High-activity hyaluronidase based on chaperonin assisted folding and preparation method thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to chaperonin assisted folding-based high-activity hyaluronidase and a preparation method thereof, and the preparation method comprises the following steps: S1, constructing a recombinant vector pET32 (a)-TrxA-6H-DDDDK-Hylp1 containing a Hylp1 fusion protein coding sequence; s2, transforming the recombinant vector into Escherichia coli host cells, screening positive engineering bacteria, and performing low-temperature induced expression to obtain expression bacteria; s3, crushing the expression thalli to obtain a cell lysis supernatant; s4, taking the cell lysis supernatant, carrying out nickel column affinity chromatography, carrying out desalination treatment on the fusion protein by adopting pretreated sephadex G-25 chromatography, and collecting a fusion protein solution; s5, performing enterokinase site-specific cleavage treatment on the fusion protein solution to obtain an enzyme-digested product; s6, carrying out nickel column affinity chromatography on the enzyme-digested product again, and collecting a flow-through liquid containing the target protein; and S7, carrying out anion exchange chromatography on the flow-through liquid to obtain the high-purity recombinant hyaluronidase Hylp1.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a highly active hyaluronidase based on chaperone protein-assisted folding and its preparation method. Background Technology

[0002] Hyaluronidase is a class of glycoside hydrolases that specifically degrade hyaluronic acid. Hyaluronic acid is a high-molecular-weight polysaccharide widely found in the extracellular matrix, playing a crucial role in maintaining tissue morphology, moisturizing, and cell signaling. Oligosaccharide fragments obtained by hydrolyzing hyaluronidase into hyaluronic acid exhibit unique bioactivities in the pharmaceutical, cosmetic, and functional food fields. For example, they can be used as drug dispersants to improve the absorption efficiency of subcutaneously injected drugs, and to promote the transdermal absorption of active ingredients in skincare.

[0003] Currently, commercially available hyaluronidase is mainly extracted from animal tissues (such as bovine testes). This method suffers from problems such as complex extraction processes, large batch-to-batch variations in enzyme activity, high costs, and potential risks of viral contamination. With the development of recombinant DNA technology, heterologous expression using engineered bacteria such as E. coli has become the mainstream research direction.

[0004] Hyaluronidase Hylp1, derived from bacteriophages, belongs to the 16th family of polysaccharide lyases. Compared to animal-derived hyaluronidases, enzymes in this family typically possess potential advantages such as smaller molecular weight and higher specific activity. Therefore, recombinant expression of phage-derived Hylp1 is considered a potentially important pathway to obtaining high-performance hyaluronidase.

[0005] However, recombinant expression of hyaluronidase often faces problems such as poor protein soluble expression and easy formation of inclusion bodies. Subsequent denaturation and renaturation steps are usually required. This process is not only lengthy and costly, but also prone to protein misfolding and irreversible inactivation, ultimately resulting in low product recovery and unsatisfactory specific activity.

[0006] Therefore, overcoming the shortcomings of existing recombinant preparation technologies, such as poor hyaluronidase soluble expression, complex purification processes, and low specific activity of the final product, is a technical problem that urgently needs to be solved in this field.

[0007] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0008] This disclosure provides at least one highly active hyaluronidase based on chaperone protein-assisted folding and its preparation method.

[0009] In a first aspect, embodiments of this disclosure provide a method for preparing a highly active hyaluronidase based on chaperone protein-assisted folding, comprising the following steps: S1, constructing a recombinant vector pET32(a)-TrxA-6H-DDDDK-Hylp1 containing the coding sequence of the Hylp1 fusion protein; S2, transforming the recombinant vector into Escherichia coli host cells, screening for positive engineered bacteria, and inducing expression at low temperature to obtain expression cells; S3, lysing the expression cells to obtain cell lysis supernatant; S4, performing nickel column affinity chromatography on the cell lysis supernatant, and desalting the fusion protein using pretreated dextran gel G-25 chromatography to collect the fusion protein solution; S5, performing enterokinase site-specific cleavage on the fusion protein solution to obtain the enzyme digestion product; S6, performing nickel column affinity chromatography again on the enzyme digestion product to collect the flow-through containing the target protein; S7, performing anion exchange chromatography on the flow-through to obtain high-purity recombinant hyaluronidase Hylp1.

[0010] In one optional embodiment, the specific process of low-temperature induced expression in S2 includes: S21, selecting single colonies from the screened positive engineered bacterial strains and culturing them as seed culture; S22, transferring an appropriate amount of seed culture to Good LB medium for culture; S23, waiting for the bacterial culture in S22 to reach OD200. 600 When the value reaches 1.2-1.3, add IPTG to the bacterial culture to a final concentration of 0.2-1.0 mM; S24, adjust the culture temperature of the bacterial culture in S23 to 16℃ and 150 rpm, and culture with shaking for 18-20 h to obtain the expressed bacterial cells.

[0011] In an optional embodiment, the specific process of nickel column affinity chromatography in S4 includes: S411, loading the supernatant into the equilibrated nickel affinity chromatography column at a low flow rate to ensure that the binding time between the sample and the packing material is not less than 10 min; S412, washing with Buffer A and Buffer B for 10 CVs respectively; S413, eluting with Buffer C and collecting the target protein eluent; S414, adding EDTA to the eluent to achieve a final concentration of 2 mM.

[0012] In one optional embodiment, Buffer A consists of 20 mM Tris-HCl, 0.3 M NaCl, 30 mM imidazole, and pH 7.8; Buffer B consists of 20 mM Tris-HCl, 0.3 M NaCl, 100 mM imidazole, and pH 7.8; and Buffer C consists of 20 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, and pH 7.8.

[0013] In one optional embodiment, the desalting process in S4 specifically includes: S421, pre-swelling the dextran gel G-25 with pure water for 24 h, and packing it onto a column using a wet method; S422, equilibrating the chromatography column with 5 CV of Buffer D, wherein Buffer D is 20 mM Tris-HCl, 0.05 M NaCl, pH 7.8; S423, after loading the sample onto the column, continuing to wash with Buffer D; S424, collecting and combining the eluent containing protein to complete the desalting process.

[0014] In one optional embodiment, the mass ratio of the fusion protein to enterokinase in S5 is 1:3000, and after being mixed evenly, it is incubated in a water bath at 27°C for 16 h.

[0015] In one optional embodiment, the amino acid sequence of the target protein Hylp1 is SEQ ID NO:1, the amino acid sequence of the fusion protein TrxA-6H-DDDDK-Hylp1 is SEQ ID NO:2, the nucleotide sequence of Hylp1 optimized for E. coli codon preference is SEQ ID NO:3, and the nucleotide sequence of TrxA-6H-DDDDK-Hylp1 is SEQ ID NO:4.

[0016] Secondly, this disclosure also provides a highly active hyaluronidase based on chaperone protein-assisted folding, wherein the hyaluronidase Hylp1 has a pH of 4.0 to 8.0 and an enzyme activity of over 60% at a temperature of 30°C to 50°C.

[0017] In one optional embodiment, the hyaluronidase Hylp1 has an optimal specific activity at a pH of 6.0 and a temperature of 45°C.

[0018] Thirdly, this disclosure also provides an application of a highly active hyaluronidase based on chaperone protein-assisted folding in the degradation of high molecular weight hyaluronic acid.

[0019] The beneficial effects of this invention are that the recombinant hyaluronidase preparation method based on chaperone protein-assisted folding achieves the controllable preparation of high-purity, high-activity target proteins through the synergistic implementation of fusion expression and multi-step chromatography techniques. Specifically, Hylp1 and the chaperone protein are constructed into a fusion form, and a low-temperature induction strategy is used to efficiently express the target protein in a soluble form. During purification, a combination of nickel column affinity chromatography, enterokinase site-directed cleavage, secondary nickel column affinity chromatography, and ion exchange chromatography is used sequentially to finally obtain high-purity Hylp1 protein, with a purity of up to 96.73%. Enzymatic characterization shows that the obtained recombinant hyaluronidase has high specific activity, and its enzyme activity can be increased by Ca2+. 2⁺ Significant activation; this enzyme can efficiently hydrolyze high molecular weight hyaluronic acid to generate small molecule oligosaccharides. Compared with traditional mammalian extraction processes, this invention utilizes recombinant expression technology through fully soluble expression and fine purification, avoiding problems such as large batch-to-batch variations in enzyme activity, high costs, and potential viral contamination risks in traditional extraction processes. It also avoids the complex refolding steps and activity loss in recombinant expression processes, providing a stable and efficient preparation route for obtaining highly active hyaluronidase.

[0020] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 The diagram shows the effect of nickel affinity chromatography purification and desalting of the fusion protein provided in the embodiments of this disclosure; Figure 2 An image showing the effect of enterokinase cleaving the fusion protein provided in the embodiments of this disclosure; Figure 3 An anion exchange chromatography effect diagram provided in the embodiments of this disclosure; Figure 4 This is a diagram illustrating the effect of calcium ion-induced enzyme activity in an embodiment of this disclosure. Figure 5 A graph showing the effect of pH measurement of the optimal reaction of recombinant hyaluronidase provided in the embodiments of this disclosure; Figure 6 The effect diagram of the determination of the optimal reaction temperature of recombinant hyaluronidase provided in the embodiments of this disclosure; Figure 7 This is a comparison diagram of the enzyme activity of hyaluronidase provided in the embodiments of this disclosure and that of imported products; Figure 8 The HA-cut GPC chromatogram provided in this embodiment of the disclosure. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0026] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0027] The numerical ranges used in this article should be understood to include all numbers within that range.

[0028] As used herein, the terms “comprising” or “including” mean “including, but not limited to”. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof.

[0029] As used in this article, "elution" refers to the desorption of molecules bound to the stationary phase by altering the solution conditions within the chromatographic column. This can be achieved by increasing the concentration of exchangeable counterions or by changing the pH to affect the binding affinity of the analyte. Molecules that lose their affinity for the stationary phase and enter the mobile phase are then "eluted" from the column.

[0030] As used herein, "buffer" refers to a reagent, typically a solution, used to alter the adsorption of an analyte (e.g., Hylp1 protein) onto a stationary phase and / or to remove unbound material from the stationary phase. The elution properties of an eluent can depend on factors such as pH, ionic strength, and detergent strength.

[0031] As used herein, “elution buffer” refers to a solution (e.g., a washing solution or a buffer solution) containing unbound substances (including “eluted” or desorbed analyte molecules, such as Hylp1 protein) that travel through the stationary phase and exit the column during chromatographic separation.

[0032] As used in this article, "high purity" means that the purity detected by SDS-PAGE is ≥95% (the percentage of the target band area after Coomassie brilliant blue staining).

[0033] As used herein, the term "pH" is a numerical value that indicates the degree of acidity or alkalinity of a solution. pH can be measured using a pH meter, and the pH of a buffer solution can be adjusted using an acid or base such as HCl or NaOH.

[0034] As used herein, the term "purification" refers to the process of increasing purity by removing coexisting impurities from a substance, and in this specification, purification means the isolation of the target protein from a culture of E. coli, and also refers to the process of increasing purity during the production of Hylp1 protein.

[0035] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0036] This disclosure provides a method for preparing a highly active hyaluronidase based on chaperone protein-assisted folding, comprising the following steps: S1, constructing a recombinant vector pET32(a)-TrxA-6H-DDDDK-Hylp1 containing the coding sequence of the Hylp1 fusion protein; S2, transforming the recombinant vector into E. coli host cells, screening for positive engineered bacteria, and inducing expression at low temperature to obtain expression cells; S3, lysing the expression cells to obtain cell lysis supernatant; S4, performing nickel column affinity chromatography on the cell lysis supernatant, and desalting the fusion protein using pretreated dextran gel G-25 chromatography, collecting the fusion protein solution; S5, performing enterokinase site-specific cleavage on the fusion protein solution to obtain the enzyme digestion product; S6, performing nickel column affinity chromatography again on the enzyme digestion product, and collecting the flow-through containing the target protein; S7, performing anion exchange chromatography on the flow-through to obtain high-purity recombinant hyaluronidase Hylp1.

[0037] In some embodiments, specifically, the low-temperature induced expression process in S2 includes: S21, selecting single colonies from the screened positive engineered bacterial strains and culturing them into seed culture; S22, transferring an appropriate amount of seed culture to Good LB medium for culture; S23, waiting for the bacterial culture in S22 to reach OD200. 600 When the value reaches 1.2-1.3, add IPTG to the bacterial culture to a final concentration of 0.2-1.0 mM; S24, adjust the culture temperature of the bacterial culture in S23 to 16℃ and 150 rpm, and culture with shaking for 18-20 h to obtain the expressed bacterial cells.

[0038] In some embodiments, the specific process of nickel column affinity chromatography in S4 includes: S411, loading the supernatant into the equilibrated nickel affinity chromatography column at a low flow rate to ensure that the binding time between the sample and the packing material is not less than 10 min; S412, washing with Buffer A and Buffer B for 10 CVs respectively; S413, eluting with Buffer C and collecting the target protein eluent; S414, adding EDTA to the eluent to achieve a final concentration of 2 mM.

[0039] In some embodiments, specifically, Buffer A consists of 20 mM Tris-HCl, 0.3 M NaCl, 30 mM imidazole, and pH 7.8; Buffer B consists of 20 mM Tris-HCl, 0.3 M NaCl, 100 mM imidazole, and pH 7.8; and Buffer C consists of 20 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, and pH 7.8.

[0040] In some embodiments, specifically, the desalting process in S4 includes: S421, pre-swelling dextran gel G-25 with pure water for 24 hours and packing it onto a column using a wet method; S422, equilibrating the chromatography column with 5 CV Buffer D solution, wherein Buffer D solution is 20 mM Tris-HCl, 0.05 M NaCl, pH 7.8; S423, after loading the sample onto the column, continuing to wash with Buffer D solution; S424, collecting and combining the eluent containing protein to complete the desalting process.

[0041] In some embodiments, specifically, the mass ratio of the fusion protein to enterokinase in S5 is 1:3000, and after being mixed evenly, it is incubated in a water bath at 27°C for 16 h.

[0042] In some embodiments, specifically, the amino acid sequence of the target protein Hylp1 is SEQ ID NO:1, the amino acid sequence of the fusion protein TrxA-6H-DDDDK-Hylp1 is SEQ ID NO:2, the nucleotide sequence of Hylp1 optimized for E. coli codon preference is SEQ ID NO:3, and the nucleotide sequence of TrxA-6H-DDDDK-Hylp1 is SEQ ID NO:4.

[0043] This disclosure also provides a highly active hyaluronidase based on chaperone protein-assisted folding, wherein the hyaluronidase Hylp1 has a pH of 4.0 to 8.0 and an enzyme activity of over 60% at a temperature of 30°C to 50°C.

[0044] In some embodiments, specifically, the hyaluronidase Hylp1 has its optimal specific activity at a pH of 6.0 and a temperature of 45°C.

[0045] Thirdly, this disclosure also provides an application of a highly active hyaluronidase based on chaperone protein-assisted folding in the degradation of high molecular weight hyaluronic acid.

[0046] Example 1: Hylp1 sequence feature analysis and vector construction The nucleotide sequence of Hylp1 as described in SEQ ID NO:3 (optimized for E. coli codon preference) was cloned into the C-terminus of the pET32a enterokinase cleavage site via homologous recombination, ultimately forming the fusion protein TrxA-6H-DDDDK-Hylp1 in an open reading frame, yielding the recombinant plasmid pET32(a)-TrxA-6H-DDDDK-Hylp1. The constructed vector was transformed into the prokaryotic expression strain ArcticExpress(DE3) using the heat shock method. After sequencing verification, the vector was stored for later use.

[0047] Select a single colony of the successfully transformed positive engineered bacteria and inoculate it into liquid LB medium containing 100 μg / mL ampicillin. Incubate overnight at 37°C with shaking at 220 rpm to obtain the seed culture.

[0048] Example 2: Induced expression of Hylp1 fusion protein The above seed culture was transferred to Good LB medium at an inoculum rate of 1-2% and cultured at 37°C with shaking at 220 rpm. The OD of the bacterial culture was monitored using a UV spectrophotometer. 600 When the pH reaches 1.2-1.3, add IPTG to the bacterial culture to a final concentration of 0.2-1.0 mM. Then adjust the culture conditions to 16℃ and 150 rpm and continue induction culture for 18-20 h.

[0049] Example 3: Extraction and multi-step purification of Hylp1 Experiment 1: Acquisition of soluble supernatant Collect the induced bacterial culture and centrifuge at 10,000 rpm for 10 min at low temperature to collect the bacterial cells, discarding the supernatant. Resuspend the bacterial cell pellet in Buffer A and wash again by centrifugation. Resuspend the washed bacterial cells in a small amount of Buffer A. Disrupt the bacterial suspension using a pre-cooled high-pressure homogenizer, repeating several times until the bacterial culture is clear. Collect the disrupted liquid and centrifuge at high speed at low temperature. Collect the supernatant and store it on ice for later use.

[0050] Experiment 2: Nickel Column Affinity Chromatography and Desalting The soluble supernatant was loaded into an equilibrated nickel affinity chromatography column at a low flow rate, ensuring that the sample-to-packing time was at least 10 min. The column was then washed with Buffer A and Buffer B for 10 CVs each, and finally eluted with Buffer C. The eluent containing the target protein was collected. Figure 1 Add EDTA to the protein eluent collected in the previous step to achieve a final concentration of 2 mM.

[0051] Dextran gel G-25, pre-swollen with pure water for 24 hours, was wet-packed onto a column, and the column was equilibrated with 5 CV Buffer D. After loading protein samples, the column was washed with Buffer D, and the protein-containing eluent was collected and combined to complete the desalting process. Figure 1 ).

[0052] Experiment 3: Enterokinase cleavage and fine purification The concentration of the desalted fusion protein was determined using the Coomassie brilliant blue method. Based on the determined protein concentration, enterokinase was added to the protein solution at a mass ratio of 1:3000, and after thorough mixing, the solution was incubated in a water bath at 27°C for 16 h.

[0053] The enzyme-digested protein solution was re-loaded onto a nickel ion affinity chromatography column, and the flow-through was collected. This flow-through contained the target protein Hylp1, whose TrxA tag had been removed. Figure 2 Subsequently, this flow-through solution was loaded onto an anion exchange chromatography column for final purification, and the flow-through was collected to obtain high-purity recombinant hyaluronidase Hylp1.

[0054] Example 4 Purity and Enzyme Activity Assessment Experiment 1: Purity Detection The purified Hylp1 protein was analyzed by SDS-PAGE. After scanning with a gel imaging system and software analysis, the purity of the target protein band reached 96.73%. Figure 3 ).

[0055] Experiment 2: Enzyme activity and Ca 2 ⁺ Activation effect assay Enzyme activity was determined using the DNS method. The experiment showed that the specific activity of recombinant hyaluronidase Hylp1 reached 6.9 × 10⁻⁶. 4 U / mg. When 1 mmol / L Ca is added to the reaction system beforehand. 2 ⁺ After pre-incubation at 37°C for 30 min, its specific activity increased to 1.17 × 10⁻⁶. 5 U / mg, an increase of 69.6% ( Figure 4 The specific activity of the imported control enzyme, measured at the same time, was 2.95 × 10⁻⁶ under the same conditions. 4U / mg, Hyaluronidase Hylp1 in Ca 2 ⁺ The specific activity after activation is approximately 4.0 times that of the imported enzyme. Figure 5 ).

[0056] Experiment 3: Determination of Optimal pH and Optimal Temperature Enzyme activity was measured under pH ranges of 2-12. The results showed that the optimal pH for the enzyme was 6.0, under which the specific activity was 1.5 × 10⁻⁶. 5 At a pH range of 4.0-8.0, this enzyme can maintain more than 60% of its activity (U / mg). Figure 6 ).

[0057] Enzyme activity was measured at different temperatures (20℃-70℃) under optimal pH conditions. The results showed that the optimal temperature for the enzyme was 45℃, and the specific activity under this condition was 2.7 × 10⁻⁶. 5 U / mg, within the temperature range of 30℃-50℃, the enzyme activity remains above 60% ( Figure 7 ).

[0058] Experiment 4: Analysis of Hydrolysis Products The products of recombinant hyaluronidase Hylp1 hydrolyzing hyaluronic acid were detected using the HPGPC method. The weight-average molecular weight of the substrate before hydrolysis was 2858 kDa. After 1 h of reaction, hyaluronic acid was completely hydrolyzed, with the main products being oligosaccharides with a weight-average molecular weight of 5.4 kDa and oligosaccharides with a weight-average molecular weight of 593 kDa. Further extension of the reaction time resulted in a more stable molecular weight distribution of the products. Figure 8 ).

[0059] In the above embodiments, specifically, SEQ ID NO:1 is the amino acid sequence of Hylp1, SEQ ID NO:2 is the amino acid sequence of the fusion protein (TrxA-6H-DDDDK-Hylp1) after adding the chaperone protein TrxA and the enterokinase cleavage sequence to the N-terminus of Hylp1, SEQ ID NO:3 is the Hylp1 nucleic acid sequence optimized for E. coli codon preference, and SEQ ID NO:4 is the TrxA-6H-DDDDK-Hylp1 nucleic acid sequence optimized for E. coli codon preference.

[0060] Specifically, the culture medium and buffer formulations involved in the above embodiments are as follows: LB medium: 10 g NaCl, 5 g yeast extract, 10 g tryptone, diluted with pure water to 1 L, adjust pH to 7.5, sterilize at 121℃ and use for later use; Good LB medium: 10 g NaCl, 24 g yeast extract, 12 g tryptone, 2.31 g KH2PO4, 12.54 g K2HPO4, 4 g glycerol, diluted with pure water to 1 L, adjust pH to 7.5, sterilize at 121℃ and use for later use; Buffer A: 20 mM Tris-HCl, 0.3 M NaCl, 30 mM imidazole, pH 7.8; Buffer B: 20 ​​mM Tris-HCl, 0.3 M NaCl, 100 mM imidazole, pH 7.8; Buffer C: 20 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, pH 7.8; Buffer D solution: 20 mM Tris-HCl, 0.05 M NaCl, pH 7.8.

[0061] In summary, this method for preparing highly active hyaluronidase based on chaperone protein-assisted folding successfully achieved efficient soluble expression and purification of recombinant hyaluronidase Hylp1 through a synergistic approach of fusion expression and multi-step chromatographic purification. This method effectively avoids the inclusion body refolding process, is free of animal-derived pathogens, and yields a final product with a purity of 96.73%. Enzyme activity assays confirmed that the obtained recombinant hyaluronidase Hylp1 possesses high specific activity (6.9 × 10⁻⁶). 4 (U / mg), and its enzyme activity can be detected by Ca... 2 ⁺Significant activation (69.6% increase); this enzyme can efficiently hydrolyze ultra-high molecular weight hyaluronic acid. This method is stable and provides a reliable preparation route for obtaining highly active hyaluronidase products.

[0062] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for the preparation of a high-activity hyaluronidase based on chaperone-assisted folding, characterized in that, The method comprises the following steps: S1, constructing a recombinant vector pET32(a)-TrxA-6H-DDDDK-Hylp1 containing a Hylp1 fusion protein coding sequence; S2, transforming the recombinant vector into an E. coli host cell, screening positive engineering bacteria, low-temperature inducing expression, and obtaining expression bacteria; S3, crushing the expression bacteria to obtain cell lysate supernatant; S4, taking the cell lysate supernatant for nickel column affinity chromatography, and using pretreated dextran gel G-25 chromatography to desalt the fusion protein, and collecting the fusion protein solution; S5, performing enterokinase site-specific cleavage treatment on the fusion protein solution to obtain a cleavage product; S6, performing nickel column affinity chromatography on the cleavage product again, and collecting the flow-through liquid containing the target protein; S7, performing anion exchange chromatography on the flow-through liquid to obtain high-purity recombinant hyaluronidase Hylp1.

2. The preparation method of claim 1, wherein the specific process of low-temperature induction expression in S2 comprises: S21, culturing a single colony of the selected positive engineering bacteria as a seed liquid; S22, taking an appropriate amount of seed liquid and transferring it into Good LB medium for culture; S24, adjusting the culture temperature of the bacterial liquid in S23 to 16°C, 150 rpm, and oscillating culture for 18-20 h to obtain expression bacteria. S23, when the OD of the bacterial liquid in S22 reaches 0.6-0.8, IPTG is added to the bacterial liquid to a final concentration of 0.2-1.0 mM 600 When the value reaches 1.2-1.3, IPTG is added to the bacterial liquid to a final concentration of 0.2-1.0 mM; 3. The preparation method of claim 1, wherein the specific process of nickel column affinity chromatography in S4 comprises: S411, loading the supernatant into an equilibrated nickel affinity chromatography column at a low flow rate, ensuring that the sample and filler combination time is not less than 10 min; S412, sequentially using Buffer A liquid and Buffer B liquid to flush 10 CV respectively; S413, using Buffer C liquid for elution, and collecting the target protein eluate; S414, adding EDTA to the eluate to make the final concentration reach 2 mM.

4. The preparation method of claim 3, wherein the Buffer A liquid is 20 mM Tris-HCl, 0.3 M NaCl, 30 mM imidazole, pH 7.8; the Buffer B liquid is 20 mM Tris-HCl, 0.3 M NaCl, 100 mM imidazole, pH 7.8; the Buffer C liquid is 20 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, pH 7.

8.

5. The preparation method of claim 1, wherein the specific process of desalting treatment in S4 comprises: S421, swelling the dextran gel G-25 with pure water for 24 h, and wet packing the column; S422, equilibrating the chromatography column with 5 CV of Buffer D liquid, which is 20 mM Tris-HCl, 0.05 M NaCl, pH 7.8; S423, after the sample is loaded, continue to use Buffer D liquid for flushing; S424, collect and combine the protein-containing effluent to complete the desalting treatment. ​ ​ ​ ​ 6. The preparation method of claim 1, wherein the mass ratio of the fusion protein to enterokinase in S5 is 1:3000, and the mixture is incubated at 27℃ for 16 hours.

7. The preparation method of any one of claims 1-6, wherein the amino acid sequence of the target protein Hylp1 is SEQ ID NO: 1, the amino acid sequence of the fusion protein TrxA-6H-DDDDK-Hylp1 is SEQ ID NO: 2, the nucleotide sequence of the codon-optimized Hylp1 for E. coli is SEQ ID NO: 3, and the nucleotide sequence of TrxA-6H-DDDDK-Hylp1 is SEQ ID NO:

4.

8. A high-activity hyaluronidase based on chaperone-assisted folding, wherein the pH of the hyaluronidase Hylp1 is 4.0-8.0, and the enzyme activity remains above 60% at a temperature of 30-50℃.

9. The high-activity hyaluronidase based on chaperone-assisted folding of claim 8, wherein the pH of the hyaluronidase Hylp1 is 6.0, and the specific activity reaches the optimal value at a temperature of 45℃.

10. Use of a high-activity hyaluronidase based on chaperone-assisted folding in the field of high-molecular-weight hyaluronic acid degradation. ​ ​ ​ ​

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