Specific inhibitor mimic peptide-based efficient separation and purification method for serratiolysin metalloproteinase

Abstract

The invention discloses a method for efficiently separating and purifying serratiolysin metalloproteinase based on a specific inhibitor mimic peptide, the amino acid sequence of the inhibitor mimic peptide is Ser-Ser-Leu-Val-Leu-Pro, Gly-Ser-Leu-Ile-Leu-Pro or Ser-Ser-Leu-Val-Leu-Leu, the inhibitor mimic peptide is coupled to a magnetic microsphere with a flexible arm to construct a bionic affinity carrier, and the serratiolysin metalloproteinase is separated and purified by using the bionic affinity carrier to obtain the serratiolysin metalloproteinase. And the steps of gradient elution and the like are combined, so that the high-purity serratiolytic metalloproteinase is finally obtained. Through the precise recognition effect of the specific inhibitor mimic peptide, the purification multiple reaches 5.40 times, the recovery rate is 72.4%, the electrophoresis purity is larger than or equal to 99%, operation is mild, consumed time is short, enzyme activity can be effectively reserved, and the method has remarkable industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme separation and purification technology, specifically relating to a protease purification method using specific inhibitor mimicking peptides as the core recognition element, which is particularly suitable for the separation of serrazinoside metalloproteinases with moderate solubility and slightly acidic isoelectric point. Background Technology

[0002] Serranolytic acid ( Serralysin Metalloproteinases (EC 3.4.24.40) are proteases derived from microorganisms, first discovered in Serratia marcescens (Serratia marcescens). Serratia marcescens Its molecular weight is between 40 and 60 kDa, and its isoelectric point is 4.5 to 5.0. It is a single-subunit metalloproteinase containing zinc ions, with each subunit having seven calcium ion binding sites and one zinc ion binding site. The conserved zinc ion binding amino acid sequence is HEXXHXXGXXHP, where histidine binds to the zinc ion, and glutamate is a potential catalytic residue. This enzyme usually has a specific inhibitor, the Inh polypeptide, with a molecular weight of 10 kDa, located in the periplasmic space, unlike the location of serrazinolytic metalloproteinase. Its physiological function is to inhibit the activity of proteases that may enter the periplasmic space, protecting periplasmic proteins from degradation.

[0003] Serranolytic metalloproteinase possesses excellent anti-inflammatory, anti-swelling, analgesic, and expectorant properties, promoting the dissolution and excretion of sputum and pus. Clinically, it is used for postoperative and traumatic inflammation, sinusitis, mastitis, cystitis, epididymitis, peri-peritoneal inflammation, and alveolar abscess, as well as for treating bronchitis, tuberculosis, and bronchial asthma with difficulty in expectorating sputum. Studies have also found that this enzyme has fibrinolytic activity, suggesting potential applications in the treatment of atherosclerosis. Its unique mechanism of action and wide range of applications hold significant research value in the biomedical and industrial fields.

[0004] At present, Sareolysin metalloprotease is produced by Serratia marcescens fermentation, and separation and purification is an important link, accounting for more than 70% of the total production cost. The separation methods of protease in fermentation broth include ammonium sulfate precipitation, acetone precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography and the like, but there are some shortcomings, for example, the operation process of step-by-step ammonium sulfate precipitation and organic solvent fractionation precipitation is relatively cumbersome and consumes a lot of time; a variety of chromatography methods are combined, and the operation conditions are relatively harsh, and the enzyme is prone to inactivation in the separation and purification process, thereby reducing the separation and purification efficiency and recovery rate of the enzyme. For example, CN105969691A discloses a method for separating and purifying Sareolysin crude enzyme solution, and the Sareolysin crude enzyme solution obtained by fermenting Serratia marcescens LL-413 strain is subjected to ammonium sulfate precipitation, dialysis desalting and freeze-drying to obtain Sareolysin. However, the method disclosed in the patent has the problems of low separation and purification efficiency and recovery rate, and the purity of the separated Sareolysin is low. CN119662609A discloses a Sareolysin purification method using two-step organic reagent-salt extraction method, and the proportion of salt and organic solvent is optimized to realize the preliminary purification of the protease, but there are still problems such as insufficient specificity, incomplete removal of impurities and the like. At the same time, the organic solvent extraction method is easy to cause the denaturation and inactivation of the protease, thereby affecting the activity retention. In the prior art, affinity technology is also used for the separation of protease, but natural antigens or antibodies are mostly used as affinity ligands, and there are problems such as complex ligand preparation, high cost and poor stability. Therefore, it is of great practical significance to develop a protease separation and purification method with strong specificity, low cost, simple operation and suitable for industrial production. SUMMARY

[0005] The purpose of the present application is to overcome the defects of the existing protease separation and purification method, and to provide a Sareolysin metalloprotease efficient separation and purification method based on biomimetic affinity. The method uses Sareolysin metalloprotease specific inhibitor mimic peptide modified magnetic microspheres as an affinity carrier, utilizes the specific recognition of the inhibitor mimic peptide and the target protease to realize the directional capture of the target protease, removes impurities through gradient elution, combines ultrafiltration refining and freeze-drying steps, and obtains a high-purity protease product.

[0006] In order to achieve the above purpose, the present application first provides a Sareolysin metalloprotease specific inhibitor mimic peptide, and the amino acid sequence of the inhibitor mimic peptide is any one of SEQ ID NO. 1 to SEQ ID NO. 3: SEQ ID NO. 1: Ser-Ser-Leu-Val-Leu-Pro (i.e. SSLVLP); SEQ ID NO. 2: Gly-Ser-Leu-Ile-Leu-Pro (i.e. GSLILP); SEQ ID NO.3: Ser-Ser-Leu-Val-Leu-Leu (i.e. SSLVLL). The amino acid sequence of the above-mentioned inhibitor mimetic peptide is designed based on the sequence characteristic structure of a specific inhibitor binding to the active site of Sareolysin metalloprotease, and comprises the following characteristics: the first amino acid is Ser or Gly, which forms a hydrogen bond with a zinc ion, a protease Glu177 and a Tyr216; the second amino acid is stable Ser, which forms a hydrogen bond with the protease Glu177; the third to sixth amino acids are non-polar amino acids without a benzene ring, the third to fifth amino acids are branched amino acids, and the sixth amino acid is Pro or Leu, so as to ensure the hydrophobicity of the peptide segment; the overall sequence length is 6 amino acids, which not only ensures the binding specificity, but also is easy to be chemically synthesized.

[0007] The present application provides a biomimetic affinity carrier based on the above-mentioned inhibitor mimetic peptide, wherein the carrier is any one of the inhibitor mimetic peptides shown in SEQ ID NO.1 to SEQ ID NO.3 coupled to a surface-aminated flexible arm Fe3O4 magnetic microsphere through an amide bond, wherein the surface-aminated flexible arm Fe3O4 magnetic microsphere is a flexible arm (-NH-(CH2) n -NH2, n = an integer of 6 to 8) Fe3O4 magnetic bead with 6 to 8 methylene groups, and the particle size is 50 to 500 nm.

[0008] Further, the preparation method of the above-mentioned biomimetic affinity carrier is as follows: the inhibitor mimetic peptide and the surface-aminated flexible arm Fe3O4 magnetic microsphere are added into 0.1 mol / L pH = 6.0 2-morpholinoethanesulfonic acid buffer in a mass ratio of 1:80 to 1:100, and a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 2:1 to 3:1 is added as an activator, and the coupling reaction is carried out at 25°C for 2 to 3 hours.

[0009] The present application also provides the application of the inhibitor mimetic peptide or the biomimetic affinity carrier in the separation and purification of Sareolysin metalloprotease.

[0010] The present application further provides a separation and purification method of Sareolysin metalloprotease, which comprises the following steps: Step 1: biomimetic affinity capture The Sareolysin metalloprotease fermentation broth is centrifuged and filtered to obtain a clear crude extract, the above-mentioned biomimetic affinity carrier is added into the crude extract, and the mixture is stirred and incubated at 28 to 32°C for 30 to 90 min, and then the biomimetic affinity carrier is collected by magnetic field adsorption at 0.4 to 0.6T.

[0011] Step 2: gradient elution The collected biomimetic affinity carrier surface weakly combined impurities are washed by a washing impurity buffer, and the serralysin metalloprotease is eluted by an elution buffer, and the serralysin metalloprotease eluent is collected; wherein the washing impurity buffer is a 50mmol / L pH=7.2 Tris-HCl buffer containing 0.2mol / L NaCl and 0.02wt% Tween-80; and the elution buffer is a 50mmol / L pH=7.2 Tris-HCl buffer containing 0.5-0.7mol / L NaCl and 0.1-0.5mmol / L EDTA.

[0012] Further, in the step 1, preferably, the added amount of the biomimetic affinity carrier in the crude extract is 0.6-10mg / L.

[0013] Further, in the step 2, preferably, the washing volume of the washing impurity buffer or the elution buffer is 10-20 times of the volume of the biomimetic affinity carrier precipitate. The method for separating and purifying serralysin metalloprotease can be used as a general method for separating and purifying proteases, and the specific scheme comprises the following steps: 1. Constructing a biomimetic affinity carrier containing a mimic peptide A mimic peptide specifically combined with a target protease is selected, and is fixed on the surface of a magnetic microsphere by chemical coupling to prepare a biomimetic affinity magnetic microsphere with specific recognition ability.

[0014] 2. Affinity capturing of the target protease The biomimetic affinity magnetic microsphere is added to a pretreated crude extract of the protease, and is incubated under suitable conditions to make the target protease specifically combined with the substrate mimic peptide on the surface of the magnetic microsphere, and then is separated by magnetic field adsorption to realize the preliminary enrichment of the target protease.

[0015] 3. Gradient elution for removing impurities The magnetic microsphere adsorbed with the protease is gradient eluted by elution buffers with different concentrations to remove weakly combined impurities, and then the target protease is eluted to improve the product purity. Subsequently, the target protease solution eluted can be concentrated and further impurities removed by the rejection of an ultrafiltration membrane to improve the concentration and purity of the protease. The protease solution concentrated by ultrafiltration can be freeze-dried to obtain a protease product with good stability.

[0016] The method has the following beneficial effects: 1、The present application is based on the highly specific interaction characteristics selected by long-term co-evolution of Sareolysin metalloprotease and inhibitor peptide, using protease and inhibitor peptide crystal structure, molecular docking simulation optimization and computer-aided design of specific inhibitor simulation peptide (SEQ ID NO. 1 to SEQ ID NO. 3) as biomimetic affinity ligand, which optimally fits the active center of the target protease (Sareolysin metalloprotease) in spatial structure and interaction site, thereby achieving a binding strength and specificity far beyond natural substrates or traditional ligands. The simulation peptide has high affinity for Sareolysin metalloprotease and can accurately "fish" out the target protein from complex mixtures (such as cell lysate, fermentation broth), and has a cross-reactivity of < 2% for other non-target proteins. It ensures high selectivity in the purification process, greatly reduces the co-purification of impurities, and lays a solid foundation for obtaining ultra-pure products. The peptide ligand designed based on molecular simulation in the present application provides predictable and stable binding performance, avoids batch-to-batch differences of biological ligands (such as antibodies), ensures high consistency and reliability of the purification results, and meets the requirements of product quality control (QA / QC).

[0017] 2、The present application couples the simulation peptide to magnetic microspheres to construct a biomimetic affinity carrier. The magnetic property allows the "capture-washing-elution" purification process to be completed quickly under the action of a magnetic field, without the need for complex centrifugation or filtration steps, easy to automate, and greatly improves efficiency. The introduction of a flexible arm connection arm eliminates the steric hindrance effect of the solid-phase carrier itself, allowing the simulation peptide to freely adjust its conformation, fully bind to the target protease, and maximize its high affinity advantage, improving binding capacity and efficiency. The use of magnetic microspheres makes solid-liquid separation exceptionally simple and can be quickly completed by magnetic separation, saving time and effort. The entire purification process is significantly shortened in time, which not only improves production efficiency but also reduces the risk of protein inactivation during prolonged purification. Compared with traditional monoclonal antibody affinity media, synthetic peptide ligands have lower production costs, better chemical stability, and longer service life, and can withstand multiple in-situ cleaning and repeated use, greatly reducing the purification cost of large-scale production. The method is clear in principle and simple in process, and relatively direct from laboratory scale to industrial production process. Magnetic separation technology is also easy to integrate into large-scale bioreactors, with significant large-scale industrial application value.

[0018] 3、The separation and purification method combines "directed capture" (using the specificity of a mimic peptide), "gradient elution" (gently changing conditions to stepwise elute impurities with different adsorption strengths and the target protein, optimizing purity) and "ultrafiltration refinement and freeze-drying", forming a complete and efficient downstream processing flow. The electrophoretic purity of the serralysin metalloprotease product obtained by the separation and purification method is ≥ 99%, meeting the requirements of high-end scientific research, diagnosis and pharmaceutical grade applications; the recovery rate of 72.4% is much higher than that of many traditional purification methods (such as ammonium sulfate precipitation, ion exchange, etc.), which means less loss of target protein, reduces the cost of raw materials and improves the yield; since mild operating conditions (specific affinity binding and gradient elution, avoiding the use of strong acid, strong base or high concentration of salt) are used, the natural conformation and enzyme activity of the target protease are maximally protected, which is crucial for obtaining functional proteins with biological activity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a serralysin metalloprotease sequence alignment result graph, * indicates a conserved site amino acid.

[0020] Figure 2 is a mature inhibitor N-terminal sequence alignment result graph.

[0021] Figure 3 is a logo analysis result graph.

[0022] Figure 4 is a crystal structure graph of metalloprotease and inhibitor of RCSB PDB database (left), molecular docking shows that the 5 amino acid residues of the N-terminal of the inhibitor will penetrate into the active site of the metalloprotease (right), green represents the structure of serralysin metalloprotease, cyan represents the structure of the inhibitor, red represents the mimic peptide segment, and the gray round ball represents the zinc ion, and the green round ball represents the calcium ion.

[0023] Figure 5 is an SDS-PAGE analysis of serralysin metalloprotease. In the figure, M is a protein marker; 1 is the supernatant of the fermentation broth; 2 is the serralysin metalloprotease sample purified by adding 0.02wt% Tween-80 to the impurity washing buffer and adding 0.2mmol / L EDTA to the elution buffer; 3 is the serralysin metalloprotease sample purified by the biomimetic affinity carrier constructed by SEQ ID NO. 1. DETAILED DESCRIPTION

[0024] The application will be further described in detail below in combination with the drawings and examples, but the protection scope of the application is not limited to these examples. The raw materials and equipment used in the application are conventional raw materials and equipment in the art if not specifically stated; the methods used in the application are conventional methods in the art if not specifically stated. Example 1

[0025] 1. Analysis and design of Sarecyclin metalloprotease specific inhibitor mimetic peptides (1) Sarecyclin metalloprotease specific inhibitor Inh protein sequence acquisition In the NCBI database, with the sequence of Sarecyclin metalloprotease specific inhibitor WP_116403987.1 as the query sequence, run Protein BLAST, and obtain the following representative microorganism Sarecyclin metalloprotease specific inhibitor Inh protein sequence: >WP_430289157.1 protease inhibitor Inh / omp19 family protein [Pseudomonas paraeruginosa] MLASAKLSRMVCLLTGFFSTGISMASSLILPSATDLAGRWTLQQDEAPAICQLELRASEVAEASGYALGGDTACLKRWLPSEPSAWRPTPAGIALLERDGLTLMLLGRQGEGDYRVQKGDGGQLVLRRVAP >WP_139115716.1 AprI / Inh family metalloprotease inhibitor [Pseudomonas sp. 25 R 14] MASSLVLPSAQQLAGQWSVHVQQNAEQRCSFTLQVFGHALTGGAECLSRWLEGSVAGWIPTPDGIAVVSEEGRKLLFFSRQGDHYSSKVSADLTLLLTRTAP >WP_070802652.1 AprI / Inh family metalloprotease inhibitor [Salmonella enterica] MTSSLILPSPVSLSGHWIVVDKINECDIYLGGERIEQANGYKLIFSSPCAFDIFSEIPEAWRPAPDGISLLNRDGRTVLFFSRENENYSSRVWECSGIMLKKKQKIITVKKDITSIENVYTA >MFI0488239.1 MAG: protease inhibitor Inh / omp19 family protein [Yersinia sp. (in: enterobacteria)] MASSLVLPTPESLSGQWTVVEQANSCVVHLRAESFKQANGYKLSVSPNCAHDLLPEATEAWRPTPDGIAFLDRDGLTVLFFSREGEHYRSQIWEETGKVLKKKEL >WP_235033901.1 AprI / Inh family metalloprotease inhibitor [Pantoeasp. 18069] MFTATSMGSSLPLPHPRDFVGDWVVFDATRFCGIHLWVEDMPEANGFRLTIDPHVAACNFAMDAVAWRPAPDGISLLDKDGTTLVFFSQEGDGYRSEIASDNGMRLCRKTGGQ >QJQ56248.1 Proteinase inhibitor precursor [Erwinia amylovora] MRTSLFVVISLFFSGVSMASSLVLPSAASLSGQWTVVDKSSSCDVQLTAERFEAANGYKLSISPGCDPSVLPETPEAWRPAPDGIALLNGDGLTVLFFSREGEHYRSQIWQQTGKILKKSKN >HFK7185988.1 TPA: AprI / Inh family metalloprotease inhibitor [Serratia odorifera] MIKRIFYAAMMAGSILMTGVVMASSLVLPSAQSLAGQWQLANGERQCRLELLADTQRETNGYQLRDRQQCLKAIFNAEVIGWRAAPDGIALLQTDGSTLAFFSRDGEVYRHPIGAADGLTLKPLR >WP_261231509.1 protease inhibitor Inh / omp19 family protein [Serratia entomophila] MKSTLTRTALTAGGMMVTGAVMAGSLVLPTAQSLAGQWQVADREQQCRIEFLASEQGETNGYQLVDKQRCLKSLFAAEVVGWRPAPDGIALLQADGSTLAFFSRDGEVYRNQLSADDGLTLRALA >WP_261166044.1 protease inhibitor Inh / omp19 family protein [Serratia quinivorans] MKGSLARAAIMAGNMMVTGAAMASSLVLPSAQSLAGQWQVADAQQQCQIEFLASEQSATNGYQLVDRQNCLKNVLTAEVVGWRPAPDGIALLRADGSTLAFFSRDGDVYRNQLGADDGLTLKALA >WP_061798790.1 AprI / Inh family metalloprotease inhibitor [Serratia ficaria] MKNTLARTAMTAGGMMVTGAVMASSLVLPTAQSLAGQWQVADRERQCRIEFLASEQSAANGYRLVDRQRCLNKLFAAEVVGWRPAPDGIALLQADGSTLAFFSRDGEVYRNRLGADDGLTLKALA >WP_061808144.1 AprI / Inh family metalloprotease inhibitor [Serratia grimesii] MKGSLACAVAMAGGMIVTGTVMASSLILPTAQSLAGQWQVADAEQQCQIVFSASEQSETNGYQLVDQQGCLKKVLAAEVVGWRPAPDGIALLQADGSTLVFFSRDGDIYRNQLGAADGLTLKALA >UTN96901.1 protease inhibitor Inh / omp19 family protein [Serratia plymuthica] MASSLVLPSAKSLAGQWLIADAEQQCQIEFLAGEQSEINGYQLVDSQHCLKKVFTAEVAGWRPAPDGIALLQADGSTLAFFSRDGESYRNQLGADDGLTLKALA >WP_153858723.1 AprI / Inh family metalloprotease inhibitor [Serratia proteamaculans] MKGSLACAAIMAGNMMVTGAAMASSLVLPSAQSLAGQWQVANAQQQCQIEFLASEQSATNGYHLVDRQGCLKKVLTAEVVGWRPAPDGIALLQADGSTLAFFSRDGDVYRNQPGADGGLTLKALA >GAK29193.1 metalloprotease inhibitor [Serratia liquefaciens FK01] MASSLVLLSAQSLAGQWQVANAEQQCQIQFLADEQPETHGYVLVDRQRCLEKVLTAEVVGWRPAPDGIALLRADGSTLAFFSRDGDIYRNQLGADDGLTLKALV >WP_346826719.1 AprI / Inh family metalloprotease inhibitor [Serratia inhibens] MKGSLAWVAVMAGGMMVTGAAMAGSLVLPSAKSLAGQWLIADAEQQCQIEFLTGEQNETNGYRLVDPQHCLQKVFSAEVVGWRPAPDGIALLQADGSTLAFFSRDGESYRNQLGAEDGLTLKALR >WEO89834.1 protease inhibitor Inh / omp19 family protein [Serratia proteamaculans] MASSLVLPTAGSLAGQWQVADAQQQCQIEFLASEQRATNGFQLVDRQNCLKKVLMAEVVGWRPAPDGIALLQADGSTLAFFSRDGDIYRNQPGAGDGLTLKALV > WP_116403987.1 AprI / Inh family metalloprotease inhibitor [Serratia marcescens] MKGNLTRTALAAGGMMVTSAVMAGSLALPTAQSLAGQWQVADSERQCQIEFLASEQSETNGYQLVDRQRCGWRPAPDGIALLREDGSTLAFFSRDGDLYRNQLGADDALTLKALA (2) Running Clustal Omega program, the above sequences were aligned to obtain the conserved sequence sites, the results are shown in Figure 1 . The signal peptide sequence was analyzed, and the periplasmic space secretion signal peptide sequence was removed to obtain the mature inhibitor N-terminal sequence (see Figure 2 ), running sequence logo analysis, visual representation of the sequence pattern of different positions of the N-terminal sequence of the preparation (see Figure 3 ), it can be seen that in the first 15 amino acids, the first 6 amino acids are highly conserved, especially the 2nd, 3rd, 5th and 6th positions. Combined with the crystal structure data of serralysin metalloprotease and inhibitor in RCSB PDB database, molecular docking showed that the 5 amino acid residues at the N-terminal of the inhibitor would penetrate into the active site of serralysin metalloprotease, and the first amino acid would form a hydrogen bond with zinc ion and the second amino acid with protease Glu177 (see Figure 4 ). The inhibitor has a very low dissociation constant with serralysin metalloprotease, from 4 pmol / L to 1 µmol / L.

[0026] According to the above analysis, the peptide segments were designed: SEQ ID NO. 1: Ser-Ser-Leu-Val-Leu-Pro (i.e. SSLVLP); SEQ ID NO. 2: Gly-Ser-Leu-Ile-Leu-Pro (i.e. GSLILP); SEQ ID NO. 3: Ser-Ser-Leu-Val-Leu-Leu (i.e. SSLVLL). SEQ ID NO. 4: Thr-Ser-Ala-Ala-Ala-Ala (i.e. TSAAAA) SEQ ID NO. 5: Gly-Thr-Ala-Ala-Ala-Ala (i.e. GTAAAA) SEQ ID NO.1 to SEQ ID NO.3 are the target peptides, and SEQ ID NO.4 and SEQ ID NO.5 are the mutation verification peptides. The peptide sequences were synthesized by Genscript Biotech and purified by high performance liquid chromatography (HPLC) with a purity ≥98%.

[0027] 2. Preparation of biomimetic affinity carriers 100 mg of commercially available, surface-aminated flexible-arm Fe3O4 magnetic microspheres (Fe3O4 magnetic beads with a particle size of 500 nm and 7 methylene flexible arms (-NH-(CH2)7-NH2), amino density 35 μmol / g, provided by Zhongke Keyou) were weighed and added to 5 mL of 0.1 mol / L pH 6.0 2-morpholine ethanesulfonic acid buffer. The mixture was ultrasonically dispersed for 10 min. 1.2 mg of the synthesized peptide was added to the dispersion, and after stirring until homogeneous, 18 mg of EDC and 9 mg of NHS were added. The mixture was stirred at 25 °C for 2 hours. After the reaction, the magnetic microspheres were adsorbed using a 0.5 T magnetic rack. The supernatant was discarded, and the microspheres were washed at least 5 times with PBS buffer to thoroughly remove unbound peptides and activators, yielding a biomimetic affinity carrier. This carrier was resuspended in 10 mL of 50 mmol / L pH 7.2 Tris-HCl buffer and stored at 4 °C protected from light for later use.

[0028] 3. Bionic affinity purification of Serratia serratia metalloproteinase The fermentation broth of Serratia serratia metalloproteinase originated from strain Serratia surfactantfaciens The fermentation broth of strain YD25 is a publicly available strain. Serratia surfactantfaciens The preparation method of strain YD25 fermentation broth is as follows: After activation culture on an agar slant, the bacterial cells are inoculated into 100 mL of seed culture medium and cultured at 30℃ and 200 rpm for 12 h to obtain the seed culture. The seed culture is then transferred to 200 mL of fermentation medium at an inoculation rate of 2% (v / v) and cultured at 30℃ and 200 rpm for 24 h to obtain the serrazinolytic metalloproteinase fermentation broth.

[0029] The method for isolating and purifying serrazinoprotein metalloproteinase is as follows: Step 1: Bionic Affinity Capture Take 1000 mL of Serratia serrata metalloproteinase fermentation broth, centrifuge at 12000 rpm for 30 min at 4 °C, collect the supernatant, filter the supernatant through a 0.45 μm filter membrane to obtain a clear crude extract; add the prepared biomimetic affinity carrier (final concentration 10 mg / L) to the crude extract, stir and incubate at 30 °C for 60 min; after incubation, place the reaction solution on a 0.5 T magnetic rack for 10 min to adsorb the biomimetic affinity carrier, discard the supernatant and collect the biomimetic affinity carrier.

[0030] Step 2: Gradient elution After stirring for 10 min, the biomimetic affinity carrier was magnetically adsorbed, and the supernatant was discarded. The washing step was repeated three times. Then, 20 mL of elution buffer (50 mmol / L Tris-HCl buffer containing 0.6 mol / L NaCl and 0.2 mmol / L EDTA, pH = 7.2) was added to the biomimetic affinity carrier. After stirring for 10 min, the biomimetic affinity carrier was magnetically adsorbed, and the supernatant was collected, which was the serralysin metalloprotease eluate. The serralysin metalloprotease eluate can be further ultrafiltrated and lyophilized to obtain serralysin metalloprotease with good stability.

[0031] Enzyme activity determination: The fluorescence polarization method was used to determine the proteolytic activity by using FITC-gelatin as a substrate. The enzyme sample was placed in a colorimetric cup containing the FITC-gelatin substrate, and the system was 1.0 mL of 50 mmol / L Tris-HCl buffer, pH = 8.0. The fluorescence polarization (FP) value was automatically determined by a fluorescence polarization spectrometer at 30°C. The protein concentration was determined by the BCA method, and bovine serum albumin (BSA) was used as a standard.

[0032] Table 1 shows the results of purifying serralysin metalloprotease by using the biomimetic affinity carrier constructed by coupling SEQ ID NO. 1: Ser-Ser-Leu-Val-Leu-Pro to magnetic microspheres with flexible arms, which achieved an enzyme activity recovery rate of 72.4% and a purification fold of 5.4, and the whole purification process took 2.5 h.

[0033] Table 1

[0034] Note: ND in the table means not determined.

[0035] Table 2 shows the results of purifying Sareolysin metalloproteinase by coupling different SEQ peptide segments to the magnetic microspheres with flexible arms to construct biomimetic affinity carriers. The purification results of Sareolysin metalloproteinase by biomimetic affinity carriers constructed using specific inhibitor mimetic peptides of SEQ ID NO. 1 to SEQ ID NO. 3 were not significantly different, indicating that the three inhibitor mimetic peptides can effectively purify Sareolysin metalloproteinase. SEQ ID NO. 4 replaces the first Ser with Thr, adding a methyl side chain to the amino acid, which may affect the formation of hydrogen bonds with zinc ions and the Glu177 and Tyr216 of the protease, and the last four amino acids are replaced from hydrophobic branched amino acids to hydrophobic small amino acid Ala, which may affect the twisting of the peptide segment and thus affect the binding to Sareolysin metalloproteinase. Similarly, SEQ ID NO. 5 replaces the second Ser with Thr, which also affects the binding to Sareolysin metalloproteinase and thus affects the purification effect.

[0036] Table 2

[0037] 4. SDS-PAGE analysis of Sareolysin metalloproteinase SDS-PAGE electrophoresis was used to characterize the purity of Sareolysin metalloproteinase. A 4% concentrated gel and a 12% separating gel were prepared in a protein electrophoresis tank. Add electrode buffer solution, adjust the voltage to 80V, wait until the sample migrates to the junction of the concentrated gel and the separating gel, adjust the voltage to 120V, and turn off the electrophoresis when the sample migrates to the bottom of the electrophoresis tank. Remove the gel and rinse it with purified water, then stain it with Coomassie Brilliant Blue R-250 for 3h, and then decolorize it with decolorizing solution (volume ratio of ethanol:acetic acid:water = 2:1:7). The molecular weight of the protein molecules is shown by protein Marker. SDS-PAGE analysis is shown in Figure 5 Figure 1, where lane 1 is the fermentation supernatant, lane 2 is the Sareolysin metalloproteinase sample purified by wash buffer without adding 0.02wt% Tween-80 and elution buffer without adding 0.2mmol / L EDTA, and lane 3 is the Sareolysin metalloproteinase sample purified by the biomimetic affinity carrier constructed using SEQ ID NO. 1. It can be found that the fermentation supernatant contains Sareolysin metalloproteinase, as well as a large amount of impurities; purification by biomimetic affinity carriers constructed using specific inhibitor mimetic peptides of SEQ ID NO. 1 to SEQ ID NO. 3 can effectively remove impurities, and the electrophoretic band of Sareolysin metalloproteinase obtained by combining optimized wash buffer and elution buffer is single and has a higher concentration, indicating that adding 0.02wt% Tween-80 to the wash buffer can more effectively elute the impurities, and adding 0.2mmol / L EDTA to the elution buffer can more effectively elute and recover Sareolysin metalloproteinase.

Claims

1. A serrazinoside metalloproteinase-specific inhibitor mimic peptide, characterized in that, The amino acid sequence of the inhibitor mimic peptide is any one of SEQ ID NO.1 to SEQ ID NO.

3.

2. A biomimetic affinity carrier, characterized in that, The carrier is formed by coupling any one of the inhibitor mimic peptides shown in SEQ ID NO.1 to SEQ ID NO.3 of claim 1 to a surface-amino-coated flexible arm Fe3O4 magnetic microsphere via an amide bond.

3. The biomimetic affinity carrier according to claim 2, characterized in that, The carrier is prepared by adding the inhibitor mimic peptide and the surface-aminated flexible arm Fe3O4 magnetic microspheres at a mass ratio of 1:80 to 1:100 to 0.1 mol / L pH=6.0 2-morpholine ethanesulfonic acid buffer, and adding a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide at a mass ratio of 2:1 to 3:1 as an activator, and coupling reaction at 25°C for 2 to 3 hours.

4. The biomimetic affinity carrier according to claim 2 or 3, characterized in that, The surface-aminated flexible arm Fe3O4 magnetic microspheres have a particle size of 50–500 nm, and the flexible arm is a carbon chain containing 6–8 methylene groups.

5. The use of the inhibitor mimic peptide of claim 1 in the isolation and purification of serrazinoside metalloproteinases.

6. The application of the biomimetic affinity carrier according to claim 2 in the separation and purification of serrazinoside metalloproteinases.

7. A method for isolating and purifying serrazinolytic metalloproteinase, characterized in that, Includes the following steps: Step 1: Bionic Affinity Capture Take the fermentation broth of sarcolysin metalloproteinase, centrifuge and filter to obtain a clear crude extract, add the biomimetic affinity carrier described in claim 2 or 3 to the crude extract, stir and incubate at 28-32℃ for 30-90 min, adsorb in a magnetic field of 0.4-0.6T, and collect the biomimetic affinity carrier. Step 2: Gradient elution First, weakly bound impurities on the surface of the collected biomimetic affinity carrier were eluted with washing buffer, and then serrazinoside metalloproteinase was eluted with elution buffer. The serrazinoside metalloproteinase eluent was collected. The washing buffer is a 50 mmol / L Tris-HCl buffer with pH=7.2 containing 0.2 mol / L NaCl and 0.02 wt% Tween-80; The elution buffer is a 50 mmol / L Tris-HCl buffer solution with pH=7.2 containing 0.5–0.7 mol / L NaCl and 0.1–0.5 mmol / L EDTA.

8. The method for isolating and purifying serrata-lysin metalloproteinases according to claim 7, characterized in that, In step 1, the amount of biomimetic affinity carrier added to the crude extract is 0.6–10 mg / L.

9. The method for isolating and purifying serrata-lysin metalloproteinases according to claim 7, characterized in that, In step 2, the washing volume of each wash buffer or elution buffer is 10 to 20 times the volume of the biomimetic affinity carrier precipitate.

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