A recombinant polymyxin enzyme and methods of making and using the same

Abstract

The application provides a recombinant polymyxin enzyme and a preparation and application method thereof, an amino acid sequence of the recombinant polymyxin enzyme is shown as SEQ ID NO. 1, and the recombinant polymyxin enzyme is obtained through site-directed mutation on an amino acid sequence of a wild-type polymyxin enzyme derived from Brevibacillus laterosporus. The recombinant polymyxin enzyme can specifically recognize and cut a peptide bond between a tripeptide side chain and a cyclic heptapeptide ring in a polymyxin structure and a peptide bond between threonine and diaminobutyric acid inside the cyclic heptapeptide ring, and further can specifically degrade or neutralize the recombinant polymyxin enzyme of polymyxin.

Description

Technical Field

[0001] This invention relates to the field of degrading enzymes, and in particular to a recombinant polymyxinase and its preparation and application methods, which can be used to efficiently and specifically degrade polymyxins. Background Technology

[0002] Polymyxins are a class of basic linear cyclic cationic polypeptide antibiotics produced by Bacillus polymyxa. Based on structural differences, they can be divided into polymyxin A, B, C, D, and E components. Among them, polymyxin B and polymyxin E are of great clinical significance. They have a strong bactericidal effect against Gram-negative bacteria (including most multidrug-resistant bacteria) and are regarded as the "last line of defense" against multidrug-resistant Gram-negative bacterial infections.

[0003] Because polymyxins are clinically used for severe infections (such as sepsis and burn infections), it is essential to ensure that the product is free from microbial contamination. Therefore, sterility testing is a critical step in drug quality control, aiming to ensure that the drug does not contain live microorganisms. For antibiotic preparations, especially potent antibiotics like polymyxins, sterility testing faces a significant challenge: residual antibiotic activity in the sample may inhibit microbial growth, leading to false negative results. Even if the sample is actually contaminated, it may not be detectable in the test.

[0004] Currently, sterility testing for polymyxin antibiotics mainly follows pharmacopoeia methods, typically employing membrane filtration combined with vigorous rinsing to remove the antibiotic's antibacterial activity. However, this rinsing and neutralization method is cumbersome and time-consuming: the use of large amounts of rinsing solution (≥1000ml) makes the process tedious, time-consuming, and inefficient. Even after vigorous rinsing, there is still a risk of antibiotic residue, which may inhibit microbial growth, leading to false negatives and posing risks to product quality and medication safety. Furthermore, this process not only requires complex validation experiments to prove the rinsing effect but also consumes large amounts of rinsing solution and laboratory consumables, increasing costs and generating more laboratory waste.

[0005] It is worth noting that polymyxin molecules are complex cyclic peptides containing multiple cationic amino acid residues and fatty acid tails. Their structure is highly stable, making it difficult to efficiently and specifically destroy their activity using conventional chemical methods. Although some bacteria naturally produce drug-resistant enzymes (such as certain hydrolases), and Bacillus licheniformis can naturally produce hydrolases (such as basic serine proteases), the extraction process of natural hydrolases is extremely complex and yields low amounts. Therefore, large-scale fermentation is not feasible, limiting the mass production of polymyxin enzymes and significantly increasing the cost of using polymyxin enzymes. Furthermore, the development and application of enzyme preparations that can efficiently degrade polymyxins and are suitable for pharmaceutical quality control environments remain unexplored areas.

[0006] Therefore, there is an urgent need in this field for a polymyxinase that is highly specific, highly active, and stable, capable of rapidly and thoroughly neutralizing the antibacterial activity of polymyxin antibiotics, simplifying their aseptic testing procedures, improving the accuracy and reliability of test results, and ultimately ensuring patient medication safety. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant polymyxinase and its preparation and application method, which prepares a recombinant polymyxinase that can specifically degrade or neutralize polymyxins, and can be applied to the aseptic testing of polymyxin antibiotics to simplify the aseptic testing process and improve the accuracy and reliability of test results.

[0008] To achieve the above objectives, in a first aspect, this technical solution provides a recombinant polymyxinase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the recombinant polymyxin was obtained by site-directed mutagenesis of the amino acid sequence of wild-type polymyxin derived from Bacillus laterosporus. The mutation sites were: phenylalanine at position 12 was mutated to glutamic acid, glycine at position 65 was mutated to alanine, and serine at position 156 was mutated to leucine. The advantage of this is that it can broaden the substrate spectrum while improving catalytic efficiency.

[0010] It should be noted that the amino acid sequence of wild-type polymyxinase of Bacillus laterosporus was selected from the Uniprot website Primary accession: Q5XZF9 as the parent.

[0011] Furthermore, the recombinant polymyxinase exhibited an enzyme activity as high as 137.59 U / mg, which was significantly superior to the enzyme activity of wild-type polymyxinase derived from Bacillus laterosporus.

[0012] Furthermore, under conditions of 37°C and pH=7.5, the recombinant polymyxinase achieved a degradation rate of 96.1% for polymyxin B sulfate within 30 minutes, which is 1.3-1.5 times the degradation efficiency of wild-type polymyxinase.

[0013] Furthermore, this recombinant polymyxinase specifically recognizes and cleaves the peptide bond between the tripeptide side chain and the cyclic heptapeptide ring in the polymyxin structure, as well as the peptide bond between threonine and diaminobutyric acid inside the cyclic heptapeptide ring, exhibiting advantages such as strong targeting, good stability, and high hydrolysis efficiency.

[0014] Specifically, the antibacterial activity of polymyxins depends on the spatial structure of their tripeptide side chain and cyclic heptapeptide ring. The tripeptide side chain binds to the bacterial outer membrane, while the cyclic heptapeptide ring penetrates and disrupts the cell membrane. The recombinant polymyxinase provided in this protocol directly breaks the core molecular structure by cleaving the peptide bond between the tripeptide side chain and the cyclic heptapeptide ring; simultaneously, it cleaves the peptide bond between Thr and Dab within the cyclic heptapeptide ring, further breaking the cyclic structure. This dual action completely degrades polymyxins into small, non-antibacterial peptide fragments, avoiding false negatives in aseptic tests caused by residual antibacterial activity.

[0015] Furthermore, the recombinant polymyxinase of this scheme can specifically hydrolyze polymyxin B1, polymyxin B2, polymyxin B sulfate, and polymyxin E sulfate.

[0016] Secondly, this solution provides a polynucleotide for encoding recombinant polymyxinase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0017] Furthermore, the polynucleotide is obtained by reverse translation of the amino acid sequence shown in SEQ ID NO.1, wherein the default codon is calculated using all E. coli coding sequences in GenBank.

[0018] Furthermore, the initial apr gene was obtained by reverse translation of the amino acid sequence shown in SEQ ID NO.1. After replacing the rare codons with synonymous codons frequently used by E. coli and performing N-terminal modification, the full sequence was synthesized to obtain the polynucleotide sequence shown in SEQ ID NO.2.

[0019] Furthermore, the N-end modification is as follows: the N-end is reconstructed by integrating the HIS tag, TEV, and MBP tag.

[0020] It should be noted that the polynucleotides in this scheme are obtained by reverse translation of the codons and N-terminal modification of the Escherichia coli host, in order to solve the problem of low expression level and poor solubility of the amino acid sequence of wild-type polymyxinase of Bacillus laterosporus in prokaryotic hosts.

[0021] Thirdly, this solution provides a recombinant vector carrying a polynucleotide encoding recombinant polymyxinase, as shown in SEQ ID NO.2.

[0022] Furthermore, an EcoRV restriction site was added to the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and an Eco53KI restriction site and a TAATAA stop codon were added to the 3' end. The recombinant vector was then seamlessly cloned into an expression vector.

[0023] This method involves adding restriction enzyme sites to both ends of the nucleotide sequence shown in SEQ ID NO.2 to seamlessly insert it into an expression vector. The homologous arms of the expression vector and the gene (composed of restriction enzyme sites and adjacent sequences) are precisely complementary, avoiding reverse insertion or multiple copy insertions, thus improving ligation efficiency. Simultaneously, the addition of a TAATAA stop codon at the 3' end of the nucleotide sequence shown in SEQ ID NO.2 ensures precise termination of ribosome translation at the polynucleotide terminus, preventing readthrough.

[0024] Furthermore, the expression vector is pET-41b. Preferably, the expression vector contains a T7 promoter and a T7 terminator. The T7 promoter can be specifically recognized by the T7 RNA polymerase in Escherichia coli BL21 (DE3), efficiently initiating the transcription of the apr gene; the T7 terminator can avoid transcriptional "readthrough," ensuring precise mRNA length and further improving translation efficiency.

[0025] Fourthly, this scheme provides an engineered strain comprising a recombinant vector, wherein the recombinant vector carries a polynucleotide encoding a recombinant polymyxinase as shown in SEQ ID NO.2.

[0026] Furthermore, the engineered strain was transformed into Escherichia coli BL21 competent cells by chemical transformation, and the strain was verified by colony PCR using primer sequences such as SEQ ID NO.3 and SEQ ID NO.4 and induced expression.

[0027] In other words, the host cell of the recombinant vector mentioned in the third aspect is Escherichia coli BL21 competent cells.

[0028] Specifically, this method involves reverse translating the apr gene obtained by reverse translating the amino acid sequence shown in SEQ ID NO.1 into a codon-biased reverse translation of E. coli to obtain a polynucleotide encoding recombinant polymyxinase. The N-terminus of the polynucleotide encoding recombinant polymyxinase is then reconstructed by fusing HIS, TEV, and MBP tags. After synthesizing the whole gene sequence, a recombinant vector is constructed using seamless cloning. The recombinant polymyxinase Apr is then expressed in a large quantity and in soluble form in E. coli BL21(DE3) through low-temperature induction.

[0029] Fifthly, this solution provides a composition comprising, as described above, recombinant polymyxinase, Tris buffer, glycerol, trehalose, BSA, EDTA, and Tween-80.

[0030] Furthermore, the composition, after being sterilized by irradiation, can maintain the stability of enzyme activity and achieve a sterility assurance level.

[0031] Sixthly, this solution provides a method for preparing recombinant polymyxinase, comprising the following steps:

[0032] S1: Seed culture of engineered strain obtained by activation culture, wherein the engineered strain contains a recombinant vector, wherein the recombinant vector carries a polynucleotide encoding recombinant polymyxinase with the sequence shown in SEQ ID NO.2;

[0033] S2: The seed culture was inoculated into the fermentation medium and cultured to obtain the fermentation broth. The fermentation broth was induced at low temperature using an inducer to obtain cells expressing recombinant polymyxinase.

[0034] S3: After collecting the bacterial cells, they are crushed to obtain crude recombinant polymyxinase solution.

[0035] S4: The recombinant polymyxin crude enzyme solution was separated and purified to obtain recombinant polymyxin.

[0036] Furthermore, the methods for obtaining engineered strains are as follows:

[0037] The apr gene was synthesized from a full sequence of polynucleotides as shown in SEQ ID NO.2.

[0038] An EcoRV restriction site was added to the 5' end of the apr gene, and an Eco53KI restriction site and a TAATAA stop codon were added to the 3' end. The recombinant vector was then obtained by seamless cloning into the vector.

[0039] The recombinant vector was transformed into Escherichia coli BL21 competent cells by chemical transformation and cultured. The engineered strain was obtained by colony PCR verification and induced expression using primer sequences such as SEQ ID NO.3 and SEQ ID NO.4.

[0040] Furthermore, the recombinant vector was added to E. coli BL21 competent cells, placed in an ice bath and then subjected to rapid heat shock followed by another ice bath treatment. Subsequently, the cells were added to antibiotic-free LB medium, cultured in a shaker, and centrifuged to obtain cell pellet. The supernatant was collected and the transformed cells were resuspended in antibiotic-free LB medium. The transformed cells were then plated in kanamycin-resistant medium and cultured overnight.

[0041] Furthermore, the cultured single-clone colonies were used for PCR verification using primers of SEQ ID NO.3 and SEQ ID NO.4. The verified colonies were then inoculated into kanamycin-resistant medium and cultured overnight. The cultured bacterial solution was then transferred to kanamycin LB medium for further culture, and IPTG was added for induction culture to obtain the engineered strain.

[0042] Furthermore, the inducer is isopropyl-β-D-thiogalactoside.

[0043] Furthermore, the crude recombinant polymyxin enzyme solution was subjected to ultrafiltration clarification and DEAE-Sepharose anion exchange chromatography in sequence to obtain recombinant polymyxin enzyme. This method simplifies the enzyme purification process and reduces the loss of polymyxin enzyme activity.

[0044] It is worth mentioning that the preparation method of recombinant polymyxinase provided in this scheme is simple to operate, easy to scale up for production, and has good economic benefits and practical value.

[0045] Seventhly, this scheme provides the use of the recombinant polymyxinase for degrading polymyxin antibiotics.

[0046] Specifically, the recombinant polymyxinase can be used in the sterility testing of polymyxin antibiotics. This recombinant polymyxinase can be widely used in the sterility testing and microbial limit testing of polymyxin antibiotics and their intermediates. It can be used to rapidly and thoroughly neutralize the antibacterial activity of polymyxin antibiotics, simplifying the sterility testing process and improving the accuracy and reliability of test results.

[0047] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects:

[0048] 1. Enhanced enzyme activity: This method reconstructs the N-terminus of recombinant polymyxinase by fusing HIS, TEV site, and MBP tags to the N-terminus of the nucleotide sequence after site-directed mutagenesis. The recombinant polymyxinase encoding gene is synthesized from the whole genome and a recombinant expression vector is constructed, achieving efficient and large-scale soluble expression of recombinant polymyxinase Apr in Escherichia coli. The purified and concentrated polymyxinase has high activity, with an enzyme activity of approximately 137.59 U / mg.

[0049] 2. Broadened substrate spectrum: The recombinant polymyxinase provided in this scheme can not only efficiently hydrolyze polymyxin B sulfate, but also effectively hydrolyze polymyxin B1, polymyxin B2, and polymyxin E sulfate, with hydrolysis efficiencies of 96.1%, 92.4%, 93.8%, and 87.9%, respectively.

[0050] 3. Simple preparation method: The downstream purification process is simplified. The polymyxinase provided by this invention has high soluble expression. Compared with the three-step purification process required by existing purification processes, this invention only requires two steps of ultrafiltration clarification and DEAE anion purification process to obtain a product with a purity of ≥80%, which is suitable for industrial production applications.

[0051] 4. This provides a new and economical option for the microbial sterility and microbial limit testing of highly antibacterial drugs such as polymyxin antibiotics. Recombinant polymyxinase effectively eliminates the antibacterial properties of polymyxin antibiotics, improving the accuracy of test results. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the results of SDS-PAGE detection of the supernatant and precipitate of the Apr-3 engineered strain before and after induction, corresponding to Example 2.

[0053] Figure 2 This is an electrophoretic image of the purified component under 0.15M NaCL elution conditions and its concentration after buffer replacement, corresponding to Example 3, detected by SDS-PAGE.

[0054] Figure 3 It represents the degradation rate of different types of antibiotic samples by different enzymes. Detailed Implementation

[0055] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0056] Example 1: Construction and transformation of recombinant vectors:

[0057] Bacillus retrosporum ( Brevibacillus laterosporus The amino acid sequence of the gene was subjected to site-directed mutagenesis, as shown in SEQ ID No. 1. The sequence of SEQ ID No. 1 was reverse translated to obtain the apr gene of the sequence shown in SEQ ID No. 2. The full-sequence apr gene was synthesized, and an EcoRV restriction site was added to the 5' end, an Eco53KI restriction site and a TAATAA stop codon were added to the 3' end. The apr gene was then seamlessly cloned into the vector pET-41b (kanamycin) to obtain the recombinant plasmid pET-41b-apr.

[0058] Add 4 μL of the recombinant plasmid pET-41b-apr constructed in Example 1 to 100 μL of *E. coli* BL21(DE3) competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and incubate on ice for 3 minutes. Then add 900 μL of LB medium without antibiotics and incubate at 37°C and 150 rpm for 1 hour. Subsequently, centrifuge at 6000 rpm for 2 minutes to obtain cell pellet, aspirate 800 μL of supernatant, resuspend the transformed cells in the remaining LB medium, spread on a medium containing 50 μg / mL kanamycin resistance, and incubate upside down at 37°C overnight.

[0059] On the second day, 20 single-clone colonies were picked and then colony PCR was performed for verification. The primer sequences used for PCR verification are shown in SEQ ID No. 3. The colonies that were confirmed to be cloning correctly were inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight in a shaker at 37°C and 150 rpm.

[0060] On the third day, 200 μL of bacterial culture from each test tube was transferred to 6 mL of LB medium containing 50 μg / mL kanamycin. The tubes were incubated at 37°C and 150 rpm for 4 hours. 3 mL of the incubated culture was transferred to another empty test tube as a pre-induction sample. 100 mM IPTG was added to each test tube to a final concentration of 0.5 mM. Both tubes were incubated at 37°C and 150 rpm for 6 hours. 1 mL of the bacterial culture was taken from each colony before and after induction. The remaining bacterial culture was centrifuged, resuspended in PBS, washed twice, and then resuspended in 1 mL of LB medium. 200 μL of the resuspended bacterial culture was then transferred to 24-well plates containing 1 mL of LB medium at final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The plates were incubated overnight at 37°C.

[0061] On the fourth day, OD was measured using an ELISA reader. 600 The growth of each colony before and after induction at different concentrations of polymyxin B sulfate was reconstructed to obtain the induction status of the target protein Apr. From the colonies that were confirmed to have the correct clone, Apr-3, which had the highest expression level, was selected as the high-expression engineered strain and denoted as "Apr-3".

[0062] Example 2: Shake flask culture of Apr-3 engineered bacteria

[0063] 200 μL of the engineered strain Apr-3 screened in Example 1 was inoculated into 20 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 150 rpm. The next day, the entire culture was inoculated into 2 L of LB medium containing 50 μg / mL kanamycin and cultured for 6 hours at 37°C with shaking at 150 rpm. 10 mL of the culture was taken as a pre-induction sample, and 100 mM IPTG was added to a final concentration of 0.25 mM. Expression was induced overnight at 25°C with 150 rpm. The induced bacterial culture was centrifuged at 8000 rpm and 4°C for 15 minutes, and then washed three times with 20 mM Tris buffer (pH=9.0) to obtain 20 g of wet cells. The cells were then resuspended in 400 mL of Tris buffer (pH=9.0).

[0064] The bacterial cells were homogenized three times under high pressure at 400 bar, and the supernatant, containing polymyxinase, was obtained by centrifugation at 12000 rpm and 4℃ for 20 minutes. SDS-PAGE was used to detect the supernatant and precipitate after Apr-3 induction. The results are as follows: Figure 1 As shown. Figure 1 This displays the expression results of Apr-3 shake-flask culture. Lanes 1-5 from left to right represent: protein molecular weight standard (marker), pre-induction supernatant, pre-induction precipitate, post-induction supernatant, and post-induction precipitate electrophoresis images. Figure 1 It is evident that the target protein Apr in lane 4 has a higher content than that in lane 5, indicating that the expressed target protein exists in the host cell in the form of a soluble protein.

[0065] Example 3: Purification of crude enzyme solution containing polymyxinase

[0066] First, activate the membrane column by circulating 200 mL of pure water at low speed for 5 minutes, then drain the water. The molecular weight of the membrane column is 10 KD, and the tangential flow rate is 100 LMH. Then, replace the water with 200 mL of 20% ethanol and circulate for 10 minutes, then drain the water. Finally, circulate with 500 mL of equilibration buffer (20 mM Tris + 150 mM NaCl, pH=8.5) for 15 minutes until the outlet conductivity stabilizes (≈15 mS / cm).

[0067] Take 250 mL of the crude enzyme solution containing polymyxinase obtained in Example 2, adjust its pH to 9.0, and pre-clarify it through a 0.45 μm depth filter. Then start the initial circulation with a tangential flow rate of 150 LMH and a transmembrane pressure of 0.25 bar, followed by a clarification process with a tangential flow rate of 200 LMH and a transmembrane pressure of 0.75 bar. Repeat this process until the filtrate is clear (NTU < 10). Finally, perform a concentration process with a tangential flow rate of 250 LMH and a transmembrane pressure of 1.5 bar. Repeat this process until the volume is reduced to 1 / 5 of the original volume.

[0068] Take 50 mL of the concentrated enzyme solution and add 1 M NaCl solution to adjust the final concentration to 10 mM. Perform anion exchange chromatography purification using a DEAE-Sepharose Fast Flow column. The equilibration buffer used is 20 mM Tris buffer (pH 9.0) containing 10 mM NaCl. Wash the column for 5-10 column volumes to equilibrate it. Start loading the sample at a flow rate of 2 mL / min. After loading, wash the column for 3-5 column volumes with the equilibration buffer to remove contaminating proteins. The elution buffer is 20 mM Tris buffer (pH 8.5), with the NaCl concentration gradually increased to 0.1 M, 0.15 M, 0.2 M, 0.4 M, 0.6 M, 0.8 M, and 1 M at a flow rate of 3 mL / min. Collect the activity peak during elution. Analyze the purified fraction at each elution concentration using SDS-PAGE. Take 15 mL of the 0.15 M NaCl purified fraction solution and place it in a 10 mL container. Centrifuge in KD ultrafiltration centrifuge tubes at 4°C and 3000×g for 10 minutes until concentrated to the target volume.

[0069] Then, the pre-cooled enzyme protection solution was gradually added and centrifuged again until the original buffer was completely replaced by the enzyme protection solution. Finally, the volume was concentrated to 5 mL. This step was repeated 5-10 times to obtain 50 mL of concentrated polymyxin enzyme with a purity of over 80%. The sample was placed on dry ice (about -78℃) for 30 kGy irradiation sterilization. After completion, it was stored at -20℃ for later use. The enzyme protection solution contained Tris buffer (20 mM, pH=8.0), glycerol (5%, v / v), trehalose (5%, w / v), BSA (0.1%, w / v), EDTA (0.1 mM), and Tween-80 (0.05%, v / v).

[0070] The components with UV-Vis values ​​≥1000 mAU and stable during purification, as well as the compositions after concentration and subsequent solvent exchange, were detected using SDS-PAGE. The results are as follows: Figure 2As shown, lanes 1-6 are, in order, the supernatant obtained after the Apr-3 engineered bacteria were cultured in shake flasks and then broken up; the flow-through solution of the supernatant after loading with AKTA; the protein molecular weight standard (marker); the elution buffer under 0.15M NaCl conditions; the concentrated elution buffer under 0.15M NaCl conditions; and the combined solution of the concentrated elution buffer under 0.15M NaCl conditions after buffer replacement. It can be seen that using the elution buffer containing 0.15M NaCl can yield polymyxinase with high purity.

[0071] Example 4: Polymyxin Base substrate range and catalytic efficiency test

[0072] The substrate range of the mutated recombinant polymyxin, wild-type polymyxin I from *Bacillus laterosporus*, and Apr wild-type polymyxin II from *Bacillus licheniformis* was tested. The substrates included polymyxin B sulfate, polymyxin B1, polymyxin B2, and polymyxin E sulfate. 500 μL of polymyxin antibiotics (5 mg / mL) and 500 μL of the three polymyxins (mutated polymyxin, wild-type polymyxin I, and wild-type polymyxin II) were placed in two identical test tubes. One tube was placed at 37°C and reacted for 1 hour. Then, 1 mL of Tris buffer, 1 mL of 0.1% (w / v) ninhydrin, and 1 mL of 0.02% (v / v) phenylacetaldehyde were added to the other tube and mixed thoroughly. The mixture was heated in an 80°C water bath for 15 minutes and then cooled in an ice-water bath. Accurately transfer the solution from the test tube to a 10 mL colorimetric tube and dilute to the mark with ethanol. Finally, perform fluorescence measurement. After the reaction time in the first test tube is complete, remove it and repeat the same procedure as for the second test tube. Then, use a standard curve to calculate the content of polymyxin antibiotics before and after the reaction in the same sample well, and further calculate the degradation rate of different enzymes on different types of antibiotic samples. Figure 3 ,in Figure 3 The mutant polymyxinase in the text corresponds to the mutated recombinant polymyxinase.

[0073] Figure 3 Experimental results show that the recombinant polymyxinase of this invention has the broadest substrate range, and the degradation rate of polymyxin B sulfate, polymyxin B1, polymyxin B2 and polymyxin E sulfate is all above 87%, which is significantly higher than that of wild-type polymyxinase I and wild-type polymyxinase II.

[0074] Example 5: Application of recombinant polymyxinase in sterility testing of polymyxin antibiotics

[0075] The concentrated polymyxin enzyme solution from Example 3 was used for sterility testing of polymyxin B sulfate for injection and polymyxin E sulfate for injection. The diluent used was sterile sodium chloride-peptone buffer at pH 7.0. The culture media used were thioglycolate fluid medium (FTM, for anaerobic / aerobic bacteria) and tryptic soybean liquid medium (TSB, for aerobic / fungi). The validation strains were Bacillus subtilis, Clostridium sporogenes, and Candida albicans, respectively.

[0076] Polymyxin samples were diluted to 5 mg / mL using sterile sodium chloride-peptone buffer (pH 7.0), and then filtered for sterilization. The polymyxin sample volume was then added to sterile test tubes at a 1:1 ratio to the enzyme working solution volume, and mixed thoroughly. The solutions were incubated at 37°C for 30 minutes. FTM was inoculated with Bacillus subtilis and Clostridium sporogenes (anaerobic bacteria), while TSB was inoculated with Bacillus subtilis and Candida albicans (fungi). Reagents were added according to the groupings in Table 1, with three replicates per group. FTM was incubated anaerobicly at 30°C for 14 days, and TSB was incubated aerobicly at 25°C for 14 days. The condition of the culture medium was observed and recorded on days 3 and 7 (see Table 2). The final results were recorded on day 14. 1 mL of culture was taken from each antibiotic group and the positive control group and placed into 48-well plates for OD measurement using a microplate reader. 600 The neutralization efficiency was calculated, and the experimental results are shown in Table 3. A neutralization efficiency of ≥80% is considered a pass.

[0077] Table 1: Different Grouping Situations

[0078]

[0079] Table 2 shows the results for different groups under different incubation days.

[0080]

[0081] Note: Turbidity levels: clear (-), slightly turbid (±), turbid (+), significantly turbid (++), heavily turbid (+++).

[0082] Table 3. Results of the experimental group and positive control group after 14 days of culture under different antibiotics.

[0083]

[0084] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0085] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A recombinant polymyxin enzyme, characterized in that, The amino acid sequence of the recombinant polymyxin enzyme is shown as SEQ ID NO.

1.

2. The recombinant polymyxin enzyme of claim 1, wherein, The recombinant polymyxin enzyme is obtained by site-directed mutagenesis on the amino acid sequence of the wild-type polymyxin enzyme derived from Brevibacillus laterosporus, and the mutation sites are: the 12th phenylalanine is mutated to glutamic acid, the 65th glycine is mutated to alanine, and the 156th serine is mutated to leucine.

3. The recombinant polymyxin enzyme of claim 1, wherein, The recombinant polymyxin enzyme specifically recognizes and cuts the peptide bond between the tripeptide side chain and the cyclic heptapeptide ring in the polymyxin structure, as well as the peptide bond between the threonine and the diaminobutyric acid in the cyclic heptapeptide ring.

4. A polynucleotide for encoding the recombinant polymyxin enzyme of claim 1, characterized in that, The nucleotide sequence of the polynucleotide is shown as SEQ ID NO.

2.

5. The recombinant polynucleotide of a polymyxin enzyme of claim 4, wherein, The polynucleotide is obtained by reverse translation and N-terminal modification of the amino acid sequence shown as SEQ ID NO.

1.

6. A recombinant vector, characterized in that, The recombinant vector carries the polynucleotide for encoding the recombinant polymyxin enzyme shown as SEQ ID NO. 2, which can be used to encode the recombinant polymyxin enzyme with the amino acid sequence shown as SEQ ID NO.

1.

7. The recombinant vector of claim 6, wherein, The recombinant vector is obtained by adding an EcoRV enzyme cutting site at the 5' end and an Eco53KI enzyme cutting site and a TAATAA stop codon at the 3' end of the polynucleotide sequence shown as SEQ ID NO. 2, and then performing seamless cloning into an expression vector.

8. An engineered bacterial strain, characterized in that, The recombinant vector comprises a polynucleotide for encoding the recombinant polymyxin enzyme shown as SEQ ID NO. 2, which can be used to encode the recombinant polymyxin enzyme with the amino acid sequence shown as SEQ ID NO.

1.

9. The engineered bacterial strain of claim 8, characterized in that, The engineering strain is obtained by chemically transforming the recombinant vector into E. coli BL21 competent cells, and then verifying by colony PCR with primers shown as SEQ ID NO. 3 and SEQ ID NO. 4, and inducing expression.

10. A composition characterized in that, The recombinant polymyxin enzyme, Tris buffer, glycerol, trehalose, BSA, EDTA, and Tween-80 are included.

11. A method of producing a recombinant polymyxin enzyme, characterized by, The method comprises the following steps: S1: activating the culture of the engineering strain to obtain a seed liquid, wherein the engineering strain comprises a recombinant vector, and the recombinant vector carries a polynucleotide for encoding the recombinant polymyxin enzyme shown as SEQ ID NO. 2, which can be used to encode the recombinant polymyxin enzyme with the amino acid sequence shown as SEQ ID NO. 1; S2: inoculating the seed liquid into a fermentation medium to culture and obtain a fermentation liquid, and then inducing the fermentation liquid at a low temperature to obtain a bacterial body expressing the recombinant polymyxin enzyme; S3: collecting the bacterial body and then crushing to obtain a crude enzyme liquid of the recombinant polymyxin enzyme; S4: separating and purifying the crude enzyme liquid of the recombinant polymyxin enzyme to obtain the recombinant polymyxin enzyme.

12. The method for preparing the recombinant polymyxin enzyme according to claim 11, wherein The apr gene is synthesized by full sequence synthesis based on the polynucleotide sequence shown in SEQ ID NO. 2, and the recombinant vector is obtained by seamless cloning into the vector after adding an EcoRV restriction site at the 5' end of the apr gene, an Eco53KI restriction site and a TAATAA termination codon at the 3' end, and culturing the recombinant vector into E. coli BL21 competent cells by a chemical transformation method, and obtaining the engineering strain by colony PCR verification and induced expression of primers with sequences shown in SEQ ID NO. 3 and SEQ ID NO.

4. The inducer is isopropyl-beta-D-thiogalactoside.

13. The method for preparing recombinant polymyxinase according to claim 11, characterized in that, The recombinant polymyxin enzyme crude enzyme solution is sequentially subjected to ultrafiltration clarification and DEAE-Sepharose anion exchange chromatography to obtain the recombinant polymyxin enzyme.

14. The method for preparing recombinant polymyxinase according to claim 11, characterized in that, 15. Use of the recombinant polymyxin enzyme according to any one of claims 1-3 for degrading polymyxin antibiotics. Use as an application in the sterile examination of polymyxin antibiotics.

16. Use of the recombinant polymyxin enzyme according to claim 15 for degrading polymyxin antibiotics, characterized in that, Use as an application in the sterile examination of polymyxin antibiotics.

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