CN106188253B - Antibacterial peptide Lexapeptide and preparation method and application thereof - Google Patents
Antibacterial peptide Lexapeptide and preparation method and application thereof Download PDFInfo
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- CN106188253B CN106188253B CN201610737649.0A CN201610737649A CN106188253B CN 106188253 B CN106188253 B CN 106188253B CN 201610737649 A CN201610737649 A CN 201610737649A CN 106188253 B CN106188253 B CN 106188253B
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- lexapeptide
- streptomyces
- streptomyces lividans
- lantipeptide
- sbt5
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- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000008650 pH stress Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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- 238000010257 thawing Methods 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention discloses an antibacterial peptide Lexapeptide and a preparation method and application thereof; the lantipeptide antibacterial peptide Lexapeptide is a new compound obtained by heterogeneously expressing a bacterial artificial chromosome library derived from a Streptomyces rochei (Streptomyces rochei) Sal35 strain in Streptomyces lividans (Streptomyces lividans) SBT5 and then separating; the L-amino acid composed of 38 amino acids is a lantipeptide which is reported as the first example and simultaneously comprises a C-terminal 2-amino alkenyl-3-methyl-cysteine structure and an N-terminal methylation modification structure, and the high modification of the structure endows Lexapeptide with better stability compared with other lantipeptide antibacterial peptides. Lexapeptides can be used as an anti-gram-positive agent and provide a solution to the problem of drug resistance commonly encountered in the clinic.
Description
Technical Field
The invention belongs to the field of application of peptide antibiotics, and particularly relates to an antibacterial peptide Lexapeptide, and a preparation method and application thereof.
Background
The lantibiotic peptides are antibiotics mainly produced by gram-positive bacteria, and are highly modified peptide antibiotics which are synthesized by ribosome and then are subjected to a series of post-translational modifications. Structurally characterized by lanthionine, methyllanthionine, dehydroalanine, and dehydroaminobutyric acid, and also named for the presence of lanthionine. The lanthionine antibacterial peptide generally has the size of 10-50 amino acids, and has a sulfur-containing lanthionine and methyl lanthionine cyclic structure formed by cysteine and dehydroalanine/dehydroaminobutyric acid on the structure. It is these structural features that endow lantibiotic peptides with strong antibacterial activity.
As the best researched Nisin in the lanthionine family of antibacterial peptides, Nisin has been used as a food preservative in the preservation of dairy products and meat products for more than 40 years, and the report of wide-range drug resistance has not yet appeared. However, Nisin has insufficient temperature resistance and exhibits good activity only at low pH values (pH < 7). The action target of the lantibiotic peptides is usually positioned on a cell wall or a cell membrane, so that the structure of the cell wall or the cell membrane is incomplete, holes are formed, and bacteria are killed, and the sterilization mechanism also causes the drug resistance of the lantibiotic peptides to rarely appear. Because the application of the traditional antibiotics has the problem of large-area drug resistance, the lantipeptide antibacterial peptide has good antibacterial activity against drug-resistant staphylococcus aureus and drug-resistant enterococcus faecalis, and the research of the lantipeptide antibacterial peptide as a substitute of the existing antibiotics is becoming a research hotspot. About 50 kinds of lantipeptide antimicrobial peptides have been discovered and reported, among which NVB302, mutacin1140, duramycin, gallidermin, lacticin 3147 and microbiosporin have been clinically studied. The high conversion rate of clinical research makes the lantipeptide antibacterial peptide have great prospect in drug research and development. However, the lantibiotide antibacterial peptides generally have the defects of narrow antibacterial spectrum and insufficient stress resistance. Therefore, the development and research of novel efficient lantipeptide antibacterial peptides with strong stress resistance have important value. The streptomyces has a plurality of biosynthetic gene clusters for coding the lantibiotide antibacterial peptides, the gene clusters are usually silent in wild strains, and the production of the lantibiotic peptides by a heterologous expression method has important significance for developing novel and efficient lantibiotic peptides.
Disclosure of Invention
The invention aims to find efficient and active antibacterial peptide from streptomyces and provide a precursor for drug development; in particular to a novel high-efficiency lantibiotic peptide Lexapeptide and a preparation method and application thereof.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention relates to a lantibiotic peptide Lexapeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1, and the primary amino acid sequence shown in the figure:
Phe-Ser-Pro-Thr-Ile-Pro-Trp-Ala-Ile-Arg-Ala-Thr-Ile-Ile-Thr-Ala-Arg-Ser-Ser-Gln-Gln-Cys-Ala-Ala-Ala-Leu-Gly-Ser-Leu-Thr-Ala-Lys-Thr-Ile-Glu-Lys-Lys-Cys; wherein a plurality of serines (Ser) and threonines (Thr) are dehydrated to dehydroalanine (Dha) and dehydroaminobutyric acid (Dhb).
Preferably, the amino acid sequence has serine at positions 2, 18 and 19 dehydrated to produce dehydroalanine, threonine at positions 4 and 12 dehydrated to produce dehydroaminobutyric acid, and serine at position 28 dehydrated and reduced to produce D-alanine.
Preferably, the lantibiotic peptide Lexapeptide contains methyl lanthionine and is formed by condensation between cysteine at position 22 and threonine at position 33 in the amino acid sequence.
Preferably, the terminal cysteine at position 38 of the amino acid sequence undergoes oxidative decarboxylation to form 2-aminoethylene thiol (2-aminoenethiol), which is further condensed with dehydroaminobutyric acid at position 30 to form the rare 2-aminoalkenyl-3-methyl-cysteine (AviMeCys) structure.
Preferably, the amino group of the phenylalanine at the N-terminus of the amino acid sequence is modified with two methyl groups.
Preferably, the serine at position 28 of the amino acid sequence is modified to alanine in the D-configuration.
The molecular formula of the lantibiotic peptide Lexapeptide is as follows:
in a second aspect, the present invention also relates to a preparation method of the lantipeptide antimicrobial peptide Lexapeptide, which comprises the following steps:
s1, heterologously expressing a bacterial artificial chromosome library derived from a Streptomyces rochei (Streptomyces rochei) Sal35 strain in Streptomyces lividans (Streptomyces lividans) SBT5 to obtain recombinant Streptomyces lividans (Streptomyces lividans) SBT5/Sal 356A 8CGMCC No. 12751;
s2, fermenting and culturing the recombinant streptomyces lividans to obtain the lanthionine antibacterial peptide Lexapeptide.
Preferably, step S2 further includes a step of separation and purification after the fermentation culture.
Preferably, step S2 includes:
a1, fermenting the recombined streptomyces lividans on a fermentation culture medium YBP for 5-8 days, and collecting a solid fermentation product;
a2, leaching the solid fermentation product for several times by methanol with the same volume, and carrying out vacuum concentration and spin drying;
a3, dissolving the methanol extract in water, extracting with n-butanol of the same volume for several times, and vacuum concentrating and spin drying;
a4, purifying the n-butanol extract by macroporous adsorption resin CHP20P and gel chromatography LH20, and separating the purified product, namely Lexapeptide, by liquid phase separation.
Preferably, the preparation conditions of the high performance liquid chromatography adopted in the separation and purification are as follows:
the mobile phase is as follows: phase A is water phase (added with 1 per mill trifluoroacetic acid (volume ratio)), phase B is acetonitrile;
the elution conditions were: the 40% B phase [ volume ratio ] was eluted isocratically for 15min at a flow rate of 0.6ml/min, and Lexapeptide was eluted at 10 min.
In a third aspect, the invention also relates to the application of the lantipeptide antibacterial peptide Lexapeptide in preparation of a preparation for resisting gram-positive bacteria.
In a fourth aspect, the invention also relates to the use of the lantipeptide antibacterial peptide Lexapeptide in any one of the above in the preparation of a medicament for treating bacterial, fungal or viral infection.
Preferably, the medicament for treating bacterial, fungal or viral infection is a medicament for treating methicillin-resistant staphylococcus aureus or staphylococcus epidermidis infection.
In a fifth aspect, the invention also relates to the application of the lantipeptide antibacterial peptide Lexapeptide in any one of the above in preparation of animal feed.
In a sixth aspect, the invention also relates to a pharmaceutical composition, which takes the lantipeptide antibacterial peptide Lexapeptide or the pharmaceutically acceptable salt thereof as an active ingredient.
Preferably, the composition further comprises a pharmaceutically acceptable excipient, carrier or diluent.
In the seventh aspect, the invention also relates to Streptomyces lividans SBT5/Sal 356A 8CGMCC No. 12751.
The preparation method of the genetic engineering recombinant streptomyces lividans comprises the following steps: bacterial artificial chromosomes derived from the Streptomyces rochei (Streptomyces rochei) Sal35 strain were screened (BAC,BacterialArtificialChromosome) library, to obtain positive BAC SAL35-6a 8; SAL35-6A8 is introduced into a heterologous expression host Streptomyces lividans (Streptomyces lividans) SBT5 to obtain a recombinant Streptomyces lividans SBT5/Sa 156A 8 which generates Lexapeptide.
In the invention, the Streptomyces lividans SBT5/Sal 356A 8 has been submitted to the China Committee for culture Collection of microorganisms at 2016, 7, 11 days (address: the code 100101 of the institute for microbiology, China academy of sciences, No. 3 of the Ministry of microbiology, North West Lu No.1 institute of south Korean, Beijing), and the strain preservation number is CGMCC No. 12751.
The novel lantipeptide antibacterial peptide Lexapeptide is further subjected to an antibacterial experiment, and the antibacterial experiment is performed on methicillin-resistant staphylococcus aureus and staphylococcus epidermidis, and the antibacterial effect is equivalent to that of vancomycin. MIC values for methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis were 0.52. mu.M and 1.03. mu.M, respectively; the MIC values for vancomycin were 0.61. mu.M and 1.21. mu.M.
Compared with the prior art, the invention has the following beneficial results:
1. the invention discloses a novel high-efficiency lantibiotic peptide Lexapeptide. A novel compound obtained by separating a bacterial artificial chromosome library derived from a Streptomyces rochei Sal35 strain Sal35 after heterologous expression in Streptomyces lividans SBT 5; lexapeptide consists of 38 amino acids and has the molecular formula: C179H286N50042S2, the structural formula is shown in figure 8, and the relative molecular mass is: 3872.61 g/mol.
2. Lexapeptide is the lantipeptide reported in the first example and simultaneously comprises a C-terminal 2-amino alkenyl-3-methyl-cysteine structure and an N-terminal methylation modified structure, and the high modification of the structure endows the Lexapeptide with better stability compared with other lantipeptide antibacterial peptides; compared with Nisin, the high-temperature-resistant Nisin has better tolerance to temperature (50 ℃) and pH (2-12).
3. The biological activity test finds that Lexapeptide has very good antibacterial activity to methicillin-resistant staphylococcus aureus and staphylococcus epidermidis which are widely clinically available, the antibacterial activity of Lexapeptide is equivalent to or even superior to vancomycin of the last line of defense of clinical antibiotics, and the Lexapeptide has no inhibitory activity to gram-negative bacteria. For Mycobacterium smegmatis mc2155 showed superior bacteriostatic activity to Nisin.
4. Lexapeptides can be used as an anti-gram-positive agent and provide a solution to the problem of drug resistance commonly encountered in the clinic.
5. The antibacterial mechanism based on the lantipeptide antibacterial peptide is different from vancomycin, and Lexapeptide has potential application value for clinical assistance or substitution of vancomycin.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a map of the expression of Lexapeptide-carrying bacterial artificial chromosomes in Streptomyces lividans; the recombinant strain Streptomyces lividans SBT5/Sal 356A 8 can obviously inhibit the growth of the bioassay indicator staphylococcus aureus on a screening plate.
FIG. 2 is a liquid phase assay profile of Lexapeptide expression; lexapeptide eluted at about 21min, and the chromatographic peak marked with an asterisk in the figure is Lexapeptide.
FIG. 3 is a liquid phase map of Lexapeptide purification; lexapeptide eluted about 10min, and the chromatographic peak marked with an asterisk in the figure is Lexapeptide.
FIG. 4 is a MALDI-FTICR mass spectrum of Lexapeptide.
FIG. 5 is an ESI-FTICR tandem mass spectrum of Lexapeptide; the b/y fragment of Lexapeptide is indicated in the figure.
FIG. 6 is a graph of Lexapeptide structure and tandem mass spectrometry fragmentation behavior.
FIG. 7 is a schematic representation of the Lexapeptide activity test and stress resistance study; wherein a is a biological activity test chart of Lexapeptide (100 mu g/ml) and Nisin (100 mu g/ml) on Mycobacterium smegmatis. And b is a biological activity test chart of Lexapeptide (100 mu g/ml) and Nisin (50 mu g/ml) on micrococcus luteus after being treated by PBS buffers with different pH values. And c is a bioactivity test chart of Lexapeptide (100 mu g/ml) and Nisin (100 mu g/ml) after being treated at 50 ℃ for different days.
FIG. 8 is a schematic diagram of the molecular structure of lantipeptide antimicrobial peptide Lexapeptide.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention relates to a novel high-efficiency lantibiotic peptide Lexapeptide and a preparation method and application thereof; wherein,
LB culture medium: culturing Escherichia coli;
10g of tryptone, 5g of yeast extract, 5g of sodium chloride and deionized water to a constant volume of 1,000ml and a pH of 7.0.
2 × YT medium: carrying out joint transfer between escherichia coli and streptomyces;
16g of tryptone, 10g of yeast extract, 5g of sodium chloride and deionized water to a constant volume of 1,000ml and a pH of 7.0.
Mueller Hinton medium: for MIC determination, Staphylococcus aureus, Staphylococcus epidermidis, etc. were cultured and purchased from 0 xoid.
SFM medium: culturing streptomycete to produce spores and performing joint transfer between escherichia coli and streptomycete;
20g of soybean cake powder, 20g of mannitol and tap water to a constant volume of 1,000ml, wherein the pH value is 7.2-7.4.
Soaking 20g of soybean cake powder in1,000 ml of tap water, sterilizing at 121 ℃ for 20min, filtering, collecting soybean cake powder leaching liquor, and preparing a culture medium.
YBP culture medium: streptomycete is used for fermentation;
10g of glucose, 2g of yeast extract, 2g of beef extract, 4g of peptone and MgSO41g, NaCl 15g and tap water to a constant volume of 1,000ml and a pH value of 7.2-7.4.
15g of agar powder is required to be added into a corresponding solid culture medium, and 20g of agar powder is required to be added into an SFM solid culture medium.
Streptomyces rochei Sal35, which is Streptomyces rochei of China Center for Type Culture Collection (CCTCC), isolated from Hippocampus, in the literature "Xu M, Wang Y, ZHaoZ, Gao G, Huang S-X, Kang Q, He X, Lin S, Pang X, Deng Z, Tao M.2016.functional genome mining for mutants encoded by b.large genes, applied. environ. Microbiol. doi: 10.1128/AEM.01383-16.
Heterologous expression hosts Streptomyces lividans SBT5 in the literature "white pavilion, shu yanfei, xu bell, dao meifeng; the construction of the streptomyces lividans high-efficiency heterologous expression host SBT5, disclosed in 2014, university of Huazhong agriculture school newspaper, is publicly available to the public from the national laboratory of microbial metabolism of Shanghai university of transportation.
The invention relates to a conjugal transfer auxiliary strain Escherichia coli ET12567/pUB307, which is disclosed in the literature' Flett F, Mersinias V, Smith CP; high efficiency microbial transfer of plasmid DNA from Escherichia coli to methyl DNA-restriction plasmids, 1997, FEMS Microbiol Lett 155 (2): 223, 229.
The strain Staphylococcus aureus ATCC 29213 for bioactivity assay according to the present invention was obtained from ATCC, and publicly available from American Type Culture Collection (ATCC).
Enterococcus faecalis ATCC29212 is a gift from Ju Jian Hua teacher of south China sea oceanographic institute of Chinese academy of sciences, and has ampicillin and kanamycin resistance. The public is available from the American Type Culture Collection (ATCC).
Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Resistant Staphylococcus epidermidis (MRSE) were offered by the Kinghua teacher, the south-sea Marine institute of Chinese academy of sciences.
Mycobacterium smegmatis mc2155 is offered by professor prof. Micrococcus luteus ATCC4698 Micrococcus luteus of Fujian province is given by Pengfei teacher of Fujian microorganism, and public can culture Micrococcus luteus from American modeCollection stock (ATCC).
Example 1 heterologous expression of bacterial artificial chromosomes containing the Lexapeptide biosynthetic Gene Cluster
(I), the bacterial artificial chromosome library of Streptomyces rochei (Streptomyces rochei) Sal35 (8 pieces of 96-well plate) was taken out from a refrigerator at-80 ℃ and left in an incubator at 37 ℃ for 2 hours until thawing was completed.
And (II) adding 130 mu l of fresh LB culture medium into a 96-well plate, inoculating the bacterial artificial chromosome library of Streptomyces rochei (Streptomyces rochei) Sal35 into the 96-well plate by using a duplicator, and culturing at 37 ℃ and 220rpm for 4-6 hours.
At the same time, ET12567/pUB307 strain was inoculated into 100ml of 250ml triangular flask containing 50. mu.g/m 1 kanamycin LB medium, and cultured at 37 ℃ and 220rpm for 4-6 hours.
(IV) centrifuging at 4,000rpm for 10min to collect ET12567/pUB307 cells, washing with fresh LB medium for 3 times, and resuspending with 15ml of fresh LB.
(V) taking 15. mu.l of concentrated ET12567/pUB307 bacterial liquid by using a discharging gun to add into each well of a 96-well plate of a bacterial artificial chromosome library of Streptomyces rochei (Streptomyces rochei) Sal 35. Culturing at 200rpm for 5min, and mixing.
And (VI) utilizing a duplicator to photocopy the mixed bacterial liquid on an SFM plate uniformly coated with a layer of thermally excited Streptomyces lividans (SBT 5) spores, drying the SFM plate in a biological safety cabinet, and culturing the SFM plate for 12 to 16 hours at the temperature of 30 ℃.
And (seventhly) covering trimethoprim and apramycin on an SFM (small form-factor pluggable) flat plate until the final concentration is 50 mu g/ml, drying in a biological safety cabinet, and culturing at 30 ℃ for 4-6 days until the conjugal metastases grow out.
(VIII) the grown conjugative transferor is photocopied on an SFM plate containing nalidixic acid (25 mu g/ml) and apramycin (50 mu g/ml) by using a duplicator to remove residual escherichia coli and a receptor bacterium streptomyces lividans SBT5, and the plate is cultured for 4-6 days at 30 ℃ until sporulation is completed.
And (ninthly), transferring the zygospores produced to a fermentation medium YBP by using a duplicator, and performing fermentation culture at 30 ℃ for 4-6 days until the production of the elements is complete.
(Ten) cover a layer of soft agar (0.5% agar) containing 1% Staphylococcus aureus on the plate with complete production of the antibiotic, incubate overnight at 37 ℃ and observe the formation of the zone of inhibition. The observed inhibition zone is shown in figure 1, and the compound produced by the fermentation of the 6A8 clone can obviously inhibit the growth of the bioassay indicator staphylococcus aureus. Obtaining an expression strain of Streptomyces lividans SBT5/Sal 356A 8CGMCC No.12751 of bacterial artificial chromosome containing Lexapeptide.
Example 2 detection of Lexapeptide
Firstly, 6A8 clone is selected from a bacterial artificial chromosome library of Streptomyces rochei (Streptomyces rochei) Sal35 and cultured. Then, the bacterial artificial chromosome is introduced into a heterologous expression host Streptomyces lividans SBT5 by escherichia coli-Streptomyces intergeneric conjugative transfer to obtain a strain Streptomyces lividans SBT5/Sal 356A 8CGMCC No.12751 capable of producing Lexapeptide.
And (II) inoculating bacterial artificial chromosome heterologous expression strains for producing Lexapeptide to a solid YBP culture medium, fermenting 3 plates for each strain, and performing fermentation culture at 30 ℃ for 4-6 days.
And (III) after the fermentation is finished, collecting the solid fermentation product, extracting for 3 times by using methanol with the same volume, and performing vacuum concentration and spin drying to obtain a methanol crude extract.
And (IV) dissolving the methanol crude extract by using 10ml of sterile water, extracting for 3 times by using n-butanol with the same volume, concentrating and spin-drying in vacuum to obtain the n-butanol crude extract, and dissolving a sample by using 2ml of methanol.
(V) centrifugation is carried out at 12,000rpm for 10min, and after filtering a methanol sample with a 0.22 μm filter, high performance liquid chromatography analysis is carried out with a sample volume of 10 μ l. The liquid phase analysis chromatographic column model is as follows: agilent Zorbax 300SB-C18(5 μm, 4.6X 250 mm).
The mobile phase is as follows:
phase A is water phase (added with 1 ‰ trifluoroacetic acid),
and the phase B is acetonitrile.
The analysis conditions were: 0-5min, 5% B; 5-20min, increasing the concentration of phase B from 5% to 50%; 20-30min, increasing the concentration of phase B from 50% to 100%; 30-35min, maintaining the 100% concentration of the B phase for 5 min: 35-36min, reducing the concentration of phase B from 100% to 5%; 36-45min, and balancing 5% concentration of phase B for 9 min. The flow rate was 0.6ml/min, room temperature. Lexapeptide eluted at 21min, as shown in FIG. 2, with the corresponding elution peaks indicated by asterisks.
Example 3 purification and characterization of Lexapeptide
Lexapeptide was purified and identified by analysis in example 2 using the strain Streptomyces lividans SBT5/Sal 356A 8CGMCC No. 12751.
Firstly, inoculating Streptomyces lividans SBT5/Sal 356A 8CGMCC No.12751 strain to SFM plate for sporulation, and culturing at 30 ℃ for 7 days until sporulation is complete. Lexapeptide has obvious inhibition on the growth of the strain, and the sporulation time is remarkably delayed later.
And (II) inoculating fresh spores of Streptomyces lividans SBT5/Sal 356A 8CGMCC No.12751 strain to a 30L solid YBP culture medium for large-scale fermentation and accumulation of Lexapeptide, and performing fermentation culture at 30 ℃ for 4-6 days.
And (III) collecting solid fermentation products of Streptomyces lividans SBT5/Sal 356A 8CGMCC No.12751 strain, leaching for 3 times by using methanol (30L) with the same volume, and performing vacuum concentration and spin drying to obtain methanol crude extracts.
And (IV) dissolving the methanol crude extract in 1L of sterile water, extracting for 3 times by using 1L of n-butyl alcohol, and carrying out vacuum concentration and spin drying on the n-butyl alcohol phase to obtain the n-butyl alcohol crude extract.
And (V) carrying out dry loading, namely uniformly stirring the n-butanol crude extract with CHP20P filler, loading the stirred filler on a CHP20P open column (50X 500mm), and carrying out gradient elution on methanol and water. When the methanol elution concentration increased to 80%, Lexapeptide began to elute until the methanol elution concentration increased to 100%. Collecting 80-100% methanol elution component, vacuum concentrating and spin drying.
Sixthly, dissolving the eluted components of the CHP20P column by using methanol again, and filtering by using a 0.22 mu m filter membrane to obtain a clear methanol solution. A sample of methanol was applied to a chromatographic column (20X 1500mm) packed with LH20 packing, pure methanol was eluted at 10 drops/s and fractions were collected in test tubes of 5ml per tube. The collected fractions were subjected to HPLC detection, and fractions containing Lexapeptide were pooled for further analysis.
Seventhly, collecting the component containing Lexapeptide eluted by the LH20 column, concentrating in vacuum, spin-drying, and dissolving again by using methanol. Filtering with a 0.22 mu m filter membrane to obtain a clear methanol solution, and carrying out high performance liquid chromatography separation and purification, wherein the sample volume is 10-20 mu l. The liquid phase analysis chromatographic column model is as follows: agilent Zorbax 300SB-C18(5 μm, 4.6X 250mm)
The mobile phase is as follows:
phase A is water phase (added with 1 ‰ trifluoroacetic acid),
and the phase B is acetonitrile.
The analysis conditions were 40% B isocratic elution for 15min, with a flow rate of 0.6 ml/min. Lexapeptide begins to elute in about 10min and finishes eluting in about 11.5min, and Lexapeptide single peak elution components are collected for analysis. Lexapeptide elution chromatographic peaks are shown in figure 3, and the chromatographic peaks marked with asterisks in the figure are Lexapeptides.
(VIII) carrying out mass spectrum analysis on the purified Lexapeptide to determine the molecular weight, and measuring the molecular weight of the Lexapeptide to be 3872.16Da by using MALDI-FTICR MS. The molecular weight determinations of Lexapeptide are shown in figure 4.
(nine) the structure of Lexapeptide is determined by ESI-FTICR-MS/MS through tandem mass spectrometry. Selection of parent ion [ M +4H]4+The secondary mass spectrometry was carried out at 969.00Da and the fragment ions from Lexapeptide 'b/y' obtained by tandem mass spectrometry are shown in FIG. 5 and the fragment ions corresponding to the Lexapeptide fragment in FIG. 6.
Example 4 determination of the biological Activity of Lexapeptide
The Minimum Inhibitory Concentration (MIC) values of Lexapeptide were determined according to the microplate method, and the strains to be tested were Staphylococcus aureus ATCC25923, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and enterococcus faecalis ATCC 29212. MIC values were defined as the lowest antibiotic concentration that inhibited the growth of the strain by more than 90% of the concentration. Ampicillin, kanamycin and vancomycin were used as controls, with 3 replicates per sample.
Firstly, Mueller-Hinton (MH) broth culture medium is selected to culture experimental bacteria, and the experimental bacteria are cultured for 12 to 16 hours at 37 ℃ overnight. The sample solution was prepared before the experimental strain grew.
(II) preparing a sample solution, dissolving samples (Ampicillin Ampicillin, Kanamycin Kanamycin, Vancomycin and Lexapeptide) by DMSO to prepare mother liquor with the concentration of 32mg/ml, 3.2mg/ml and 6.4mg/ml respectively.
(III) Add sterile MH broth medium to 96-well plates, 184. mu.l sterile MH broth to column 1, and 100. mu.l sterile MH broth to each of the remaining columns. Columns 11 and 12 serve as positive and negative controls, respectively.
And (IV) sucking 16 mul of prepared mother solution, adding the mother solution into a 96-well plate in the row 1, and gently blowing and beating the mother solution by using a pipette to mix the mother solution and the 96-well plate uniformly.
And (V) sucking 100 mu l of the culture solution mixed with the antibiotics from the row 1 to the row 2, gently blowing and mixing the culture solution by using a pipette, sucking 100 mu l of the culture solution to the row 3, and diluting the culture solution to the row 10 by analogy. After mixing in column 10, 100. mu.l of the culture medium was aspirated and discarded.
(VI) overnight cultured cells were diluted 1000-fold with fresh MH broth, 100. mu.l of the diluted cell suspension was added to the wells from column 1 to column 11, and 100. mu.l of fresh MH broth was added to column 12 as a blank. The drug concentration gradient of ampicillin and kanamycin thus obtained was: 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 μ g/ml; the drug concentration gradient of vancomycin is as follows: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μ g/ml; the drug concentration gradient for Lexapeptide was: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5. mu.g/ml.
And (seventhly) covering a 96-well plate cover, and culturing in an incubator at 37 ℃ overnight for 16-24 hours.
(eighth) using 11 th column as positive control and 12 th column as blank control, and using microplate reader to OD600The concentrations were read down and the MIC values for each sample were determined as shown in table 1 below.
TABLE 1 minimum inhibitory concentrations (MIC. mu.g/ml) of Lexapeptide and related antibiotics
MIC data in Table 1 are expressed in mass concentration; when converted to molar concentrations, the MIC concentrations of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were 0.61. mu.M and 1.21. mu.M, respectively; the MIC concentrations of Lexapeptide against methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis were 0.52. mu.M and 1.03. mu.M, respectively. Therefore, the Lexapeptide bacteriostatic effect was comparable to that of vancomycin.
Lexapeptide on Mycobacterium smegmatis (Mycobacterium smegmatis mc)2155) And (3) determination of antibacterial activity:
lexapeptide and Nisin solutions were prepared at 100. mu.g/ml with 100mM Tris-HCl (pH 7.0). 30 μ l of the suspension was added to a growth plate of Mycobacterium smegmatis on which a small hole was punched, and cultured at 37 ℃ for 24 hours to observe the formation of a zone of inhibition. Equiconcentrations of Lexapeptide were superior to Nisin in activity against Mycobacterium smegmatis, see FIG. 7a, in which case Lexapeptide had inhibitory activity against Mycobacterium smegmatis, whereas Nisin had no activity.
Example 5 stress resistance study of Lexapeptide.
(I) research on pH stress resistance: PBS buffer is prepared and adjusted to different pH values with phosphoric acid or hydrochloric acid for later use (2, 4, 6, 8, 10, 12). Lexapeptide 100. mu.g/ml and Nisin 50. mu.g/ml stock solutions were prepared in PBS buffer. Mu.l of Lexapeptide mother liquor and Nisin mother liquor are respectively added into 24 mu.l of buffer solutions with different pH values and are kept stand for 2 hours at room temperature. Mu.l of the sample was added to a bioassay plate of Micrococcus luteus perforated with a small hole, and incubated at 30 ℃ for 24 hours to observe the formation of a zone of inhibition.
The bioassay results are shown in FIG. 7b, which shows that the Nisin activity is significantly reduced in the alkaline pH range, and the bacteriostatic activity is completely lost at a pH value of 12. However, Lexapeptide showed comparable bacteriostatic activity to the control over the tested pH range. Indicating that Lexapeptide has stronger tolerance to acid and alkali.
(II) temperature stress resistance research: lexapeptide and Nisin solutions were prepared in 100mM Tris-HCl (pH 7.0). Lexapeptide (100. mu.g/ml) and Nisin (100. mu.g/ml) were incubated in a water bath at 50 ℃ for 8 days, and 30. mu.l of the remaining sample was taken every day for bioactivity assay. And adding the treated sample to a bioassay plate of micrococcus luteus with a small hole, and culturing at 30 ℃ for 24 hours to observe the formation of a bacteriostatic ring.
The results of bioassay are shown in FIG. 7c, which shows that the antibacterial activity of Nisin gradually decreases after being treated at 50 ℃ for one day, and the activity is basically completely lost after 5 days. Lexapeptide showed good tolerance to temperature and activity after 8 days of treatment was still comparable to that of the untreated Lexapeptide control sample. The activity of Lexapeptide is not affected when the warm bath time is prolonged to 10 days. It can be seen that Lexapeptide is more temperature tolerant than Nisin.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (5)
1. A lanthionine antibacterial peptide Lexapeptide is characterized in that the framework sequence is shown as SEQ ID NO: 1; wherein, the serine at the 2 nd, 18 th and 19 th positions in the amino acid sequence is dehydrated to generate dehydroalanine, the threonine at the 4 th and 12 th positions is dehydrated to generate dehydroaminobutyric acid, and the serine at the 28 th position is dehydrated and reduced to generate D-alanine; the lanthionine antibacterial peptide Lexapeptide contains methyl lanthionine and is formed by condensation between cysteine at position 22 and threonine at position 33 in the amino acid sequence; the 38 th cysteine at the tail end of the amino acid sequence undergoes oxidative decarboxylation reaction to form 2-aminoethylene thiol, and is further condensed with 30 th dehydroaminobutyric acid to form a rare 2-aminoalkenyl-3-methyl-cysteine structure; the amino group of the phenylalanine at the N-terminal of the amino acid sequence is modified by two methyl groups.
2. A method for preparing the lantipeptide antimicrobial peptide Lexapeptide according to claim 1, wherein the method comprises the following steps:
s1, heterologous expression of a bacterial artificial chromosome library derived from a Streptomyces rochei (Streptomyces rochei) Sal35 strain in Streptomyces lividans (Streptomyces lividans) SBT5 to obtain recombinant Streptomyces lividans (Streptomyces lividans) SBT5/Sal 356A 8 with a preservation number: CGMCC No. 12751;
s2, fermenting and culturing the recombinant streptomyces lividans to obtain the lanthionine antibacterial peptide Lexapeptide.
3. Use of the lantipeptide antimicrobial peptide Lexapeptide of claim 1 in the preparation of a gram-positive bacterial preparation, an animal feed or a medicament for the treatment of methicillin-resistant staphylococcus aureus or staphylococcus epidermidis infections.
4. A pharmaceutical composition comprising the lantipeptide antimicrobial peptide Lexapeptide or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.
5. A Streptomyces lividans SBT5/Sal 356A 8, deposited under accession number: CGMCC No. 12751.
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