CN106188253A - Antibacterial peptide Lexapeptide and its production and use - Google Patents
Antibacterial peptide Lexapeptide and its production and use Download PDFInfo
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- CN106188253A CN106188253A CN201610737649.0A CN201610737649A CN106188253A CN 106188253 A CN106188253 A CN 106188253A CN 201610737649 A CN201610737649 A CN 201610737649A CN 106188253 A CN106188253 A CN 106188253A
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- lexapeptide
- lanthiopeptin
- antibacterial peptide
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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)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a kind of antibacterial peptide Lexapeptide and its production and use；This lanthiopeptin class antibacterial peptide Lexapeptide is by coming from the Bacterial Artificial Chromosome Library of Lou Che Shi streptomycete (Streptomyces rochei) the Sal35 bacterial strain noval chemical compound of isolated after heterogenous expression in muta lead mycillin (Streptomyces lividans) SBT5；It is by 38 Amino acid profiles, being to comprise C-terminal 2 aminoalkenyl 3 methyl cysteine structure while the first report and N-terminal methylates modification structure lanthiopeptin, the height in this structure is modified and is imparted Lexapeptide relative to other lanthiopeptin more preferable stability of class antibacterial peptide.Lexapeptide can serve as resisting gram-positive bacteria preparation, and the drug resistance problems commonly encountering clinic provides solution.
Description
Technical field
The invention belongs to the application of peptide antibiotics, be specifically related to a kind of antibacterial peptide Lexapeptide and preparation thereof
Method and purposes.
Background technology
Lanthiopeptin class antibacterial peptide is the antibiotic that a class is mainly produced by gram positive bacteria, is by Ribosome biogenesis
After through a series of post translational modifications and then generation height modify peptide antibiotics.With L-lanthionine, methyl in structure
L-lanthionine, dehydroalanine and dehydrogenation aminobutyric acid are characterized, and also gain the name because of the existence of L-lanthionine simultaneously.Sheep
The hair sulfur general size of peptides antibacterial peptide, at 10-50 aminoacid, structure exists by cysteine and dehydroalanine/dehydrogenation ammonia
The sulfur-bearing L-lanthionine of base butanoic acid formation and methyllanthionine circulus.Marked feature in these structures just,
Impart the antibacterial activity that lanthiopeptin class antibacterial peptide is the strongest.
As the most thorough nisin (Nisin) of research in lanthiopeptin class antibacterial peptide family, make
Apply in milk product and food preservation up to more than 40 years for food preservative, and drug resistance on a large scale does not occurs the most yet
Report.But Nisin exists temperature tolerance not enough, and (pH ＜ 7) just can demonstrate the most at the low ph
Good activity.Lanthiopeptin class antibacterial peptide action target spot is usually located on cell wall or cell membrane, causes cell wall or film
Structure is imperfect, forms hole thus kills antibacterial, and this bactericidal mechanism also causes the drug resistance pole of lanthiopeptin class antibacterial peptide
Few occur.Owing to the application of conventional antibiotic occurs in that large-area drug resistance problems, and lanthiopeptin class antibacterial peptide is to drug resistance
The staphylococcus aureus of property, the enterococcus faecalis of drug resistance demonstrates good antibacterial activity, lanthiopeptin class antibacterial peptide conduct
The research of the succedaneum of existing antibiotic is becoming study hotspot.The lanthiopeptin class antibacterial peptide having now been found that and report
At about 50 kinds, wherein NVB302, mutacin1140, duramycin, gallidermin, lacticin 3147 and
Microbisporicin comes into clinical research.The highest clinical research conversion ratio makes lanthiopeptin class antibacterial peptide exist
Great prospect in medicament research and development.But lanthiopeptin class antibacterial peptide is usually present the shortcoming that antimicrobial spectrum is narrower and resistance is not enough.
Therefore the lanthiopeptin class antibacterial peptide that developmental research is new and effective resistance is stronger has important value.Streptomycete exists
A lot of biological synthesis gene clusters encoding lanthiopeptin class antibacterial peptide, these gene clusters are typically silence at wild-type strain,
The production of these lanthiopeptin class antibacterial peptides is realized for researching and developing new and effective lanthiopeptin class by the method for heterogenous expression
Antibacterial peptide is significant.
Summary of the invention
It is contemplated that find efficient activated antibacterial peptide from streptomycete, provide precursor for medicament research and development；Specifically
A kind of new and effective lanthiopeptin class antibacterial peptide Lexapeptide and its production and use is provided.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of lanthiopeptin class antibacterial peptide Lexapeptide, and it comprises such as SEQ ID NO:
Primary amino acid sequences shown in 1:
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, multiple serines (Ser) and threonine (Thr) dehydrated generation dehydroalanine (Dha) and dehydrogenation amino
Butanoic acid (Dhb).
Preferably, the serine dehydratase generation dehydroalanine of the 2nd, 18,19 in described aminoacid sequence, the 4th, 12
Threonine generate dehydrogenation aminobutyric acid through dehydration, the 28th serine dehydratase, reduction generate D-alanine.
Preferably, described lanthiopeptin class antibacterial peptide Lexapeptide contains a methyllanthionine, by described ammonia
Base acid sequence is condensed between cysteine and the threonine of 33 of 22 and forms.
Preferably, the cysteine of described aminoacid sequence end the 38th experienced by oxidative deamination reaction and forms 2-amino
Ethylene mercaptan (2-aminoenethiol), further the dehydrogenation aminobutyric acid condensation with 30 formed rare 2-aminoalkenyl-
3-methyl-cysteine (AviMeCys) structure.
Preferably, the amino of the phenylalanine of described aminoacid sequence N-end is modified by two methyl groups.
Preferably, the serine of described aminoacid sequence the 28th is modified to as D-form alanine.
The molecular formula of described lanthiopeptin class antibacterial peptide Lexapeptide is:
Second aspect, the invention still further relates to the system of lanthiopeptin class antibacterial peptide Lexapeptide described in any of the above-described item
Preparation Method, described method comprises the steps:
S1, derive from Lou Che Shi streptomycete (Streptomyces rochei) Sal35 bacterial strain bacterial artificial chromosome literary composition
Storehouse is heterogenous expression in muta lead mycillin (Streptomyces lividans) SBT5, it is thus achieved that the muta lead mycillin of restructuring
(Streptomyces lividans)SBT5/Sal35 6A8 CGMCC No.12751；
S2, the muta lead mycillin fermentation culture of described restructuring, it is thus achieved that described lanthiopeptin class antibacterial peptide
Lexapeptide。
Preferably, in step S2, after fermentation culture, also include isolated and purified step.
Preferably, step S2 includes:
A1, restructuring muta lead mycillin fermentation medium YBP top fermentation 5～8 days, collect solid fermentation product；
Some times of A2, equal-volume methanol leached solids fermented product, be concentrated in vacuo and be spin-dried for；
A3, methanol extract with after water-soluble, some times of equal-volume n-butanol extraction, be concentrated in vacuo and be spin-dried for；
A4, n-butyl alcohol extract after macroporous adsorbent resin CHP20P, gel chromatography LH20 purification through liquid phase separation to pure
Product Lexapeptide.
Preferably, the high performance liquid chromatography preparation condition of described isolated and purified middle employing is:
Flowing is mutually: A phase is aqueous phase (adding 1 ‰ trifluoroacetic acids [volume ratio]), and B phase is acetonitrile；
Elution requirement is: 40%B phase [volume ratio] isocratic elution 15min, and flow velocity is that 0.6ml/min, Lexapeptide exist
10min eluting.
The third aspect, the invention still further relates to the lanthiopeptin class antibacterial peptide Lexapeptide described in a kind of any of the above-described item
Purposes in preparing resisting gram-positive bacteria preparation.
Fourth aspect, the invention still further relates to the lanthiopeptin class antibacterial peptide Lexapeptide described in a kind of any of the above-described item
In preparation for treating the purposes in antibacterial, fungus or virus infective medicament.
Preferably, described treatment antibacterial, fungus or virus infective medicament are the Staphylococcus aureus treating methicillin-resistant
The medicine that bacterium or staphylococcus epidermidis infect.
5th aspect, the invention still further relates to the lanthiopeptin class antibacterial peptide Lexapeptide described in a kind of any of the above-described item
Purposes in preparing animal feed.
6th aspect, the invention still further relates to a kind of pharmaceutical composition, and said composition is with the Pilus Caprae seu Ovis sulfur described in above-mentioned any one
Peptides antibacterial peptide Lexapeptide or its pharmaceutically acceptable salt are as active component.
Preferably, described compositions is possibly together with pharmaceutically acceptable excipient, carrier or diluent.
7th aspect, the invention still further relates to a kind of muta lead mycillin (Streptomyces lividans) SBT5/
Sal35 6A8 CGMCC No.12751。
The muta lead mycillin preparation method of this genetic engineering restructuring is: screening derives from Lou Che Shi streptomycete
The bacterial artificial chromosome of (Streptomyces rochei) Sal35 bacterial strain (BAC,Bacterial ArtificialCHromosome) library, it is thus achieved that positive BAC SAL35-6A8；SAL35-6A8 imports heterogenous expression host's muta lead mycillin
In (Streptomyces lividans) SBT5, it is thus achieved that produce the restructuring muta lead mycillin of Lexapeptide
Streptomyces lividans SBT5/Sa15 6A8。
In the present invention, described muta lead mycillin (Streptomyces lividans) SBT5/Sal35 6A8 in
On July 11st, 2016 submits China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (address: court of Beijing
No. 3 Institute of Microorganism, Academia Sinica's postcodes 100101 of sun North Star West Road 1 institute of district), the numbered CGMCC of culture presevation
No.12751。
Further Novel wool sulfur peptides antibacterial peptide Lexapeptide is carried out antibacterial experiment, the gold to methicillin-resistant
Staphylococcus aureus and staphylococcus epidermidis carry out bacteriostatic experiment, suitable with vancomycin fungistatic effect.To methicillin-resistant
The MIC value of staphylococcus aureus and staphylococcus epidermidis is respectively 0.52 μM and 1.03 μMs；The corresponding MIC value of vancomycin is
0.61 μM and 1.21 μMs.
Compared with prior art, the present invention has a following useful achievement:
1, the invention discloses a kind of new and effective lanthiopeptin class antibacterial peptide Lexapeptide.Come from Lou Che Shi chain
The Bacterial Artificial Chromosome Library of mycete (Streptomyces rochei Sal35) Sal35 bacterial strain is at muta lead mycillin
The noval chemical compound of isolated after heterogenous expression in (Streptomyces lividans) SBT5；Lexapeptide is by 38 ammonia
Base acid is constituted, and molecular formula is: C179H286N50042S2, and as shown in Figure 8, relative molecular mass is structural formula: 3872.61g/
mol。
2, Lexapeptide comprises C-terminal 2-aminoalkenyl-3-methyl-cysteine knot while being first case report
Structure and N-terminal methylate the lanthiopeptin of modification structure, the height in this structure modify impart Lexapeptide relative to
Other lanthiopeptin more preferable stability of class antibacterial peptide；Compared to Nisin, temperature (50 DEG C) and pH (2～12) are had the most resistance to
By property.
3, biological activity determination finds the golden yellow Fructus Vitis viniferae of the Lexapeptide methicillin-resistant to being widely present clinically
Coccus and staphylococcus epidermidis have extraordinary bacteriostatic activity, its bacteriostatic activity and clinical practice antibiotic last together with prevent
Line vancomycin is quite even better than vancomycin, and gram negative bacteria is not had inhibitory activity.To mycobacterium smegmatis
mc2155 show the bacteriostatic activity being better than Nisin.
4, Lexapeptide can serve as resisting gram-positive bacteria preparation, and the drug resistance problems commonly encountering clinic carries
For solution.
5, antimicrobial mechanism based on lanthiopeptin class antibacterial peptide is different from vancomycin, and Lexapeptide has potential use
In clinic auxiliary or the using value of replacement vancomycin.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is that the bacterial artificial chromosome carrying Lexapeptide expresses raw mapping in muta lead mycillin；Recombinant bacterium
It is golden yellow that strain Streptomyces lividans SBT5/Sal35 6A8 can significantly inhibit raw survey indicator bacteria on screening flat board
The staphylococcic growth of color.
Fig. 2 is the Liquid Detection collection of illustrative plates that Lexapeptide expresses；Lexapeptide at about 21min eluting, in figure with
The chromatographic peak of asterisk mark is Lexapeptide.
Fig. 3 is the chromatograms of Lexapeptide purification；Lexapeptide is at about 10min eluting, with asterisk in figure
The chromatographic peak of mark is Lexapeptide.
Fig. 4 is that the MALDI-FTICR of Lexapeptide detects mass spectrum.
Fig. 5 is the ESI-FTICR tandem mass spectrum figure of Lexapeptide；The b/y fragment of Lexapeptide is shown in mark in figure.
Fig. 6 is Lexapeptide structure and tandem mass spectrum fragmentation behavior figure.
Fig. 7 is Lexapeptide active testing and resistance research schematic diagram；Wherein, a is Lexapeptide (100 μ
And the Nisin (100 μ g/ml) the biological activity test figure to mycobacterium smegmatis g/ml).B is Lexapeptide (100 μ g/ml)
And Nisin (50 μ g/ml) biological activity test figure to micrococcus luteus after the PBS of different pH processes.C is
The Lexapeptide (100 μ g/ml) and Nisin (100 μ g/ml) biological activity test figure after 50 DEG C process different number of days.
Fig. 8 is the schematic arrangement of lanthiopeptin class antibacterial peptide Lexapeptide.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in the technology of this area
Personnel are further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into the present invention
Protection domain.
The present invention relates to a kind of new and effective lanthiopeptin class antibacterial peptide Lexapeptide and preparation method thereof and use
On the way；Wherein,
LB culture medium: for colibacillary cultivation；
Tryptone 10g, yeast extract 5g, sodium chloride 5g, deionized water is settled to 1,000ml, pH 7.0.
2 × YT culture medium: escherichia coli one streptomycete Conjugal transfer；
Tryptone 16g, yeast extract 10g, sodium chloride 5g, deionized water is settled to 1,000ml, pH 7.0.
Cultivate the use such as staphylococcus aureus, staphylococcus epidermidis when Mueller Hinton culture medium: MIC measures, purchase
From 0xoid.
SFM culture medium: streptomycete is cultivated to produce and engages transfer between spore and Escherichia coli-Streptomyces；
Soybean cake powder 20g, mannitol 20g, tap water is settled to 1,000ml, pH 7.2～7.4.
Soybean cake powder 20g is first with 1, and 000ml tap water soaks, and then 121 DEG C of sterilizing 20min are collected by filtration soybean cake powder
Lixiviating solution preparation culture medium.
YBP culture medium: streptomycete fermentation is used；
Glucose 10g, yeast extract 2g, beef extract 2g, peptone 4g, MgSO41g, NaCl 15g, tap water
It is settled to 1,000ml, pH 7.2～7.4.
Corresponding solid medium needs to add 15g agar powder, and SFM solid medium adds 20g agar powder.
Lou Che Shi streptomycete Streptomyces rochei Sal35 involved in the present invention, is Chinese Typical Representative culture
The AA97007 S. tenebrarius at preservation center (CCTCC), is isolatable from Shennongjia mountain area, at document " Xu M, Wang Y, Zhao
Z, Gao G, Huang S-X, Kang Q, He X, Lin S, Pang X, Deng Z, Tao are M.2016.Functional
genome mining for metabolites encoded by large gene clusters through
heterologous expression of a whole-genome bacterial artificial chromosome
Library in Streptomyces spp.Appl.Environ.Microbiol.doi:10.1128/AEM.01383-16. "
Disclosed in mistake.
Heterogenous expression host Streptomyces lividans SBT5 is at document " Bai Tingli, Yu Yanfei, Xu Zhong, Tao Mei
Phoenix；The structure of muta lead mycillin efficient heterogenous expression host SBT5,2014, Hua Zhong Agriculture University's journal " disclosed in mistake, the public
Can obtain from microbial metabolism National Key Laboratory of Shanghai Communications University.
Joint transfer supplementary strain Escherichia coli ET12567/pUB307 involved in the present invention is at document
" Flett F, Mersinias V, Smith CP；Highefficiencyintergenericconjugal transfer of
plasmid DNA from Escherichia coli to methyl DNA-restricting
Streptomycetes.1997, FEMS Microbiol Lett 155 (2): 223-229. " disclosed in mistake.
The strain golden color staphylococcus Staphylococcus aureus of biological activity determination involved in the present invention
ATCC 29213 is obtained by ATCC, and the public can obtain from American Type Culture collection warehousing (ATCC).
Enterococcus faecalis Enterococcus faecalis ATCC 29212 is brought up by Chinese Academy of Science Nanhai Ocean Research Institute
Jian Hua teacher give, and has resistance to ampicillin and the character of resistance to kanamycin.The public can deposit from American Type Culture collection
Storehouse (ATCC) obtains.
Methicillin-resistant staphylococcus aureus (Methicillin Resistant Staphylococcus aureus,
And Methicillin-resistant Staphylococcus epidermidis (Methicillin Resistant Staphylococcus MRSA)
Epidermis, MRSE) given by Chinese Academy of Science Nanhai Ocean Research Institute teacher Ju Jianhua.
Mycobacterium smegmatis Mycobacteria smegmatis mc2155 are given by Shandong University professor Pang Xiuhua.Rattan
Yellow micrococcus luteus Micrococcus luteus ATCC4698 is given by Fujian Institute of Micro-biology teacher Peng Fei, and the public can be from the U.S.
Type culture collection warehousing (ATCC) obtains.
Embodiment 1, the heterogenous expression of bacterial artificial chromosome containing Lexapeptide biological synthesis gene cluster
(1) from-80 DEG C of refrigerators, take out the antibacterial people of Lou Che Shi streptomycete (Streptomyces rochei) Sal35
Work chromosome library (8 piece of 96 orifice plate), 37 DEG C of incubators place 2 hours to melting completely.
(2) in 96 orifice plates, add 130 μ l fresh LB, utilize duplicator by Lou Che Shi streptomycete
The Bacterial Artificial Chromosome Library of (Streptomyces rochei) Sal35 is seeded in 96 orifice plates, 37 DEG C, and 220rpm cultivates 4
～6 hours.
(3) meanwhile, inoculate in filling the 100ml 250ml triangular flask containing 50 μ g/m1 kanamycin LB culture medium
ET12567/pUB307 bacterial strain, 37 DEG C, 220rpm cultivates 4～6 hours.
(4) 4,000rpm are centrifuged 10min and collect ET12567/pUB307 thalline, wash 3 times by fresh LB culture medium,
Resuspended with the fresh LB of 15ml again.
(5) volley of rifle fire is utilized to take the ET12567/pUB307 bacterium solution addition cultivation Lou Che Shi streptomycete that 15 μ l concentrate
In each hole of 96 orifice plates of the Bacterial Artificial Chromosome Library of (Streptomyces rochei) Sal35.200rpm continues training
Support 5min, fully mix.
(6) utilize duplicator by the bacterium solution photocopy of mixing to even spread the muta lead mycillin of layer of thermally actuated mistake
The SFM flat board of (Streptomyces lividans) SBT5 spore, dries up in Biohazard Safety Equipment, and 30 DEG C of cultivations 12～16 are little
Time.
(7) on SFM flat board, trimethoxy benzyl pyridine and apramycin extremely final concentration of 50 μ g/ml are covered, in bio-safety
Cabinet dries up, cultivates 4～6 days for 30 DEG C and grow to engaging transfer.
(8) utilize duplicator that the joint grown is shifted sub-photocopy to drawing mould containing nalidixic acid (25 μ g/ml) He Abo
Escherichia coli and the recipient bacterium muta lead mycillin (Streptomyces of residual is removed on the SFM flat board of element (50 μ g/ml)
Lividans) SBT5,4～6 days extremely product spores of 30 DEG C of cultivations are complete.
(9) utilize duplicator by produce spore joint transfer son be transferred on fermentation medium YBP, 30 DEG C of fermentation culture 4～
6 days to producing element completely.
(10) covering one layer soft agar (0.5% fine jade containing 1% staphylococcus aureus on element flat board completely is being produced
Fat), 37 DEG C of overnight incubation, observe the generation of inhibition zone.The inhibition zone change produced as it is shown in figure 1,6A8 clone is fermented observed
Compound can significantly inhibit the raw growth surveying indicator bacteria staphylococcus aureus.Obtain the bacteria artificial containing Lexapeptide
Expression strain muta lead mycillin (Streptomyces lividans) the SBT5/Sal35 6A8 CGMCC of chromosome
No.12751。
Embodiment 2, the detection of Lexapeptide
(1) choose from the Bacterial Artificial Chromosome Library of Lou Che Shi streptomycete (Streptomyces rochei) Sal35
Select 6A8 clone to cultivate.Then bacterial artificial chromosome is imported different by joint transfer between Escherichia coli-Streptomyces
Source expressive host Streptomyces lividans SBT5 obtains producing the bacterial strain lead-changing penicillium strepto-of Lexapeptide
Bacterium (Streptomyces lividans) SBT5/Sal35 6A8 CGMCC No.12751.
(2) the bacterial artificial chromosome heterogenous expression inoculation of Lexapeptide will be produced in solid YBP culture medium,
3 pieces of flat boards of each strain fermentation, 30 DEG C of fermentation culture 4～6 days.
(3) after fermentation ends, collect solid fermentation thing, with equal-volume methanol extraction 3 times, be concentrated in vacuo and be spin-dried for obtaining first
Alcohol crude extract.
(4) methanol crude extract 10ml sterilized water is dissolved, with equal-volume n-butanol extraction 3 times, be concentrated in vacuo and be spin-dried for
To n-butyl alcohol crude extract, by 2ml methanol sample dissolution.
(5) 12,000rpm is centrifuged 10min, after 0.22 μm membrane filtration methanol sample, carries out high performance liquid chromatography and divides
Analysis, sample size is 10 μ l.Liquid phase analysis chromatographic column model is: Agilent Zorbax 300SB-C18 (5 μm, 4.6 ×
250mm)。
Flowing is mutually:
A phase is aqueous phase (adding 1 ‰ trifluoroacetic acids),
B phase is acetonitrile.
Analysis condition is: 0-5min, 5%B；5-20min, B phase is risen to 50% by 5% concentration；20-30min, B phase by
50% concentration rises to 100%；30-35min, B phase 100% concentration maintains 5min:35-36min, B phase to be down to by 100% concentration
5%；36-45min, B phase 5% concentration balance 9min.Flow velocity is 0.6ml/min, room temperature.Lexapeptide elutes at 21min
Coming, as shown in Figure 2, Lexapeptide correspondence eluting peak indicates with asterisk.
Embodiment 3, the purification of Lexapeptide and qualification
Analyze in embodiment 2 with muta lead mycillin (Streptomyces lividans) SBT5/Sal35 6A8
CGMCC No.12751 bacterial strain set out accumulation Lexapeptide be purified and identify.
(1) by Streptomyces lividans SBT5/Sal35 6A8 CGMCC No.12751 inoculation extremely
Carrying out producing spore on SFM flat board, 7 days extremely product spores of 30 DEG C of cultivations are complete.Lexapeptide has substantially suppression to strain growth, produces spore
Time postpones the most backward.
(2) by Streptomyces lividans SBT5/Sal35 6A8 CGMCC No.12751 bacterial strain Fresh spores
It is seeded to carry out in 30L solid YBP culture medium the large scale fermentation accumulation of Lexapeptide, 30 DEG C of fermentation culture 4～6 days.
(3) solid of Streptomyces lividans SBT5/Sal35 6A8 CGMCC No.12751 bacterial strain is collected
Fermented product, equal-volume methanol (30L) extracts 3 times, is concentrated in vacuo and is spin-dried for obtaining methanol crude extract.
(4) methanol crude extract is dissolved in 1L sterilized water, with 1L n-butanol extraction 3 times, n-butyl alcohol is concentrated in vacuo mutually rotation
Dry, obtain n-butyl alcohol crude extract.
(5) n-butyl alcohol crude extract is mixed thoroughly by dry method loading with CHP20P filler, the filler mixed installs to CHP20P open
On post (50 × 500mm), first alcohol and water carries out gradient elution.When methanol-eluted fractions concentration rises to 80%, Lexapeptide starts to wash
It is de-until methanol-eluted fractions concentration rises to 100%.Collect 80-100% methanol-eluted fractions component, be concentrated in vacuo and be spin-dried for.
(6) CHP20P post elution fraction is dissolved with methanol again, methanol solution must be clarified with 0.22 μm membrane filtration.
Methanol sample is loaded to (20 × 1500mm) on the chromatographic column equipped with LH20 filler, pure methanol with the speed eluting of 10/s,
Collect fraction in test tube, often pipe 5ml.The fraction collected carries out high performance liquid chromatography detection, merges containing Lexapeptide's
Fraction carries out next step and analyzes.
(7) collect the component that LH20 post eluting contains Lexapeptide, be concentrated in vacuo and be spin-dried for, again dissolve with methanol.
Methanol solution must be clarified with 0.22 μm membrane filtration, carry out high performance liquid chromatography isolated and purified, sample size 10～20 μ l.Liquid phase is divided
Analysis chromatographic column model is: Agilent Zorbax 300SB-C18 (5 μm, 4.6 × 250mm)
Flowing is mutually:
A phase is aqueous phase (adding 1 ‰ trifluoroacetic acids),
B phase is acetonitrile.
Analysis condition is 40%B equality eluting 15min, and flow velocity is 0.6ml/min.Lexapeptide is at about 10min
Starting eluting, about 11.5min eluting terminates, and collects Lexapeptide simple spike elution fraction and is analyzed.
Fig. 3 is shown at Lexapeptide elution chromatography peak, is Lexapeptide with the chromatographic peak of asterisk mark in figure.
(8) Lexapeptide to purification carries out mass spectral analysis and determines its molecular weight, utilizes MALDI-FTICR MS to survey
The molecular weight obtaining Lexapeptide is 3872.16Da.The molecular weight of Lexapeptide determines as shown in Figure 4.
(9) utilize ESI-FTICR-MS/MS that Lexapeptide is carried out Tandem Mass Spectrometry Analysis and determine its structure.Select mother
Ion [M+4H]4+=969.00Da carries out second mass analysis, ' b/y ' fragment of the obtained Lexapeptide of tandem mass spectrum from
Son is shown in that Fig. 5, fragment ion correspondence Lexapeptide fragment are shown in Fig. 6.
Embodiment 4, the biological activity determination of Lexapeptide
Minimum inhibitory concentration (MIC) value of Lexapeptide measures according to Microdilution plate method, and bacterial strain to be tested is golden yellow
Staphylococcus A TCC25923, methicillin-resistant staphylococcus aureus (MRSA), Methicillin-resistant Staphylococcus epidermidis (MRSE)
And enterococcus faecalis ATCC29212.MIC value is defined as being suppressed over the minimum antibiotic concentration of 90% concentration strain growth.With
Ampicillin, kanamycin and vancomycin are as comparison, and 3, each sample is parallel.
(1) Mueller-Hinton (MH) broth bouillon culture experiment bacterium, 37 DEG C of incubated overnight 12-16 hour are selected.
Sample solution was got out before experimental bacteria length is good.
(2) preparation sample solution, with DMSO sample dissolution (ampicillin Ampicillin, kanamycin
Kanamycin, vancomycin Vancomycin and Lexapeptide) be respectively prepared concentration be 32mg/ml, 32mg/ml,
The mother solution of 3.2mg/ml and 6.4mg/ml.
(3) adding aseptic MH broth bouillon in 96 orifice plates, the 1st row add 184 μ l aseptic MH meat soup, and remaining respectively arranges
It is separately added into 100 μ l aseptic MH meat soup.11st row and the 12nd row are respectively as positive control and negative control.
(4) draw in mother solution addition the 1st row 96 orifice plate that 16 μ l prepare, utilize pipettor to blow and beat mixing gently.
(5) from the 1st row, draw 100 μ l be mixed with the culture fluid of antibiotic in the 2nd row, blow and beat with pipettor mixed gently
During after even, sucking-off 100 μ l to the 3rd arranges again, the rest may be inferred is diluted to the 10th row.The 100 μ l that after 10th row mixing, sucking-off is unnecessary cultivate
Liquid discards.
(6) thalline of incubated overnight is diluted 1000 times with fresh MH broth bouillon, take the bacterium solution of 100 μ l dilutions
Adding in the hole of the 1st row to the 11st row, the 12nd row add 100 MH broth bouillons fresh for μ l as blank.Such
Drug concentration gradient to ampicillin and kanamycin is: 1280,640,320,160,80,40,20,10,5,2.5 μ g/
ml；The drug concentration gradient of vancomycin is: 128,64,32,16,8,4,2,1,0.5,0.25 μ g/ml；Lexapeptide's
Drug concentration gradient is: 256,128,64,32,16,8,4,2,1,0.5 μ g/ml.
(7) 96 orifice plate lids are covered, in 37 DEG C of incubator incubated overnight 16-24 hour.
(8) with the 11st row as positive control, the 12nd is classified as blank, utilizes microplate reader at OD600Lower reading bacterium is dense, really
The MIC value of fixed each sample, as shown in table 1 below.
The minimum inhibitory concentration (MIC μ g/ml) of table 1 Lexapeptide and associated antibiotic
In table 1, MIC data system represents with mass concentration；If the molar concentration of being converted into, vancomycin is to methicillin-resistant gold
The MIC concentration of Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus epidermidis (MRSE) is respectively 0.61 μM and 1.21
μM；The MIC concentration of methicillin-resistant staphylococcus aureus and Methicillin-resistant Staphylococcus epidermidis is divided by Lexapeptide
It it is not 0.52 μM and 1.03 μMs.Therefore, Lexapeptide fungistatic effect is suitable with vancomycin.
Lexapeptide is to mycobacterium smegmatis (Mycobacteria smegmatis mc2155) survey of bacteriostatic activity
Fixed:
Lexapeptide and the Nisin solution of 100 μ g/ml is prepared with 100mM Tris-HCl (pH 7.0).Take 30 μ l to add
Enter on the raw master plate of the mycobacterium smegmatis being with holes, cultivate the formation observing inhibition zone for 24 hours for 37 DEG C.Isoconcentration
Lexapeptide the activity of mycobacterium smegmatis is better than Nisin, see Fig. 7 a, Lexapeptide is to shame dirt in this example
Mycobacteria has inhibitory activity, and Nisin does not has activity.
Embodiment 5, the resistance research of Lexapeptide.
(1) pH resistance research: preparation PBS, with phosphoric acid or hydrochloric acid regulate to different pH value stand-by (2,4,6,
8,10,12).Lexapeptide and the 50 μ g/ml Nisin mother solutions of 100 μ g/ml are prepared with PBS.Take 6 μ l
Lexapeptide and Nisin mother solution is separately added in the buffer of 24 μ l difference pH value, and room temperature stands 2 hours.By 30 μ l samples
Product join on the raw master plate of the micrococcus luteus being with holes, and cultivate the formation observing inhibition zone for 24 hours for 30 DEG C.
Raw result of surveying is shown in Fig. 7 b, and in alkaline pH range, the activity of Nisin is obviously reduced as seen from the figure, to pH value be 12 time
Completely lose bacteriostatic activity.But Lexapeptide all demonstrate in the pH value range tested with compare suitable antibacterial
Activity.Show that Lexapeptide all has higher toleration to acid, alkali.
(2) temperature resistance research: prepare the molten of Lexapeptide and Nisin with 100mM Tris-HCl (pH 7.0)
Liquid.Lexapeptide (100 μ g/ml) and Nisin (100 μ g/ml) is placed in 50 DEG C of water-bath temperature bathe 8 days, takes 30 μ l every day and keep sample
Carry out biological activity determination.Sample after processing adds on the raw master plate of the micrococcus luteus being with holes, 30 DEG C of cultivations
The formation observing inhibition zone in 24 hours.
Raw result of surveying is shown in Fig. 7 c, and Nisin bacteriostatic activity after 50 DEG C process a day gradually weakens, to 5 space-baseds as seen from the figure
Originally activity is completely lost.Lexapeptide then shows good toleration to temperature, after processing 8 days activity still with not
The Lexapeptide control sample activity processed is quite.The prolongation temperature bath time, the activity of Lexapeptide was not subject to 10 days
Impact.Lexapeptide has more preferable toleration than Nisin to temperature as can be seen here.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Claims (10)
1. a lanthiopeptin class antibacterial peptide Lexapeptide, it is characterised in that it comprises as shown in SEQ ID NO:1
Grade amino acid sequence；Wherein, multiple serines and threonine dehydrated generation dehydroalanine and dehydrogenation aminobutyric acid.
Lanthiopeptin class antibacterial peptide Lexapeptide the most according to claim 1, it is characterised in that described aminoacid sequence
In row, the serine dehydratase of the 2nd, 18,19 generates dehydroalanine, and the threonine of the 4th, 12 generates dehydrogenation amino through dehydration
Butanoic acid, the 28th serine dehydratase, reduction generate D-alanine.
Lanthiopeptin class antibacterial peptide Lexapeptide the most according to claim 2, it is characterised in that described lanthiopeptin
Class antibacterial peptide Lexapeptide contains a methyllanthionine, by the cysteine of 22 in described aminoacid sequence with
Between the threonine of 33, condensation forms.
Lanthiopeptin class antibacterial peptide Lexapeptide the most according to claim 3, it is characterised in that described aminoacid sequence
The cysteine that row end is the 38th experienced by oxidative deamination reaction and forms 2-amino ethylene mercaptan, the further dehydrogenation with 30
Aminobutyric acid condensation forms rare 2-aminoalkenyl-3-methyl-cysteine structure.
Lanthiopeptin class antibacterial peptide Lexapeptide the most according to claim 3, it is characterised in that described aminoacid sequence
The amino of the phenylalanine of row N-end is modified by two methyl groups.
6. a preparation method of the lanthiopeptin class antibacterial peptide Lexapeptide as according to any one of Claims 1 to 5,
It is characterized in that, described method comprises the steps:
S1, the Bacterial Artificial Chromosome Library deriving from Lou Che Shi streptomycete (Streptomyces rochei) Sal35 bacterial strain exist
Heterogenous expression in muta lead mycillin (Streptomyces lividans) SBT5, it is thus achieved that the muta lead mycillin of restructuring
(Streptomyces lividans)SBT5/Sal35 6A8CGMCC No.12751；
S2, the muta lead mycillin fermentation culture of described restructuring, it is thus achieved that described lanthiopeptin class antibacterial peptide Lexapeptide.
The preparation method of lanthiopeptin class antibacterial peptide Lexapeptide the most according to claim 6, it is characterised in that step
In rapid S2, after fermentation culture, also include isolated and purified step；The high performance liquid chromatography of described isolated and purified middle employing prepares bar
Part is:
Flowing is mutually: A phase is to add the aqueous phase of 1 ‰ trifluoroacetic acids, and B phase is acetonitrile；
Elution requirement is: 40%B equality eluting 15min, and flow velocity is that 0.6ml/min, Lexapeptide are at 10min eluting.
8. the lanthiopeptin class antibacterial peptide Lexapeptide as according to any one of Claims 1 to 5 is preparing anti-leather orchid
Family name's positive bacteria preparation, animal feed or for treating the purposes in antibacterial, fungus or virus infective medicament.
9. a pharmaceutical composition, said composition is with the lanthiopeptin class antibacterial peptide as according to any one of Claims 1 to 5
Lexapeptide or its pharmaceutically acceptable salt are as active component.
10. muta lead mycillin (Streptomyces lividans) SBT5/Sal35 6A8CGMCC No.12751.
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| CN110684087A (en) * | 2019-10-21 | 2020-01-14 | 河北省科学院生物研究所 | Lanthionine precursor peptide amyA16, and preparation method and application thereof |
| CN112851773A (en) * | 2021-02-03 | 2021-05-28 | 河北省科学院生物研究所 | Novel antibacterial lantibide AmylocinC and preparation method and application thereof |
| CN114644688A (en) * | 2020-12-17 | 2022-06-21 | 中国科学院微生物研究所 | Bacillus licheniformis for producing lantibiotic peptide and inhaul cable peptide |
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