Method for affinity separation and purification of serratiolysin extracellular metalloproteinase based on polyglutamic acid

Abstract

The invention discloses a method for affinity separation and purification of serratiolysin extracellular metalloproteinase based on polyglutamic acid, and belongs to the technical field of bioengineering. According to the method, the characteristic that the serratiolysin extracellular metalloproteinase contains seven calcium ion binding sites is utilized, polyglutamic acid is taken as a specific polycarboxylic acid ligand, directional capture of target protease is realized through calcium ion mediated coordination, and the steps of gradient elution and the like are combined, so that the high-purity serratiolysin extracellular metalloproteinase is finally obtained. The purification multiple reaches 6.8 times, the recovery rate is 85.2%, the electrophoresis purity is larger than or equal to 99%, and the method is mild in operation, low in cost and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme separation and purification technology, specifically relating to a method for separating and purifying serrazid extracellular metalloproteinases based on polyglutamic acid (PGA) affinity, especially utilizing the calcium ion binding characteristics of the target protease to achieve efficient separation. Background Technology

[0002] Serranolytic acid ( Serralysin Extracellular metalloproteinases (EC3.4.24.40) are proteases derived from microorganisms, first discovered in Serratia marcescens (Serratia marcescens). Serratia marcescens Its molecular weight is between 40 and 60 kDa. It is a single-subunit metalloproteinase containing zinc ions, with seven calcium ion binding sites and one zinc ion binding site on each subunit. In particular, the C-terminal domain of the protease consists of an extended fold layer in which the GGXGXDX(L / I / F / V)X motif forms a parallel β-helix that can bind five calcium ions.

[0003] Seracillin, an extracellular metalloproteinase, possesses excellent anti-inflammatory, anti-swelling, analgesic, and expectorant-promoting effects, as well as 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 where expectoration is difficult. 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 extracellular 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, etc., 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 time-consuming; a variety of chromatography methods are combined, and the operation conditions are relatively harsh, which can easily cause the enzyme to be inactivated in the separation and purification process, thereby reducing the efficiency and recovery rate of enzyme separation and purification. 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 low recovery rate, and the purity of the separated Sareolysin is low. The Sareolysin purification method disclosed in CN119662609A uses a two-step organic reagent-salt extraction method to realize the preliminary purification of the protease by optimizing the proportion of salt and organic solvent, but there are still problems such as insufficient specificity and incomplete removal of impurities. At the same time, the organic solvent extraction method can easily cause the protease to be denatured and inactivated, thereby affecting the activity retention. In the prior art, affinity technology has also been used for the separation of protease, but natural antigens or antibodies are usually used as affinity ligands, which have the problems of complex ligand preparation, high cost and poor stability. Therefore, it is of great practical significance to develop a protease separation and purification method with high 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 method for separating and purifying Sareolysin extracellular metalloprotease based on polyglutamic acid (PGA) affinity, which utilizes the high affinity of PGA and calcium ions and realizes the efficient and directional capture and mild elution of the target protease through the specific bridging action of calcium ions.

[0006] The method for separating and purifying Sareolysin extracellular metalloprotease based on PGA affinity provided by the present application comprises the following steps: Step 1: Preparation of biomimetic affinity carrier The PGA is coupled to the surface-aminated flexible arm Fe3O4 magnetic microspheres through an amide bond to obtain a PGA-magnetic microsphere biomimetic affinity carrier.

[0007] Step 2: Affinity capture of Sareolysin extracellular metalloprotease The fermentation liquor of the lysin extracellular metalloprotease is centrifuged and filtered to obtain a clear crude extract; the crude extract is adjusted to pH 7.5-8.0 with NaOH and CaCl2 is added to a final concentration of 5-10 mmol / L, then the biomimetic affinity carrier of step 1 is added, and incubated at 25-30°C for 40-60 min, and the biomimetic affinity carrier is collected under a magnetic field of 0.4-0.6T.

[0008] Step 3: Gradient elution First, the weakly bound impurities on the biomimetic affinity carrier collected in step 2 are eluted with the impurity elution buffer, and then the lysin extracellular metalloprotease is eluted with the elution buffer, and the lysin extracellular metalloprotease eluate is collected; wherein the impurity elution buffer is 50 mmol / L Tris-HCl buffer at pH 7.5 containing 5-10 mmol / L CaCl2, 0.15 mol / L NaCl, and 0.02 wt% Tween-80, and the elution buffer is 50 mmol / L Tris-HCl buffer at pH 7.5 containing 0.1-0.3 mmol / L EDTA and 0.5 mol / L NaCl.

[0009] Further, in the above step 1, the surface-aminated flexible-arm Fe3O4 magnetic microspheres are added to 0.1 mol / L 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5-6.5 and ultrasonically dispersed for 10-20 min, and then collected under a magnetic field of 0.4-0.6T and the supernatant is discarded; the above process is repeated 2-3 times, and then the surface-aminated flexible-arm Fe3O4 magnetic microspheres are resuspended in 0.1 mol / L MES buffer at pH 5.5-6.5 to obtain a pretreated magnetic microsphere dispersion of 8-15 mg / mL; PGA is completely dissolved in 0.1 mol / L MES buffer at pH 5.5-6.5, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added as activators, and the mixture is stirred and activated at 25°C for 20-40 min to obtain an activated PGA solution; the pretreated magnetic microsphere dispersion is added to the activated PGA solution, and the mixture is coupled at 25°C for 2-3 hours to obtain a PGA-magnetic microsphere biomimetic affinity carrier.

[0010] Further, in the above step 1, the mass ratio of PGA to surface-aminated flexible-arm Fe3O4 magnetic microspheres is preferably 1:50-1:70, and the mass ratio of EDC to NHS is preferably 2:1-3:1.

[0011] Further, the surface-aminated flexible-arm Fe3O4 magnetic microspheres have a flexible arm (-NH-(CH2) nFe3O4 magnetic beads with particle size of 100-500 nm and amino group density of 30-40 μmol / g.

[0012] Further, the PGA has a molecular weight of 5-10 kDa and a carboxyl group density of ≥2.5 mmol / g.

[0013] Further, in step 2, preferably, the final concentration of the added biomimetic affinity carrier in the crude extract is 0.5-0.7 mg / mL.

[0014] Further, in step 3, the Sarecyclin extracellular metalloproteinase fermentation broth is centrifuged at 4°C and 12000 r / min for 30 min, the supernatant is filtered through a 0.45 μm filter membrane, and the pH is adjusted to 7.5-8.0 with 1 mol / L NaOH aqueous solution to obtain a clear crude extract.

[0015] Further, in step 3, preferably, the washing volume of the impurity washing buffer or the elution buffer is 10-20 times the volume of the biomimetic affinity carrier precipitate. The PGA is coupled to the flexible arm Fe3O4 magnetic microspheres with surface amination, which ensures that the ligand is fully stretched to facilitate the simultaneous binding of multiple calcium ion sites of the enzyme molecule; in the CaCl2-containing buffer system, the PGA forms a stable “PGA-calcium ion-protease” ternary complex with the Sarecyclin extracellular metalloproteinase through calcium ions, and then the rapid enrichment is realized through magnetic field separation; first, the buffer containing Ca 2+ is used for washing to remove weakly bound non-specific impurities, and then the eluent containing EDTA is used for competitive binding of calcium ions to destroy the ternary complex, thereby gently releasing the high-purity target protease. The present application utilizes the calcium ion binding characteristics of Sarecyclin extracellular metalloproteinase and the polycarboxylic acid coordination ability of PGA to construct a high-efficiency affinity purification system, which has significant advantages in purification efficiency, cost and activity retention compared with the prior art, and is suitable for industrial application.

[0016] Compared with the prior art, the present application has the following advantages: 1. High-stability biomimetic affinity carrier: PGA is connected to the magnetic microspheres through a flexible arm, which makes the PGA chain far away from the surface of the microspheres, reduces the steric hindrance, and ensures that the carboxyl group is fully exposed to bind calcium ions; the stability of the covalent bond is high, and the shedding rate of PGA is <2% in the subsequent capture and elution process, which is significantly better than the physical adsorption method (shedding rate > 30%). Moreover, PGA has good biocompatibility and low cost, which provides an ideal solution for efficient and low-cost large-scale purification.

[0017] 2. High specificity capture mechanism: using the 7 calcium ion binding sites specific to Sareolysin extracellular metalloprotease, linear arrangement, PGA with multiple carboxyl groups as specific ligand. PGA has strong coordination ability to calcium ion (dissociation constant Kd=1.2×10 -5 mol / L), can bind to multiple sites of protease at the same time through calcium ion "bridging", high binding efficiency and strong specificity, low cross-reaction rate (<1%) to impurities without calcium ion binding sites, strong anti-interference ability, and complete impurity removal.

[0018] 3. Precise and controllable mild elution: the purification process is based on the regulation of calcium ion concentration. In the capture stage, appropriate Ca 2+ promotes complex formation; in the elution stage, only low concentration of EDTA is needed to competitively chelate calcium ions, resulting in the dissociation of the complex. This method avoids the use of high concentrations of salt (such as 0.5-0.7 mol / L NaCl) in traditional ion exchange methods, which can better maintain enzyme activity.

[0019] 4. Natural compatibility and high activity retention: through the ingenious "calcium ion mediated affinity capture" strategy, the entire purification mechanism fully adapts to the natural characteristics of the enzyme that relies on calcium ions to maintain its structure, avoiding direct interaction with the enzyme active center, thereby maximizing the protection of the enzyme's natural conformation and activity. After purification by this method, combined with ultrafiltration, freeze-drying and other steps, high-purity, high-activity Sareolysin extracellular metalloprotease products can be obtained, solving the problems of existing purification methods in efficiency, cost and enzyme activity retention. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the analysis of calcium ion binding sites of Sareolysin extracellular metalloprotease, the first two GG of calcium ion binding GGXGXDX (L / I / F / V) X motif are marked with yellow.

[0021] Figure 2 is the binding site of calcium ion on the crystal structure of Sareolysin extracellular metalloprotease, calcium ion is represented by a green ball.

[0022] Figure 3 is the SDS-PAGE analysis of Sareolysin extracellular metalloprotease. DETAILED DESCRIPTION

[0023] The present application will be further described in conjunction with specific examples and drawings, but the scope of protection of the present application is not limited thereto.

[0024] The raw materials and equipment used in the present application, if not specifically stated, are common raw materials and equipment in the art; the methods used in the present application, if not specifically stated, are conventional methods in the art. Example 1

[0025] 1. Analysis of the calcium binding sequence of serralysin extracellular metalloprotease (1) Acquisition of serralysin extracellular metalloprotease protein sequence In the NCBI database, using the sequence WP_147095173.1 of the specific inhibitor of serratia protease as the query sequence, running Protein BLAST, the following representative microbial serralysin extracellular metalloprotease protein sequences were obtained: >WP_147095173.1 MULTISPECIES: serralysin family metalloprotease [Serratia] MQSTKKAIEITESNFAAAKTGYDAVADLLHYHERGNGIQINGKDSFSNEQAGLFITRENQTWNGYKVFGQPVKLTFSFPDYKFSATNVAGDTGLSKFSAEQQQQAKLSLQSWADVANITFTEVAAGQKANITFGNYSQDRPGHYDYGTQAYAFLPNTIWQGQDLGGQTWYNVNQSNVKHPATEDYGRQTFTHEIGHALGLSHPGDYNAGEGNPTYRDATYAEDTRQFSLMSYWSETNTGGDNGGHYAAAPLLDDIAAIQHLYGANLSTRTGDTVYGFNSNTGRDFLSTTSNAQKLIFAVWDAGGNDTFDFSGYTANQRINLNEKSFSDVGGLKGNVSIAAGVTIENAIGGSGNDVIVGNAANNVLKGGAGNDVLFGGGGADELWGGSGQDTFVFTAASDSAPGASDWIRDFQKGIDKIDLSFFNKEAQSSDFIHFVDHFSGAAGEALLSYNASNNVTDLAVNIGGHQSPDFLVKIVGQVDVATDFIV >WP_269874039.1 serralysin family metalloprotease [Serratia rubidaea] MQSKKKAIETNESSLAAAKSGYDAVNDLLHYHQRGNGIQVNGKDSFSTEEAGLYITRTNQTWNGKGVFDTPVKLTFSFPDYQFNSANGGGDRGLSKFSTEQQQQAKLSLQSWSDVANITFTEVSGTQKANITFGNYSQDQYGQTDYDTQAYAWLPGSGSVSGQSWYNINQSNIQHPASEDYGRQTFTHEIGHALGLSHPGDYNAGQGNPSYRDASYAEDTRMFSLMSYWSESNTGGDNGGHYAAGPLLDDISAIQHLYGANMSTRTGDTVYGFNSNTDRDFFSTSSNAEKVIFSVWDAGGNDTFDFSGYSADQRINLNEKSFSDVGGLKGNVSIAAGVTIENAIGGSGNDVLVGNQADNVLKGGAGNDILFGGGGADELWGGAGSDTFVFSAVSDSTPGAPDWIRDFQKGIDKIDLSYFNKGGEGVHFVDHFSGAAGEALLTYDAANNVSDLAVDFGGHSTPDFLVKIVGQADVATDFIV >WP_143838116.1 serralysin family metalloprotease [Dickeya sp. ws52] MEKNLSSRDDDALHSLSAPSSSYNSIYDLLHYHERGNGSTINGKPSYSIEEAGDQITRDNVSWNGANVFGKSANLTFKFLQSARSTPDGDTGFVKFNAAQISQAKLALQSWADVANVTFTEVTGNQSANVTFGNYTRDSSGRLDYGSQAYAYLPGSGSASGTTWYNYNVDNIRSPDTMEYGRQTLTHEIGHALGLNHPGDYNAGEGNPSYSDVTYAEDTRQFSIMSYWSEKNTGGDFKGHYAAGPMLDDIAAIQRLYGANMTTRTGDTVYGFNSNTDRDFYTATSSSKALIFSAWDAGGNDTFDFSGYSNNQRINLNEGSLSDVGGLKGNVSIAEGVTIENAIGGSGNDLLIGNNADNTLRGGAGDDVLFGGSGADRLYGGSGRDTFVYTAASDSKVAAPDWILDFQTGTDKIDLSALNTGNNLHFVNQFSGSGGEIMLNWDASANTSNLYLNLDNNTSPEFLVKIVGQVSQTADFVV >WP_050074815.1 serralysin family metalloprotease [Yersinia intermedia] MKASSNKVVEQFDSAMARSGYNAVSEFFQYHARGENLVINGKPSYSNEDAGLQITRTDQTWNGKYVFDQPVKLTYSFLDSVTRIPGGDKGFVKFNPAQIATAKLSLQSWSDAANITFTEITPSQKANVTFGNFTLSHDGSLADSQAYALLPGSGSSSGSTWYNYNVDNIRHPDTMEYGRQTLTHEIGHALGLSHPGNYNAGEGTPTYKDVTYAEDTRQFSIMSYWNEKNTGGDNKGHYAAAPMLDDIAAIQHLYGANMTTRTGDTIYGFHSNTHRDYYTAADSSKALIFSVWDADGNDTFDFSGYSNDQRINLHEGSFTDVGGLKGNVSIAAGVTIENAIGGSGNDIIVGNDARNTLMGGDGNDILFGDGGADVLWGGGGKDIFVFGQVSDSTPQVADWIMDFERGIDKIDLSAFNFANSGGFHFVNSFSGKAGEAMLTYDAGSNVSDLALNNVAGDHSFSDFLVKIIGQPAQETDFIV >WP_004161034.1 serralysin family metalloprotease [Erwinia amylovora] MDNALKGKKTGWDSINDLLNYHQRGNGSSVNNKTSYDIDQAGKEIARGEQSWNGVHVTDKGATVTYSFPSWEPGKKNFNGDTIHSAFNPEQQAQTKLSLQSWSDVANIKFVEVSGDQYSNITFGNIVAPDTQAYAMLPQSTDNGKIIYDDRSFDISGQSWYSTSDPENLAPELGNYGRLTLTHEIGHTLGLNHPGDYNAGEGNPSYADATYAEDSRQFSDMSYWNEPNTGGDNGGNYSAAPLIDDIAAIQHLYGANMTTRTGDTVYGFHSNTDRDFYTAKDSNQKLIFAVWDAGGNDTLDFSGYSQNQRINLNEGSFSDVGGLKGNISIAAGATIENAIGGAGNDVIVGNAADNVIKGGAGNDVIYGGGGQDQLWGGSGNDIFVFSDLKDSSSKSPDQIRDFESGKDKIDLSFFNQGDKGSDFIHFVDHFSGQAGEALLSYDARSNLSELAFNVDGGTNPDFMVQIVGQANVASDFIV >WP_205545419.1 serralysin family metalloprotease [Pectobacterium brasiliense] MALRDEDKDTAESALHAAGTGYSDVYDLYNYHSRGDGQLNGKPSFTSDLAAKEIVRDGLTWNGTNVFGKSANLTYSFLQNVRSIPSGDQGFVKFNAAQTAQAKLSLQSWSDVANITFTEVNPSQKATITFGNYTRDSSGRLDNSTQAYGYMPGNHSAAGSTWYNYNVDNIRNPDTMEYGRQTLTHEIGHALGLNHPGDYNAGQGNPTYRDVTYAEDTRQFSLMSYWSEQNTGGDFQGHYAAGPLIDDISAIQYLYGANMNTRTGDTVYGFNSNTGRDFYSANSNSDKLIFSVWDAGGTDTFDFSGYRNDQRINLNEGGFSDVGGLKGNVSIAHGVTIENAIGGSGNDIIIGNDANNVLIGGAGDDVIYGGGGADTLTGGAGKDIFVYASASDSSYKNGYDTITDFQRGIDKIDLSALNPKGDLQFVNNFTGLGNEALLNWNAESNTTDLWLNFAGQTTPDFVVHIVGQPSAATDFIV >WP_226311672.1 serralysin family metalloprotease [Salmonella enterica] MYQAINRAGLESVNDLLDYHKCGNDILINDKPSFDIQRAGEQIARGEKTWNGENVTGKKAIITYSFPEWSTGSKNQAGDIIHSGFIPLQQAQAKLSLQSWSDVANIHLVEVKNNQEADITFGNISAQDTQAYAYLPGPGRSSGESWYSTSGTDNLQPESGNYGRLTLTHEIGHTLGLNHPGNYNAGNGNPTYENATYAEDTRQFSVMSYWNESNTDGDNGGYYAAAPLVDDIAAIQYLYGANMTTRTEDTVYGFNSSTNREFYTAEDNKQALIFSVWDAGGNDTLNFSGYTQNQRININEGSFSDAGGLRGNVSIAAGVTIENAIGGKGDDVIVGNHADNIIKGNGGNDVIYDGGGQDQLWGGKDNDIFLFSGMNDSPVFSPDKIMDFESGKDRVDLSFFNEGNSSGSDFIRFVENLSGREGEALLSYDNRNNLSELELNIDGGPSPDILVHVVGEIDIATDFIV >WP_248802466.1 serralysin family metalloprotease [Pseudomonas sp. MWU13-2100] MSKVKTKAIVSSDAALAANGTSSAFNQIDSFSHQYDRGDNLTVNGKPSYSVDQAATQLLRDGAAWHDSNGDGRIDLSYTFLTSPTSNFSGLGVTGFSQFSALQKSQAVLSMQSWADVANVTFTEAAKGGDGHMTFGNYSGGQEGAAAFAFLPGTEPGFDGQSWYLTGSGYNVNKTPGLNNYGRQTLTHEIGHTLGLAHPGDYNAGEGNPTYNDATYGQDTRGYSVMSYWSESNTSQNFSKGGVEAYSSGPLMDDIAAIQKLYGANYSTRSGDTTYGFNSNAGRDYMSATSSADKLVFSVWDGGGNDTLDFSGFTQNQKINLHDGSFSDVGGMVGNISIAQGVTIENAIGGSGNDLLIGNDAANELRGGAGNDILYGAGGADKLWGGSGNDTFVFAAVSDSAPKAVDRIMDFTSGQDKIDLSGITHGAGLSFVNAFTGHAGDAVLTYASGTNLGTLAVDFSGHGVADFLVTTVGQAAVTDIVA >WP_159811309.1 serralysin family metalloprotease [Pseudomonas sp. 18058] MSKVKTNAIGAAEQAFQPVGAVQPLAAASSAWNQINSFSHQYDRGGNLTVNGKPSFSVDQAATQLLRDGAAYQDKDGSGKIELTYTFLTSASSSTMNKHGISGFSQFSTQQKAQAALAMQSWADVANVTFTEKASGGDGHMTFGNYSGGQDGAAAFAYLPGTGAGYDGTSWYLINSGYTQNKNPDLNNYGRQTLTHEIGHSLGLAHPGDYNAGNGNPTYNDASYGQDTRGYSLMSYWSESNTNQNFSKGGVEAYASGPLMDDIAAIQKLYGANTNIRTGDTTYGFNSNAGRDFLSASSSSDKLVFSVWDAGGKDTLDFSGFTQNQKINLNDASFSDVGGMVGNVSIAKGAIIENAIGGSGNDLLIGNSVANELKGGAGNDIIWGAGGADKLWGGAGSDTFVFAASSDSKPGAVDQILDFVSGLDKIDLTGITNGAGLHFVSSFTGAVGDAVLTSSGGNSLLSVDFSGHGVADFIVSTVGQAATSDIVA (2) Running Clustal Omega program to perform multiple sequence alignment of the above sequences to obtain conserved sequence sites, the results are shown in Figure 1 The first two GGs of the calcium ion binding GGXGXDX (L / I / F / V) X motif are marked with yellow. By combining the RCSB PDB database metalloprotease crystal structure data, the binding site of calcium ion on the 3D structure of the protein is determined, and the calcium ion is represented by a green ball. The results are shown in Figure 2 It can be determined that the sarcolysin extracellular metalloprotease is a single subunit metalloprotease, which provides sufficient calcium ion binding sites, and one protease binds 7 calcium ions, which provides a basis for using calcium ion affinity to purify the protease.

[0026] 2. Isolation and purification of sarcolysin extracellular metalloprotease Step 1: Preparation of biomimetic affinity carrier (1) Magnetic microsphere pretreatment Take 100 mg of flexible arm Fe3O4 magnetic microspheres (particle size 500 nm, flexible arm 7 methylene chain, amino group density 35 μmol / g, provided by Kexuekeyou) on the surface of amino, add 20 mL of 0.1 mol / L pH=6.0 MES buffer, and place it in an ultrasonic cleaner (power 300 W) for 15 min to ensure uniform suspension and no aggregation. Use a 0.5T permanent magnet to adsorb the magnetic microspheres for 10 min, and discard the supernatant. Repeat the above process for 3 times to remove the residual free amino groups and impurities on the surface of the microspheres. Finally, resuspend with 10 mL of 0.1 mol / L pH=6.0 MES buffer to obtain a 10 mg / mL pretreated magnetic microsphere dispersion.

[0027] (2) Activation of polyglutamic acid Take 2 mg of PGA with a molecular weight of 7 kDa (carboxyl group density 2.8 mmol / g, provided by Sigma-Aldrich), dissolve in 5 mL of 0.1 mol / L pH=6.0 MES buffer, and stir until completely dissolved. Add 15 mg of EDC and 7.5 mg of NHS to the resulting solution, and stir at 25°C for 30 min. At this time, the carboxyl group of PGA is activated to a reactive ester (-CO-NHS), which enhances the reactivity with amino groups.

[0028] (3) Coupling reaction Slowly add the activated PGA solution to the pretreated magnetic microsphere dispersion, and stir at 25°C (speed 200 r / min) for 2.5 h. During the reaction, the activated carboxyl group of PGA reacts with the amino group (-NH2) at the end of the flexible arm of the magnetic microsphere to form a stable -CONH- covalent bond, achieving the fixation of PGA on the surface of the microsphere. In the reaction system, the mass ratio of PGA to magnetic microspheres is 1:50, which ensures a moderate ligand density (about 50 μg PGA / mg microspheres) and avoids steric hindrance between ligands.

[0029] (4) Removal of unbound PGA After the reaction, use a 0.5T permanent magnet to adsorb the coupled magnetic microspheres for 10 min, and discard the supernatant (containing unreacted PGA and activators). Add 20 mL of 0.1 mol / L pH=7.4 PBS buffer, and ultrasonically disperse for 5 min before magnetic separation. Repeat the washing for 5 times until there is no absorption at 220 nm in the supernatant under ultraviolet detection (confirming no free PGA residue). Finally, resuspend with 10 mL of 50 mmol / L pH=7.5 Tris-HCl buffer to obtain a 10 mg / mL PGA-magnetic microsphere biomimetic affinity carrier suspension, and store it at 4°C in the dark.

[0030] The residual amount of amino groups on the surface of the magnetic microspheres before and after coupling was determined by the TNBS method (2,4,6-trinitrobenzenesulfonic acid): 0.1 mL of the biomimetic affinity carrier was taken, 0.2 mL of 0.1% TNBS solution was added, and the reaction was carried out at 37°C for 1 h. The absorbance was determined at 570 nm. The coupling rate of PGA and magnetic microspheres was calculated to be 82.5%±3.2%, i.e., 41.2 μg of PGA was successfully immobilized per mg of magnetic microspheres.

[0031] Step 2: Affinity capture of sarenase extracellular metalloprotease The sarenase extracellular metalloprotease fermentation broth is derived from a strain Serratia surfactantfaciens strain The fermentation broth of YD25 is a public strain. Serratia surfactantfaciens strain The preparation method of the YD25 fermentation broth is as follows: after the slope activation culture, the bacterial cells are inoculated into 100 mL of seed culture medium, and the seed liquid is obtained by culturing at 30°C under the condition of 200 rpm oscillation for 12 h. The seed liquid is inoculated into 200 mL of fermentation medium at a inoculation amount of 2% (v / v), and the sarenase metalloprotease fermentation broth is obtained by culturing at 30°C under the condition of 200 rpm oscillation for 24 h.

[0032] Take 1000 mL of sarenase extracellular metalloprotease fermentation broth, centrifuge at 4°C and 12000 r / min for 30 min, filter the supernatant through a 0.45 μm filter membrane to obtain a clear crude extract; adjust the pH of 940 mL of the crude extract to 7.8 with 1 mol / L NaOH aqueous solution, then add CaCl2 to a final concentration of 7.5 mmol / L, stir for 10 min, add 60 mL of PGA-magnetic microsphere biomimetic affinity carrier suspension, the final concentration of the biomimetic affinity carrier added in the crude extract is 0.6 mg / mL, and incubate at 28°C under the condition of 180 r / min stirring for 60 min; after incubation, the reaction solution is placed in a 0.5T magnetic stand for adsorption for 10 min, the biomimetic affinity carrier is aggregated, the supernatant is discarded, and the biomimetic affinity carrier is collected.

[0033] Step 3: Gradient elution Add 30 mL of washing buffer (containing 5 mmol / L CaCl2, 0.15 mol / L NaCl, and 0.02 wt% Tween-80 in 50 mmol / L Tris-HCl buffer at pH 7.5) to the biomimetic affinity carrier, stir for 10 min, then perform magnetic adsorption. Discard the supernatant and repeat the washing process three times. Add 20 mL of elution buffer (containing 0.5 mol / L NaCl and 0.2 mmol / L EDTA in 50 mmol / L Tris-HCl buffer at pH 7.5), stir for 15 min, perform magnetic adsorption, and collect the supernatant, which is the serrazidin extracellular metalloproteinase eluent. The serrazidin extracellular metalloproteinase eluent can be further ultrafiltered and lyophilized to obtain serrazidin extracellular metalloproteinase with good stability.

[0034] Enzyme activity was determined using fluorescence polarization spectroscopy with FITC-gelatin as the substrate. The enzyme sample was placed in a cuvette containing FITC-gelatin substrate in 1.0 mL of 50 mmol / L Tris-HCl buffer (pH 8.0), and the fluorescence polarization (FP) value was automatically measured at 30 °C using a fluorescence polarization spectrometer. Protein concentration was determined using the BCA method, with bovine serum albumin (BSA) as the standard.

[0035] The entire affinity purification process took 2.5 hours. Table 1 shows the purification results, with a purification fold of 6.8 times and a serrazinolytic extracellular metalloproteinase recovery rate of 85.2%.

[0036] Table 1

[0037] Note: ND in the table indicates not measured.

[0038] 3. SDS-PAGE analysis of serrazinolytic extracellular metalloproteinases The purity of serrazinoside metalloproteinase was characterized by SDS-PAGE electrophoresis. A 4% stacking gel and a 12% separating gel were prepared in a protein electrophoresis tank. Electrode buffer was added, and the voltage was adjusted to 80V. Once the sample migrated to the interface between the stacking and separating gels, the voltage was increased to 120V. Electrophoresis was stopped when the sample migrated to the bottom of the electrophoresis tank. The gel was removed, rinsed with purified water, stained with Coomassie Brilliant Blue R-250 for 3 hours, and then destained with a destaining solution (ethanol:acetic acid:water = 2:1:7, v / v). The molecular weight of the protein molecules was displayed using protein markers. SDS-PAGE analysis is as follows: Figure 3As shown in the figure, lane 1 is the fermentation supernatant, lane 2 is the Sarecyclin extracellular metalloprotease sample purified without adding 0.2 mmol / L EDTA in the elution buffer, and lane 3 is the Sarecyclin extracellular metalloprotease sample obtained in the above-mentioned experiment 2. It can be found that the fermentation supernatant contains Sarecyclin extracellular metalloprotease, and there are also a large amount of impurities; PGA affinity purification can effectively remove the impurities, and the Sarecyclin extracellular metalloprotease obtained by combining the washing and impurity removal buffer and the elution buffer has a single electrophoretic band and a higher concentration, indicating that the addition of 0.02 wt% Tween-80 in the washing and impurity removal buffer can more effectively elute the impurities, and the addition of 0.2 mmol / L EDTA in the elution buffer can more effectively elute and recover the Sarecyclin extracellular metalloprotease.

Claims

1. A method for purifying Sareolysin extracellular metalloprotease based on polyglutamic acid affinity separation, characterized by, The method comprises the following steps: Step 1: preparation of a biomimetic affinity carrier Polyglutamic acid is coupled to surface-aminated flexible-arm Fe3O4 magnetic microspheres through an amide bond to obtain a polyglutamic acid-magnetic microsphere biomimetic affinity carrier; Step 2: affinity capture of serralysin extracellular metalloproteinase Take the serralysin extracellular metalloproteinase fermentation liquor, centrifuge and filter to obtain a clear crude extract; adjust the pH of the crude extract to 7.5-8.0 with NaOH, add CaCl2 to a final concentration of 5-10 mmol / L, then add the biomimetic affinity carrier of step 1, stir and incubate at 25-30°C for 40-60 min, adsorb under a magnetic field of 0.4-0.6 T, and collect the biomimetic affinity carrier; Step 3: gradient elution First, elute the weakly bound impurities on the biomimetic affinity carrier collected in step 2 with a washing impurity buffer, then elute the serralysin extracellular metalloproteinase with an elution buffer, and collect the serralysin extracellular metalloproteinase eluate; The washing impurity buffer is 50 mmol / L Tris-HCl buffer at pH 7.5 containing 5-10 mmol / L CaCl2, 0.15 mol / L NaCl and 0.02 wt% Tween-80; The elution buffer is 50 mmol / L Tris-HCl buffer at pH 7.5 containing 0.1-0.3 mmol / L EDTA and 0.5 mol / L NaCl.

2. The method for purifying Sareolysin extracellular metalloprotease based on polyglutamic acid affinity separation according to claim 1, characterized in that, In step 1, the surface-aminated flexible-arm Fe3O4 magnetic microspheres are added to 0.1 mol / L 2-morpholinoethanesulfonic acid buffer at pH 5.5-6.5, ultrasonically dispersed for 10-20 min, adsorbed under a magnetic field of 0.4-0.6 T, and the supernatant is discarded; the foregoing process is repeated 2-3 times, then the surface-aminated flexible-arm Fe3O4 magnetic microspheres are resuspended in 0.1 mol / L 2-morpholinoethanesulfonic acid buffer at pH 5.5-6.5 to obtain a pretreated magnetic microsphere dispersion of 8-15 mg / mL; polyglutamic acid is completely dissolved in 0.1 mol / L 2-morpholinoethanesulfonic acid buffer at pH 5.5-6.5, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added as activators, the mixture is stirred and activated at 25°C for 20-40 min to obtain an activated polyglutamic acid solution; the pretreated magnetic microsphere dispersion is added to the activated polyglutamic acid solution, and a coupling reaction is carried out at 25°C for 2-3 hours to obtain a polyglutamic acid-magnetic microsphere biomimetic affinity carrier.

3. The method for purifying Sareolysin extracellular metalloprotease based on polyglutamic acid affinity separation according to claim 2, characterized by, The mass ratio of the polyglutamic acid to the surface-aminated flexible-arm Fe3O4 magnetic microspheres is 1:50-1:70, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to N-hydroxysuccinimide is 2:1-3:

1.

4. The method for purifying Sarecyclin extracellular metalloprotease based on polyglutamic acid affinity separation according to any one of claims 1-3, characterized in that, The particle size of the surface-aminated flexible-arm Fe3O4 magnetic microspheres is 100-500 nm, and the amino group density is 30-40 μmol / g; the flexible arm is a carbon chain containing 6-8 methylene groups.

5. The method for purifying Sarecyclin extracellular metalloprotease based on polyglutamic acid affinity separation according to any one of claims 1-3, characterized in that, The molecular weight of the polyglutamic acid is 5-10 kDa, and the carboxyl group density is ≥2.5 mmol / g.

6. The method for purifying Sarecyclin extracellular metalloprotease based on polyglutamic acid affinity separation according to claim 1, characterized in that, In step 2, the final concentration of the added biomimetic affinity carrier in the crude extract is 0.5-0.7 mg / mL.

7. The method for purifying Sareolysin extracellular metalloprotease based on polyglutamic acid affinity separation according to claim 1, characterized in that, In step 3, the Sarecyclin extracellular metalloprotease fermentation broth is centrifuged at 12000 r / min for 30 min at 4 ℃, the supernatant is filtered through a 0.45 μm filter membrane, and the pH is adjusted to 7.5-8.0 with 1 mol / L NaOH aqueous solution to obtain a clear crude extract.

8. The method for purifying Sareolysin extracellular metalloprotease based on polyglutamic acid affinity separation according to claim 1, characterized in that, In step 3, the washing volume of the impurity washing buffer or the elution buffer is 10-20 times the volume of the biomimetic affinity carrier precipitate.

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