissolved oxygen feedback feeding. Feeding is started when the DO value is above 30%, and the induction is continued for 96 hours to obtain the fermentation bacterial liquid.

[0050] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and water as solvent.

[0051] BSM medium: glycerol 40 g / L, K 2 SO 4 18 g / L, KOH 4.13 g / L, 85% H 3 PO 4 26.7 mL / L, CaSO 4 ·2H 2 O 0.93 g / L, MgSO 4 7H 2 O 14.9 g / L, 4.4 mL / L filter-sterilized PTM1, solvent is water.

[0052] PTM1 formula: CuSO 4 ·5H 2 O 6 g / L, KI 0.09 g / L, MnSO4·H 2 O 3 g / L, H 3 BO 3 0.02 g / L, MoNa 2 O 4 ·2H 2O 0.2 g / L, CoCl 2 6H 2 O 0.92 g / L, ZnCl 2 20 g / L, FeSO 4 7H 2 O 65 g / L, biotin 0.2 g / L, H 2 SO 4 5.0 mL / L, the solvent is water.

[0053] Example 2: Preparation and purification of hyaluronidase The buffer solution formula is: Equilibration buffer: 20 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole, pH 7.5.

[0054] Wash buffer: 20 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 7.5.

[0055] Elution buffer: 20 mM phosphate buffer, 300 mM sodium chloride, 250 mM imidazole, pH 7.5.

[0056] Regeneration buffer: 8 M urea.

[0057] First, perform magnetic bead pretreatment. Each milliliter of magnetic bead solid volume can adsorb about 30 mg of target protein. Estimate the content of target protein in the sample to ensure that the amount of magnetic beads added is sufficient.

[0058] After the fermentation of the recombinant E. coli of hyaluronidase A in Example 1 was completed, the fermentation liquid was centrifuged at 5000 rpm for 15 min to collect the bacterial sludge, and the bacterial sludge was subjected to the following operations to obtain pure hyaluronidase: ① Resuspend the bacterial sludge in phosphate buffer (20 mM, pH 7.5) at a solid-liquid mass volume ratio of 1:10 g / mL, and crush it three times with a homogenizer at a high pressure of 800 bar to prepare the bacterial cell crushing liquid.

[0059] ② Use the balancing buffer to balance the magnetic beads. Take a certain amount of magnetic bead suspension in a triangular flask, put in a magnetic bar, the magnetic beads will immediately adsorb on the surface of the magnetic bar, and remove the supernatant. Add an appropriate amount of balancing buffer, remove the magnetic bar, resuspend the magnetic beads, mix well, put in the magnetic bar, and remove the supernatant. Repeat the magnetic bead balancing step once.

[0060] ③ Resuspend the balanced magnetic beads in the above bacterial disruption solution. o Incubate at 80 rpm on a C shaker for 30 min to fully suspend the magnetic beads and bacterial disruption solution to ensure maximum binding of the magnetic beads and the target protein.

[0061] ④After the incubation, place the magnetic bar in the above incubation system. After the magnetic beads are adsorbed to the surface of the magnetic bar, transfer the magnetic bar to another triangular flask, add 5 times the volume of the magnetic beads solid washing buffer, remove the magnetic bar to resuspend the magnetic beads, mix well, place the magnetic bar again, after the magnetic beads are adsorbed to the surface of the magnetic bar, remove the supernatant and keep a small amount of sample for subsequent detection. Repeat this washing step 2 times.

[0062] ⑤ Add elution solution 5 times the solid volume of magnetic beads to the washed magnetic beads. o Incubate at 80 rpm for 5 min on a shaker at C, add a magnetic bar, and collect the eluted target protein after the magnetic beads are adsorbed to the surface of the magnetic bar. To obtain a higher recovery rate, repeat the elution step twice and combine the eluates to obtain the hyaluronidase enzyme solution.

[0063] ⑥ Add 5 times the volume of magnetic beads solid to the regeneration buffer in the flask to resuspend the magnetic beads. o Incubate on a shaker at C for 10 min, place the magnetic bar in a triangular flask, and discard the supernatant after the magnetic beads are adsorbed to the surface of the magnetic bar. Repeat the alkaline washing step once. Wash the regenerated magnetic beads twice with distilled water 5 times the solid volume of the magnetic beads.

[0064] ⑦ Add 25% ethanol solution 3 times the volume of magnetic beads to completely immerse the beads and store at 4 o C. Refrigerator.

[0065] Example 3: Experimental conditions study As a protein with special activity, purity and activity are the most important indicators to measure the quality standard of enzyme. How to obtain highly active enzyme is an important part of purification work.

[0066] ① Effect of high-speed centrifugation on the activity of hyaluronidase in bacterial cell disruption fluid The bacterial fermentation was carried out using the method of Example 1, and the hyaluronidase was purified using the method of Example 2.

[0067] Take a certain mass of hyaluronic acid A recombinant Escherichia coli mud, resuspend the mud with phosphate buffer (20 mM, pH 7.5) according to the ratio of solid-liquid mass volume ratio of 1:10 g / mL, and crush it 3 times with a homogenizer at high pressure of 800 bar to prepare a bacterial cell crushing liquid. The bacterial cell crushing liquid was divided into 2 parts, one part was directly tested for enzyme activity, and the other part was centrifuged at 12000 rpm for 30 min. The obtained supernatant and precipitate were respectively tested for enzyme activity, and the results are shown in Table 1. The enzyme activity of Escherichia coli crushing liquid was 319,000 U / mL, the enzyme activity of the supernatant obtained by centrifugation of the bacterial crushing liquid was 261,000 U / mL, and the enzyme activity of the precipitate obtained by centrifugation was 23,000 U / mL. The inventor found that the enzyme activity was lost by 18.1% after high-speed centrifugation. Using the hyaluronidase purification method provided by the present invention, the step of high-speed centrifugation can be omitted, which not only simplifies the steps and saves costs, but also protects the enzyme activity from loss as much as possible.

[0068] Table 1 Effect of high-speed centrifugation on the activity of hyaluronidase in bacterial cell disruption solution

[0069] ②The effect of incubation time on the activity of purified hyaluronidase The length of the incubation time directly affects the binding efficiency of the target protein and the magnetic beads. The inventors found that if the incubation time is insufficient, the target protein may not be fully bound to the magnetic beads, thereby affecting the purification efficiency and enzyme activity; and too long an incubation time may increase nonspecific binding, affecting the purity and activity of the purified enzyme. During the purification process, different incubation times were used for the magnetic beads and the bacterial cell disruption solution (other conditions were the same as in Example 2), and the enzyme activity purified under different incubation times was detected, and the results are shown in Table 2. The enzyme activity obtained by purifying the magnetic beads and the bacterial cell disruption solution for 15 min was 9.528 million U / mL, the enzyme activity obtained by purifying the magnetic beads for 30 min was 18.601 million U / mL, and the enzyme activity obtained by purifying the magnetic beads for 45 min was 15.839 million U / mL, so the preferred incubation time is 30 min.

[0070] Table 2 Effects of different binding incubation times on hyaluronidase activity (final combined eluate)

[0071] ③Effect of elution incubation time on the activity of purified hyaluronidase The length of the elution incubation time affects the elution efficiency of the target protein. The inventors found that too long an elution time may cause excessive interaction between the enzyme and the purification medium, thereby changing the spatial structure of the enzyme and causing a loss of enzyme activity, while insufficient elution time may cause the enzyme to be insufficiently eluted, thereby affecting the purification effect and enzyme activity. The control of the elution time is also related to reducing nonspecific adsorption. Appropriate elution incubation time can reduce the nonspecific binding of the enzyme to other non-target molecules, thereby improving the purity and activity of the purified enzyme. Different elution incubation times (other conditions are the same as in Example 2) were taken, and the enzyme activity obtained by purification under different incubation times was detected, and the results are shown in Table 3. The enzyme activity obtained by eluting and incubating the eluent with the magnetic beads bound to the target protein for 1 min was 8.953 million U / mL, the enzyme activity obtained by eluting and incubating for 5 min was 19.032 million U / mL, and the enzyme activity obtained by eluting and incubating for 10 min was 16.247 million U / mL, so the preferred elution incubation time is 5 min.

[0072] Table 3 Effects of different elution incubation times on hyaluronidase activity (final combined eluate)

[0073] ④ Selection of pH of purification system Under the same conditions, an equal amount of hyaluronidase A bacterial cell fragmentation liquid was prepared, purified based on the magnetic bead method under the following conditions, and the enzyme activity after purification at different pH values ​​was determined (other conditions were the same as in Example 2). Theoretical prediction results show that hyaluronidase A has good activity under a neutral environment, and a 20 mM phosphate buffer of pH 6.0-9.0 is prepared respectively, the sodium chloride concentration contained in the buffer is 300 mM, the imidazole concentration contained in the equilibrium buffer is 10 mM, the imidazole concentration contained in the washing buffer is 20 mM, and the imidazole concentration contained in the elution buffer is 250 mM, the incubation time is 30 min, and the elution incubation time is 5 min. The results are shown in Table 4, so the purification system preferably has a pH of 7.5.

[0074] Table 4 Effects of different pH conditions on hyaluronidase activity (final combined eluate)

[0075] Example 4: Detection of hyaluronidase activity and purity The DNS method was used to detect the hyaluronidase activity of bacterial cell disruption liquid, fermentation supernatant, washing liquid and elution liquid.

[0076] The enzyme activity unit (U) was defined as the amount of enzyme required to produce 1 μg of glucose reducing equivalent per minute under the optimal reaction conditions, and the determination was repeated 3 times.

[0077] Preparation of standard curve: Weigh an appropriate amount of anhydrous glucose into a plate and dry it in a 60 ℃ oven for 1-2 h. Weigh 100 mg of dry anhydrous glucose, dilute to 50 mL with ultrapure water, and prepare 2 mg / mL glucose mother solution. Use the mother solution for dilution to prepare 0, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 mg / mL glucose standard solutions. Take 1 mL of glucose standard solution into glass colorimetric tubes, add 2 mL of DNS reagent, boil in water bath for 10 min, cool and dilute to 10 mL with water, mix well, and measure the absorbance at 540 nm. Draw a standard curve with glucose content as the x-axis and corresponding absorbance as the y-axis.

[0078] Method for determination of enzyme activity: 900 μL of 2 g / L hyaluronic acid solution (1.2 million molecular weight) and 100 μL of crude enzyme solution (after appropriate dilution) were prepared into 1 mL enzyme activity measurement system, and the buffer solution was used instead of the enzyme solution as the control group. The reaction was carried out in a water bath at 37°C for 15 min, and the reaction was terminated in a boiling water bath for 2 min. 2 mL of DNS solution was added and boiled for 10 min to fully develop the color. The solution was cooled to room temperature and diluted to 10 mL with deionized water. The absorbance was measured at 540 nm to calculate the enzyme activity.

[0079] Extraction and purification were performed according to the method of Example 2. The results of enzyme activity determination are shown in Table 5. The enzyme activity of the hyaluronic acid A recombinant Escherichia coli disrupted liquid was 319,000 U / mL, the enzyme activity of the washing liquid was 6320 U / mL, and the enzyme activity of the eluent was 18.381 million U / mL. The enzyme activity was increased by more than 57 times after purification, indicating that a high-activity hyaluronidase was obtained by the purification method provided by the present invention.

[0080] The protein purity of the bacterial cell fragmentation solution, washing solution and elution solution was tested by polyacrylamide gel protein electrophoresis. The theoretical size of the recombinant hyaluronidase is 87 kDa. The SDS-PAGE results are as follows: Figure 1 As shown, the eluate has an obvious single protein band at the theoretical size (indicated by the arrow), indicating that high-purity hyaluronidase was obtained by magnetic bead purification.

[0081] By using the high-activity hyaluronidase purified and prepared by the present invention, by controlling different degradation times, small-molecule sodium hyaluronate with different molecular weights can be prepared with only one thousandth of the addition amount.

[0082] Table 5 The results of enzyme activity determination of each component of hyaluronidase purified by magnetic beads

[0083] Example 5: Purification of different hyaluronidases based on magnetic beads Under the same most preferred conditions, hyaluronidase A, hyaluronidase B and hyaluronidase C are purified based on magnetic beads. That is, the pH of the purification system is 7.5, the concentration of phosphate buffer is 20 mM, the concentration of sodium chloride contained in the buffer is 300 mM, the imidazole concentration contained in the equilibrium buffer is 10 mM, the imidazole concentration contained in the washing buffer is 20 mM, and the imidazole concentration contained in the elution buffer is 250 mM, the binding incubation time is 30 min, and the elution incubation time is 5 min. The results show that in Table 6, compared with before purification, the activity of hyaluronidase A after purification is the highest. Hyaluronidase purification is specifically performed using the method of Example 2; when hyaluronidase B and hyaluronidase C are purified, the cell crushing liquid in Example 2 is replaced by the recombinant bacterial fermentation supernatant of hyaluronidase B and the recombinant bacterial fermentation supernatant of hyaluronidase C.

[0084] Table 6 Results of activity determination of different hyaluronidases purified by magnetic beads

Claims

1. A method for separating and purifying hyaluronidase, characterized in that: The steps include: (1) fermenting the recombinant engineered bacteria expressing hyaluronidase, collecting the bacterial cells after fermentation of the recombinant engineered bacteria expressing hyaluronidase intracellularly, and disrupting the cells to obtain a bacterial cell disruption solution; Or after fermentation of the recombinant engineered bacteria expressing extracellular hyaluronidase, the bacteria are removed and the fermentation supernatant is retained; (2) Purification of intracellularly expressed hyaluronidase: Add the equilibrated magnetic beads to the bacterial cell disruption solution obtained in step (1) for 4-8 o C, incubate at 80-100 rpm for 15-45 min. After the incubation, collect the magnetic beads and remove the supernatant. Or purification of extracellularly expressed hyaluronidase: add the equilibrated magnetic beads to the fermentation supernatant obtained in step (1) for 4-8 o C, incubate at 80-100 rpm for 15-45 min. After the incubation, collect the magnetic beads and remove the supernatant. (3) Resuspend the magnetic beads collected in step (2) using a washing buffer with a volume 5-10 times the solid volume of the magnetic beads, mix well, collect the magnetic beads again, remove the supernatant, and repeat the washing step 1-2 times; (4) Resuspend the magnetic beads in elution buffer 5-10 times the volume of the magnetic beads. o C, incubate at 80-100 rpm for 1-10 min, collect the magnetic beads, and recover the eluate containing hyaluronidase.

2. The method according to claim 1, characterized in that In step (1), the recombinant engineered bacteria expressing hyaluronidase is a recombinant Escherichia coli or a recombinant yeast; Preferably, in step (1), the recombinant Escherichia coli is a host bacterium, and the hyaluronidase encoding gene sequence is transferred into Escherichia coli using a vector; The recombinant yeast uses Pichia pastoris as a host bacterium, and the hyaluronidase encoding gene sequence is transferred into Pichia pastoris using a vector.

3. The method according to claim 2, characterized in that In step (1), the fermentation method of recombinant Escherichia coli comprises the following steps: The recombinant Escherichia coli was inoculated into LB liquid medium, and cultured at 35-37°C and 200-220 rpm for 8-10 hours to obtain a primary seed solution; the primary seed solution was transferred to a secondary seed medium at an inoculation volume of 2%-3%, and cultured at 35-37°C and 200-220 rpm for 8-10 hours to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation medium at an inoculation volume of 10%-11%, and the recombinant Escherichia coli was obtained by fermentation; Preferably, the fermentation conditions are: the initial fermentation parameters are set to a fermentation temperature of 35-37 o C, initial aeration rate of 1.0-1.2 L / min, fermentation to dissolved oxygen 50%-60%, adjust aeration rate to 3.0-4.0 L / min according to dissolved oxygen, dissolved oxygen associated stirring 50%-60%, set upper speed limit 700 rpm and lower speed limit 150 rpm, automatically add ammonia water to control pH at 7.0-7.5 during fermentation, fermentation for 8-10 h; Preferably, LB liquid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, the final concentration of ampicillin is 0.05 mg / mL, and the solvent is water; Secondary seed medium: 10 g / L glycerol, 24 g / L peptone, 12 g / L yeast powder, 10 g / L NaCl, final concentration of ampicillin is 0.05 mg / mL, solvent is water; Fermentation medium: glycerol 20 g / L, peptone 24 g / L, yeast powder 12 g / L, NaCl 10 g / L, MgSO4·H2O1.25 g / L, the final concentration of ampicillin is 0.05 mg / mL, and the solvent is water.

4. The method according to claim 1, characterized in that In step (1), the amino acid sequence of the hyaluronidase contains a His tag; Preferably, the amino acid sequence of the hyaluronidase is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; Preferably, the amino acid sequence of the hyaluronidase is SEQ ID NO.1, and is expressed by recombinant Escherichia coli; The amino acid sequence of the hyaluronidase is SEQ ID NO.2 or SEQ ID NO.3, and is expressed by recombinant yeast.

5. The method according to claim 1, characterized in that In step (1), after the fermentation of the recombinant engineered bacteria expressing hyaluronidase intracellularly is completed, the fermentation broth is centrifuged at 4000-5000 rpm for 15-20 min to collect the bacterial sludge, and the bacterial sludge is resuspended in phosphate buffer at a solid-liquid mass volume ratio of 1: (10-15) g / mL, and the bacterial sludge is crushed 2-3 times with a homogenizer at a high pressure of 800-900 bar to prepare a bacterial cell crushing liquid; Preferably, the phosphate buffer is 10 mM-30 mM, pH 6.0-9.0; Preferably, the phosphate buffer is 10 mM-30 mM, pH 7.0-8.0; Preferably, the phosphate buffer is 20 mM, pH 7.

5.

6. The method according to claim 1, characterized in that In step (2), the magnetic beads are chelated Ni 2+ Cross-linked superparamagnetic agarose microspheres.

7. The method according to claim 1, characterized in that In step (2), the balancing method of the magnetic beads is as follows: Use the equilibration buffer to mix with the magnetic beads, collect the magnetic beads, remove the supernatant, then add the equilibration buffer to resuspend the magnetic beads, collect the magnetic beads, and remove the supernatant; The equilibration buffer comprises: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 5 mM-15 mM imidazole, pH 7.0-8.0; Preferably, the equilibration buffer comprises: 20 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole, pH 7.

5.

8. The method according to claim 1, characterized in that In step (2), before adding the magnetic beads, estimate the content of the target protein in the sample to determine the sufficient amount of magnetic beads to be added; Preferably, in step (2), the incubation time is 25-35 min; Preferably, in step (2), the incubation time is 30 min.

9. The method according to claim 1, characterized in that In step (3), the washing buffer is: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 15 mM-25 mM imidazole, pH 7.0-8.0; More preferably, the washing buffer is: 20 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 7.

5.

10. The method according to claim 1, characterized in that In step (4), elution buffer: 10 mM-30 mM phosphate buffer, 250 mM-350 mM sodium chloride, 200 mM-300 mM imidazole, pH 7.0-8.0; Further preferably, the elution buffer is: 20 mM phosphate buffer, 300 mM sodium chloride, 250 mM imidazole, pH 7.5; Preferably, in step (4), the incubation time is 3-7 min; Preferably, in step (4), the incubation time is 5 min; Preferably, in step (4), the elution step is repeated 1-2 times to recover the eluate containing hyaluronidase; Preferably, in step (4), after elution is completed, the collected magnetic beads are resuspended in a regeneration buffer having a volume 5-10 times that of the solid volume of the magnetic beads, and then the magnetic beads are resuspended in a regeneration buffer having a volume 4-8 o C, incubate at 80-100 rpm for 10-15 min, collect the magnetic beads and discard the supernatant, repeat this step 1-2 times; wash the regenerated magnetic beads 1-3 times with distilled water 5-10 times the solid volume of the magnetic beads, add 25% ethanol solution 3-5 times the solid volume of the magnetic beads, and store at 4-8 o Under C conditions; Further preferably, the regeneration buffer is 8 M urea; Preferably, in step (2), step (3) or step (4), the method for collecting the magnetic beads is to use a magnetic rod to collect the magnetic beads.

Images