Polymyxin B specific degrading enzyme and application thereof

Abstract

The invention belongs to the field of biology, and discloses a polymyxin B specific degrading enzyme and application thereof. The amino acid sequence of the polymyxin B specific degrading enzyme is as shown in SEQ ID NO: 1. The polymyxin B specific degrading enzyme disclosed by some embodiments of the invention can selectively degrade polymyxin B1-1 and polymyxin B3, and does not influence the activity of polymyxin B1 and polymyxin B2, so that the polymyxin B specific degrading enzyme can be used for purification in the preparation process of polymyxin B; the polymyxin can resist acid, alkali and organic solvents, and can efficiently identify and decompose polymyxin B1-1 and B3 components under various conditions. The porous microspheres are used as the solid-phase carrier, so that the surface of the microspheres can accommodate more enzyme ligands, the acid-base resistance and organic solvent resistance of the ligands can be effectively improved, and the polymyxin B purification efficiency can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a polymyxin B specific degradation enzyme and application thereof. Background Art

[0002] Polymyxin was first isolated from Bacillus polymyxa in 1947. Paenibacillus polymyxa ) or Bacillus aerosporeformis ( Bacillus aeroballus ) is a polypeptide antibiotic isolated from the wild-type fungus Pseudomonas aeruginosa, containing five components: A, B, C, D, and E. Polymyxin B (PMB) is a lipopeptide antimicrobial peptide that has an inhibitory or bactericidal effect on Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Escherichia coli parainfectious, Klebsiella pneumoniae, Bacillus acidophilus, Bordetella pertussis, and Shigella dysenteriae, and is considered to be the "last line of defense" for the clinical treatment of multidrug-resistant Gram-negative bacteria infections. PMB is mainly used clinically for infections caused by sensitive bacteria and urinary tract infections, meningitis, sepsis, burn infections, and skin and mucosal infections caused by Pseudomonas aeruginosa. Although PMB has a strong bactericidal effect on Gram-negative bacteria, its clinical application has been stopped in the 1980s and 1990s due to its strong nephrotoxicity and neurotoxicity. However, with the increasing number of severe diseases caused by multidrug-resistant Gram-negative bacterial strains in clinical practice in recent years, the clinical application of PMB has been re-emphasized, and more and more clinical studies have been conducted.

[0003] The molecular formula of PMB is C 56 H 98 N 16 O 13 , molecular weight 1301.56, melting point 122-205℃. PMB is composed of ten amino acids, seven of which are linked to each other at the N-terminus to form a fatty chain, while the amino acids at the tail are scattered and flexible, so there is no fixed conformation in the solution. PMB is a multi-component product, including polymyxin B1, B2, B3 and B1-Ⅰ, of which PMB1 (C 56 H 98 N 16 O 13 ) and PMB2(C 55 H 96 N 16 O 13) is the active ingredient, and PMB1 has the highest content, generally reaching more than 50%, while PMB3 and PMB1-Ⅰ are the main impurities. The amino acids of PMB1 and B2 include L-2,4-diaminobutyric acid, L-threonine, L-leucine and D-phenylalanine, while L-leucine in PMB1-Ⅰ is replaced by L-isoleucine. PMB is a fermentation product, and amino acid configuration transformation may occur during the production process. The change of amino acids and the difference in D- / L-configuration will cause significant changes in its antibacterial activity and nephrotoxicity. Therefore, its amino acids and their configuration must be monitored to ensure drug safety.

[0004] The PMB-producing bacteria is Bacillus polymyxa, which has a fast metabolism and a short culture cycle, and the fermentation process is difficult to accurately control. The control of related substances in PMB fermentation products is also a difficult point. The USP / EP Pharmacopoeia has clear regulations: PMB content ≥ 80%, maximum single impurity ≤ 3.0%, total impurity ≤ 17%, B3 ≤ 6.0%, B1-Ⅰ≤ 15%. At present, the highest content of domestic products can only reach about 85%. Although the content meets the requirements of the USP / EP Pharmacopoeia, it is still far from the world's highest level. The currently published polymyxin B sulfate preparation processes can all achieve a content of more than 80%, but it is relatively difficult to meet the requirements for the control of individual impurities, especially the maximum single impurity is prone to exceed the standard.

[0005] The current purification method of PMB obtained by fermentation is mainly a physical method. For example, CN104327167A discloses a process for extracting polymyxin B, which adopts a precipitation method, first precipitates the polymyxin B fermentation liquid with oxalic acid, separates the precipitated solid and adds an alkaline substance to dissolve it, then adsorbs it with a weak acid cation exchange resin, washes it with purified water, pre-washes it with dilute sulfuric acid, and analyzes it. The analytical solution is precipitated with an alkaline substance, the acidic substance is dissolved, the solid and liquid are separated, and the liquid is decolorized, nanofiltered, and dried to obtain polymyxin B. The production method of polymyxin B disclosed in CN118146314A includes the following steps: filtering the polymyxin B sulfate fermentation liquid, adsorbing it through a macroporous adsorption resin D152 column, separating and purifying the collected D152 analytical solution through a C18 resin, mixing and concentrating the effective part to obtain a purified solution; then adjusting the final pH through concentration, and finally freeze-drying it to obtain a finished powder of polymyxin B.

[0006] Yang Xiaohu. Research on separation and purification process of polymyxin B sulfate [D]. Zhejiang University, 2020. The separation and purification process (membrane method, ion exchange method, precipitation method and adsorption method) was optimized by single-factor experimental method. It has been verified that the purity of polymyxin B1+B2+B3+B1-1 reached more than 95%, polymyxin B3 was lower than 3.0%, polymyxin B1-1 was lower than 2.0%, and the extraction yield of the whole process was not less than 70%.

[0007] The existing fermentation method for producing PMB purification is complex and the purification effect needs to be further improved. Summary of the invention

[0008] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a polymyxin B-specific degrading enzyme and its application.

[0009] The technical solution adopted by the present invention is: The first aspect of the present invention provides: A polymyxin B-specific degrading enzyme has the activity of selectively degrading polymyxin B1-1 and polymyxin B3, which comprises a substrate recognition domain 1, a catalytic domain 1, a stabilizing region 1, a substrate recognition domain 2, a catalytic domain 2, a stabilizing region 2, and a substrate recognition domain 3 in sequence, wherein the amino acid sequence of the substrate recognition domain 1 is QSHVSIQWFDGVVDYT, the amino acid sequence of the catalytic domain 1 is GSHRIISVGAHESVTVRDIKLAFGAPRT, and the amino acid sequence of the substrate recognition domain 2 is AVNGTLHDFFKVAAKK GFVIDSIREALDSHCVPYTGKKDLRFEATURINGGRVVGRSGRLEFY, the amino acid sequence of catalytic domain 2 is GQEKTLYVPGCDSAVEYAIKGTSENGIWAPAAQRNISVMGSYWDAVVKQPGIALEAHTAYVVHLGNAPAIKQFGEPVDI, and the amino acid sequence of substrate recognition domain 3 is NTLLGTLKAHQWRTYDNTVGGVRVVFPPDVDVPSVSVSNAKGPYTFKN.

[0010] In some examples of polymyxin B-specific degrading enzymes, they satisfy at least one of the following characteristics: 1) Its N-terminus has a signal peptide, and preferably, the amino acid sequence of the signal peptide is MKTLLSLALAVFVSAEA; 2) The amino acid sequence of the stabilizing region 1 is EDGSMHYLAKSEFTGHGTVPSSWVTKGP; 3) The amino acid sequence of the stabilizing region 2 is SSDGSVYFAVKEKLGRTLAISLGQPDGKRLAETGLPGIYGPSAHAKEHFLDAKAYDFTGKSGAGVSGQLGAWVNRIT.

[0011] In some examples of polymyxin B-specific degrading enzymes, the amino acid sequence thereof is as shown in SEQ ID NO: 1, or has at least 90% homology to SEQ ID NO: 1.

[0012] In some examples of the polymyxin B-specific degrading enzyme, a tag sequence is connected to at least one end thereof.

[0013] The second aspect of the present invention provides: A polymyxin B enzymatic purification agent comprises the polymyxin B specific degrading enzyme described in the first aspect of the present invention.

[0014] In some examples of polymyxin B enzymatic purification agents, the polymyxin B-specific degrading enzyme is coupled to the surface of a solid support.

[0015] In some examples of polymyxin B enzymatic purification agents, the solid phase carrier is selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres, agarose microspheres, chitosan microspheres, cellulose microspheres, and dextran microspheres.

[0016] In some examples of polymyxin B enzymatic purification agents, the pore size of the solid phase carrier is 600-800 nm.

[0017] The above features can be combined arbitrarily unless they conflict with each other.

[0018] The third aspect of the present invention provides: Use of the polymyxin B-specific degrading enzyme described in the first aspect of the present invention in polymyxin B purification.

[0019] Specifically, the application operations include: S1) contacting the PMB fermentation product with the polymyxin B-specific degrading enzyme according to the first aspect of the present invention and allowing the reaction to proceed sufficiently; S2) further separating and purifying the PMB fermentation product after enzymatic hydrolysis to obtain high-purity polymyxin B.

[0020] A fourth aspect of the present invention provides: A gene encoding the polymyxin B-specific degrading enzyme described in the first aspect of the present invention.

[0021] A fifth aspect of the present invention provides: An expression vector comprising the gene described in the fourth aspect of the invention.

[0022] The beneficial effects of the present invention are: The polymyxin B-specific degrading enzymes of some examples of the present invention can selectively degrade polymyxin B1-1 and polymyxin B3 without affecting the activities of polymyxin B1 and polymyxin B2, and thus can be used for purification in the polymyxin B preparation process.

[0023] The polymyxin B-specific degrading enzymes of some examples of the present invention are resistant to acids, alkalis and organic solvents, and can efficiently recognize and decompose polymyxin B1-1 and B3 components under a variety of conditions.

[0024] The enzymatic purification agent of polymyxin B in some examples of the present invention can increase the flow rate of the fermentation broth, reduce the plugging phenomenon caused by impurities in the fermentation broth, and improve the efficiency of enzymatic decomposition of polymyxin B1-1 and B3 components by coupling the polymyxin B specific degradation enzyme to a solid phase carrier.

[0025] The enzymatic purification agent of polymyxin B in some examples of the present invention uses porous microspheres as solid phase carriers to ensure that the surface of the microspheres can accommodate more enzyme ligands, which can effectively improve the acid, alkali and organic solvent resistance of the ligands, and can effectively improve the efficiency of polymyxin B purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS PAGE electrophoresis diagram of polymyxin B-specific degrading enzyme, where M is a marker. DETAILED DESCRIPTION

[0027] The first aspect of the present invention provides: A polymyxin B-specific degrading enzyme has the activity of selectively degrading polymyxin B1-1 and polymyxin B3, which comprises a substrate recognition domain 1, a catalytic domain 1, a stabilizing region 1, a substrate recognition domain 2, a catalytic domain 2, a stabilizing region 2, and a substrate recognition domain 3 in sequence, wherein the amino acid sequence of the substrate recognition domain 1 is QSHVSIQWFDGVVDYT, the amino acid sequence of the catalytic domain 1 is GSHRIISVGAHESVTVRDIKLAFGAPRT, and the amino acid sequence of the substrate recognition domain 2 is AVNGTLHDFFKVAAKK GFVIDSIREALDSHCVPYTGKKDLRFEATURINGGRVVGRSGRLEFY, the amino acid sequence of catalytic domain 2 is GQEKTLYVPGCDSAVEYAIKGTSENGIWAPAAQRNISVMGSYWDAVVKQPGIALEAHTAYVVHLGNAPAIKQFGEPVDI, and the amino acid sequence of substrate recognition domain 3 is NTLLGTLKAHQWRTYDNTVGGVRVVFPPDVDVPSVSVSNAKGPYTFKN.

[0028] In some examples of polymyxin B-specific degrading enzymes, they satisfy at least one of the following characteristics: 1) Its N-terminus has a signal peptide, and preferably, the amino acid sequence of the signal peptide is MKTLLSLALAVFVSAEA; 2) The amino acid sequence of the stabilizing region 1 is EDGSMHYLAKSEFTGHGTVPSSWVTKGP; 3) The amino acid sequence of the stabilizing region 2 is SSDGSVYFAVKEKLGRTLAISLGQPDGKRLAETGLPGIYGPSAHAKEHFLDAKAYDFTGKSGAGVSGQLGAWVNRIT.

[0029] In some examples of polymyxin B-specific degrading enzymes, the amino acid sequence thereof is as shown in SEQ ID NO: 1, or has at least 90% homology to SEQ ID NO: 1.

[0030] The tag sequence can facilitate the purification of the enzyme. In some examples of polymyxin B-specific degrading enzymes, at least one end thereof is connected to a tag sequence. The tag sequence can be a separation tag commonly used in the art, including but not limited to His-tag, GST-tag, MBP-tag, Strep-tag, etc.

[0031] The second aspect of the present invention provides: A polymyxin B enzymatic purification agent comprises the polymyxin B specific degrading enzyme described in the first aspect of the present invention.

[0032] In some examples of polymyxin B enzymatic purification agents, the polymyxin B-specific degrading enzyme is coupled to the surface of a solid support.

[0033] In some examples of polymyxin B enzymatic purification agents, the solid phase carrier is selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres, agarose microspheres, chitosan microspheres, cellulose microspheres, and dextran microspheres.

[0034] In some examples of polymyxin B enzymatic purification agents, the pore size of the solid phase carrier is 600-800 nm.

[0035] The above features can be combined arbitrarily unless they conflict with each other.

[0036] The third aspect of the present invention provides: Use of the polymyxin B-specific degrading enzyme described in the first aspect of the present invention in polymyxin B purification.

[0037] Specifically, the application operations include: S1) contacting the PMB fermentation product with the polymyxin B-specific degrading enzyme according to the first aspect of the present invention and allowing the reaction to proceed sufficiently; S2) further separating and purifying the PMB fermentation product after enzymatic hydrolysis to obtain high-purity polymyxin B.

[0038] A fourth aspect of the present invention provides: A gene encoding the polymyxin B-specific degrading enzyme described in the first aspect of the present invention.

[0039] A fifth aspect of the present invention provides: An expression vector comprising the gene described in the fourth aspect of the invention.

[0040] The technical solution of the present invention is further illustrated below in conjunction with examples. The following description is a further detailed description of the present invention and should not be regarded as a limitation to the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

[0041] Example 1: Expression and purification of polymyxin B-specific degrading enzyme Escherichia coli constructed with a cloning vector that can express the L-amino acid oxidase (LAAO) gene was inoculated into LB liquid medium and cultured to the logarithmic growth phase (OD600 = 0.6~0.8). The bacterial suspension (1 mL) was taken and spread on an LB agar plate.

[0042] Place the plate under a UV mutagenizer (254 nm) and adjust to the appropriate distance and mutagenesis time. Scrape the UV-treated bacteria from the plate and inoculate them in fresh LB liquid medium for recovery culture. Dilute the mutagenic bacterial solution gradiently and spread it on the LB plate, pick a single clone and inoculate it in a 96-well deep-well plate. Add screening medium containing polymyxin B to each well, and after culture, determine whether the mutagenic bacteria can specifically oxidize polymyxin B3 by liquid phase. Bacteria that can specifically oxidize polymyxin B3 are sent for sequencing, and the resulting sequence is cloned into the pet28-a vector.

[0043] After analysis, the amino acid sequence of the obtained polymyxin B specific degradation enzyme fusion protein is as follows: MKTLLSLALAVFVSAEA-QSHVSIQWFDGVVDYT-GSHRIISVGAHESVTVRDIKLAFGAPRT-EDGSMHYLAKSEFTGHGTVPSSWVTKGP-AVNGTLHDFFKVAAKKGFVIDSIREALDSHCVPYTGKKDLRFEATURINGGRVVGRSGRLEFY-GQEKTLYVPGCDSAVEYAIKGTSENGI WAPAAQRNISVMGSYWDAVVKQPGIALEAHTAYVVHLGNAPAIKQFGEPVDI-SSDGSVYFAVKEKLGRTLAISLGQPDGKRLAETGLPGIYGPSAHAKEHFLDAKAYDFTGKSGAGVSGQLGAWVNRIT-NTLLGTLKAHQWRTYDNTVGGVRVVFPPDVDVPSVSVSNAKGPYTFKN, SEQID NO:1.

[0044] The different domains of the fusion protein are as follows: Signal peptide: MKTLLSLALAVFVSAEA, SEQ ID NO: 2; Substrate recognition domain 1: QSHVSIQWFDGVVDYT, SEQ ID NO: 3; Catalytic domain 1: GSHRIISVGAHESVTVRDIKLAFGAPRT, SEQ ID NO: 4; Stable region 1: EDGSMHYLAKSEFTGHGTVPSSWVTKGP, SEQ ID NO: 5; Substrate recognition domain 2: AVNGTLHDFFKVAAKKGFVIDSIREALDSHCVPYTGKKDLRFEATURINGGRVVGRSGRLEFY, SEQ ID NO: 6; Catalytic domain 2: GQEKTLYVPGCDSAVEYAIKGTSENGIWAPAAQRNISVMGSYWDAVVKQPGIALEAHTAYVVHLGNAPAIKQFGEPVDI, SEQ ID NO:7; Stable region 2: SSDGSVYFAVKEKLGRTLAISLGQPDGKRLAETGLPGIYGPSAHAKEHFLDAKAYDFTGKSGAGVSGQLGAWVNRIT, SEQ ID NO: 8; Substrate recognition domain 3: NTLLGTLKAHQWRTYDNTVGGVRVVFPPDVDVPSVSVSNAKGPYTFKN, SEQ ID NO:9.

[0045] To facilitate purification of the fusion protein, a sharing tag sequence is added to the C-terminus of the protein. The specific tag sequence is a 6-His sequence. Of course, other tag sequences can also be used.

[0046] The gene sequence corresponding to the above fusion protein was chemically connected to the pET 28a plasmid, transformed into Escherichia coli BL21 (DE3) at 42°C for 45s, and spread on a culture dish overnight. The next day, a single colony was picked for expansion culture, the bacterial precipitate was collected and ultrasonically disrupted (on for 1s, off for 2s, working time for 20 min), the supernatant was collected and filtered using a 0.45μm polyethersulfone filter membrane, and the protein was purified through a nickel column. After elution, the polymyxin B-specific enzyme was obtained. The expression level of the polymyxin B-specific enzyme was determined to be 20 mg / L.

[0047] SDS PAGE purification results of polymyxin B specific enzyme Figure 1 As shown, its molecular weight is about 45KD.

[0048] Example 2: Preparation of polymyxin B enzymatic purification agent S1) Take 5 mL of Giga PST microspheres, add 20 mL of 0.01 M sodium hydroxide and 3.0 mL of epichlorohydrin solution, react at 37°C and 120 rpm for 1 h. After the reaction is completed, rinse the filler with ten times the volume of purified water to wash away the residual solution; S2) Add 5 mL of 1X PBS solution, add 5 mL of glutaraldehyde solution, react at 37°C, 120 rpm for 2 h, and then rinse the filler with ten times the volume of purified water to wash away the residual solution; S3) adding polymyxin B specific degrading enzyme protein solution to the treated Giga PST microspheres and gently mixing; S4) Carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used for cross-linking reaction. EDC and NHS were added at a molar ratio of EDC:NHS:protein of 1:1:10, and the mixture was reacted at room temperature for 2 hours. The filler was rinsed with ten volumes of purified water to wash away the residual solution to obtain a polymyxin B-specific enzymatic purification agent, which was stored in 20% ethanol for later use.

[0049] Giga AP, Giga DEAE AP and Giga SP AP microspheres were also prepared using this method to obtain the corresponding polymyxin B enzymatic purification agents.

[0050] Example 3: Acid, alkali and organic solvent resistance determination of polymyxin B enzymatic purification agent Add equal amounts of polymyxin B-specific enzyme to the following reagents: pH determination: Acidic: 0.1 M citrate buffer (pH 3-6); Neutral: 0.1 M phosphate buffer (pH 6-8); Alkaline: 0.1 M Tris-HCl buffer (pH 8-10); Organic solvents: methanol, ethanol, acetonitrile, and DMSO at different volume fractions (10%, 20%, 30%, 40%, and 50%).

[0051] After adding polymyxin B-specific enzymes, the above solvents were incubated at 25°C for 2 hours. After the reaction was completed, the enzyme system was replaced with a 0.01M PBS system, an equal amount of polymyxin B was added, and the reaction was carried out at 25°C and 180 rpm for 5 minutes. After that, the samples were collected for liquid phase detection, and the decomposition of polymyxin B3 was recorded to obtain the enzyme activity.

[0052] Similarly, a polymyxin B-specific enzymatic purification agent was added to the above solution, and the decomposition of polymyxin B3 was recorded to obtain the enzyme activity. The results are shown in Tables 1 and 2.

[0053] Table 1. Effect of different pH on enzyme activity

[0054] Table 2. Effects of different organic solvents on enzyme activity

[0055] It can be seen from the data in Tables 1 and 2 that, after being combined with ultra-large-porous polymer microspheres, the acid, alkali and organic solvent resistance of the polymyxin B-specific enzyme is significantly improved, and it can still maintain a high activity under extreme conditions.

[0056] Example 4 Isolation and Purification of Polymyxin B 1. Take the fermentation liquid, add H2C2O4·2H2O according to the ratio of oxalic acid mass to fermentation liquid volume of 5:1 g / L, and stir for 30 minutes; then filter through ceramic membrane and collect the filtrate. Dilute and adjust pH according to the test requirements; 2. Load the Giga PST polymyxin B-specific enzymatic purification agent onto the column and load the filtrate. The purification conditions are: the flow rate of the column liquid is 1.0 mL / min, the eluent is a Gly HCl solution with a concentration of 1 mol / L, the flow rate of the Gly HCl eluent is 1.0 mL / min, and the eluent is collected; 3. Load the eluate onto the D 152 cation adsorption column. The pH of the column liquid is 6.5-7.0. The optimal flow rate of the column liquid is 1.0 mL / min. The eluate is a solution with a sulfuric acid concentration of 1 mol / L. The flow rate of the sulfuric acid eluate is 1.0 mL / min. After elution, adjust the pH of the eluate to 7.0 and filter through a 0.22 μm filter membrane to obtain the polymyxin B sulfate solution.

[0057] The same method was used to separate and purify polymyxin B based on Giga AP, Giga DEAE AP and Giga SP AP microsphere polymyxin B enzymatic purification agents. The HPLC identification process after separation and purification of polymyxin B is as follows: HPLC liquid chromatography conditions: Buffer solution: 4.46 g of anhydrous sodium sulfate was added to 900 mL of ultrapure water, and the pH was adjusted to 2.3 with phosphoric acid, and the volume was finally made up to 1000 mL with ultrapure water. The buffer solution was filtered twice through an aqueous phase filter membrane to ensure its purity.

[0058] Mobile phase: Use chromatographic grade acetonitrile, filter through an organic phase filter membrane, the ratio of acetonitrile to buffer is 20:80.

[0059] The wavelength of liquid phase UV detection was set to 215 nm, the injection volume of the detection solution was 20 μL, the flow rate of the mobile phase was 1.0 mL / min, the column temperature was maintained at 25 °C, and the chromatographic column used was a C18 silica gel column with a specification of 4.6 mm × 25 cm and a particle size of 5 μm.

[0060] The purification results of different purification methods are shown in Table 3.

[0061] Table 3. Comparison of purification effects of different polymyxin B enzymatic purification agents

[0062] As shown in Table 3, different polymyxin B enzymatic purification agents have degradation effects on B1-1 and B3 components in polymyxin B, and can fully exert their activity when combined with ultra-large-porous polymer microspheres, and can meet the requirements of the Chinese Pharmacopoeia after purification. According to the purification results, Giga PST enzymatic purification agent has a good purification effect on polymyxin B and is the preferred enzymatic purification agent.

[0063] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A polymyxin B-specific degrading enzyme having the activity of selectively degrading polymyxin B1-1 and polymyxin B3, which comprises a substrate recognition domain 1, a catalytic domain 1, a stabilizing region 1, a substrate recognition domain 2, a catalytic domain 2, a stabilizing region 2, and a substrate recognition domain 3 in sequence, wherein: The amino acid sequence of the substrate recognition domain 1 is QSHVSIQWFDGVVDYT, the amino acid sequence of the catalytic domain 1 is GSHRIISVGAHESVTVRDIKLAFGAPRT, the amino acid sequence of the substrate recognition domain 2 is AVNGTLHDFFKVAAKKGFVIDSIREALDSHCVPYTGKKDLRFEATURINGGRVVGRSGRLEFY, the amino acid sequence of the catalytic domain 2 is GQEKTLYVPGCDSAVEYAIKGTSENGIWAPAAQRNISVMGSYWDAVVKQPGIALEAHTAYVVHLGNAPAIKQFGEPVDI, and the amino acid sequence of the substrate recognition domain 3 is NTLLGTLKAHQWRTYDNTVGGVRVVFPPDVDVPSVSVSNAKGPYTFKN.

2. The polymyxin B-specific degrading enzyme according to claim 1, characterized in that It satisfies at least one of the following characteristics: 1) Its N-terminus has a signal peptide, and preferably, the amino acid sequence of the signal peptide is MKTLLSLALAVFVSAEA; 2) The amino acid sequence of the stabilizing region 1 is EDGSMHYLAKSEFTGHGTVPSSWVTKGP; 3) The amino acid sequence of the stabilizing region 2 is SSDGSVYFAVKEKLGRTLAISLGQPDGKRLAETGLPGIYGPSAHAKEHFLDAKAYDFTGKSGAGVSGQLGAWVNRIT.

3. The polymyxin B-specific degrading enzyme according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO: 1, or has at least 90% homology to SEQ ID NO:

1.

4. The polymyxin B-specific degrading enzyme according to claim 1, characterized in that At least one end of the DNA sequence is connected to a tag sequence.

5. A polymyxin B enzymatic purification agent, characterized in that The polymyxin B-specific degrading enzyme comprises the polymyxin B-specific degrading enzyme according to any one of claims 1 to 4.

6. The polymyxin B enzymatic purification agent according to claim 5, characterized in that The polymyxin B specific degradation enzyme is coupled to the surface of the solid phase carrier.

7. The polymyxin B enzymatic purification agent according to claim 6, characterized in that The solid phase carrier is selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres, agarose microspheres, chitosan microspheres, cellulose microspheres, and dextran microspheres; and / or the pore size of the solid phase carrier is 600-800 nm.

8. Use of the polymyxin B-specific degrading enzyme according to any one of claims 1 to 4 in the purification of polymyxin B.

9. A gene encoding the polymyxin B-specific degrading enzyme according to any one of claims 1 to 4.

10. An expression vector, characterized in that: Comprising the gene according to claim 9.

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