阅读说明：本技术 一种脂肽分子及其应用 (Lipopeptide molecule and application thereof ) 是由 孙超岷 张德超 刘瑞 修鹏远 于 2017-04-26 设计创作，主要内容包括：本发明属于生物技术领域,脂肽分子为子CLP1和CLP2,CLP1分子式为C<Sub>57</Sub>H<Sub>101</Sub>N<Sub>7</Sub>O<Sub>13</Sub>,CLP2分子式为C<Sub>58</Sub>H<Sub>103</Sub>N<Sub>7</Sub>O<Sub>13</Sub>。脂肽分子的制备方法,其特征在于：将海洋芽孢杆菌(Bacillus sp.)BS176接种到2216E液体培养基中,28℃过夜震荡培养；而后取菌液转接到发酵培养基中,28℃震荡培养2天；发酵结束后,离心收集发酵上清液,上清液经酸化、冷冻干燥以及旋转蒸发得海洋芽孢杆菌(Bacillus sp.)BS176胞外活性物质的粗提物(CLPs),而后经进一步的纯化,即得脂肽分子CLP1和CLP2。本发明脂肽具有抑制水产养殖病原细菌运动能力的活性,并且对多种病原细菌具有促进菌体凝集和沉降的作用。(The invention belongs to the field of biotechnology, lipopeptide molecules are CLP1 and CLP2, and the molecular formula of CLP1 is C 57 H 101 N 7 O 13 CLP2 with the molecular formula C 58 H 103 N 7 O 13 . A method of producing a lipopeptide molecule, comprising: inoculating marine Bacillus (Bacillus sp.) BS176 into 2216E liquid culture medium, and performing shake culture at 28 ℃ overnight; then taking the bacterial liquid to transfer to a fermentation medium, and carrying out shake culture at 28 ℃ for 2 days; after fermentation, centrifugally collectingFermenting the supernatant, acidifying, freeze-drying and rotary evaporating the supernatant to obtain crude extracts (CLPs) of extracellular active substances of marine Bacillus (Bacillus sp) BS176, and further purifying to obtain lipopeptide molecules CLP1 and CLP 2. The lipopeptide has the activity of inhibiting the movement capacity of aquaculture pathogenic bacteria, and has the effect of promoting thallus agglutination and settlement on various pathogenic bacteria.)

1. A lipopeptide molecule, wherein: the lipopeptide molecules are CLP1 and CLP2, CLP1 has molecular formula C₅₇H₁₀₁N₇O₁₃CLP2 with the molecular formula C₅₈H₁₀₃N₇O₁₃。

2. The lipopeptide molecule of claim 1, wherein: the lipopeptide molecules CLP1 and CLP2 polypeptide portions are bothConsists of glutamic acid (Glu) -leucine (Leu) -aspartic acid (Asp) -leucine (Leu) -isoleucine (Ile), and the fatty acid chains of the glutamic acid (Glu) -leucine (Leu) -aspartic acid (Asp) -leucine (Leu) -isoleucine (Ile) are all β hydroxy fatty acids, and the difference is only that the fatty acid chains are different by one methylene group (CH)₂-)。

3. A method of producing the lipopeptide molecule of claim 1, wherein: inoculating marine Bacillus (Bacillus sp.) BS176 into 2216E liquid culture medium, and performing shake culture at 28 ℃ overnight; then taking the bacterial liquid to transfer to a fermentation medium, and carrying out shake culture at 28 ℃ for 2 days; after fermentation, centrifugally collecting fermented supernatant, acidifying, freeze-drying and rotary evaporating the supernatant to obtain crude extracts (CLPs) of extracellular active substances of marine Bacillus (Bacillus sp) BS176, and further purifying to obtain lipopeptide molecules CLP1 and CLP 2;

the marine Bacillus (Bacillus sp.) BS176 is preserved in China general microbiological culture Collection center, and the address is as follows: west road No.1 north chen, north yang district, beijing, 3 (institute for microbiology, chinese academy of sciences), with a collection date of 2016, 12 and 29 days, and a collection number: CGMCC No. 13515.

4. Use of a lipopeptide molecule according to claim 1, wherein: the lipopeptide molecules CLP1 and CLP2 are applied to the preparation of medicines for inhibiting pathogenic bacteria.

5. Use of a lipopeptide molecule according to claim 4 wherein: the pathogenic bacteria are gram-negative bacteria or gram-positive bacteria.

6. Use of a lipopeptide molecule according to claim 5, wherein: the pathogenic bacteria are vibrio anguillarum, vibrio splendidus, vibrio vulnificus, pseudomonas aeruginosa, pseudomonas stutzeri, staphylococcus aureus, bacillus subtilis or bacillus BS 176.

Technical Field

The invention belongs to the technical field of biology, and relates to a lipopeptide molecule and application thereof.

Background

Biosurfactants (Biosurfactants) are a class of surfactants derived from living organisms, particularly microorganisms. According to the structural characteristics of biosurfactants, they can be classified into 5 major classes, such as glycolipids, lipopeptides and lipoproteins, phospholipids and fatty acids, polymeric surfactants and microparticle surfactants. Due to the characteristics of mild production conditions, low toxicity, biodegradability, environmental compatibility and the like, the biosurfactant becomes an excellent substitute of a chemical surfactant, has potential application advantages in industrial production, and can be used in the aspects of food, cosmetics, pharmaceutical industry, environmental protection, energy-saving process and the like [1 ].

Lipopeptides (lipopeptides) are an important class of biosurfactants and consist mainly of hydrophilic amino acid chains and hydrophobic fatty acid chains. At present, most lipopeptide molecules have strong biological activities such as antifungal activity, antibacterial activity, antiviral activity, antitumor activity and the like, so that the lipopeptide molecules have important application potential in the aspects of antibiotic development, environmental management, food safety prevention and control and the like [2 ]. The lipopeptide molecule is derived primarily from a microorganism; among them, lipopeptides derived from Bacillus spp. For example, surfactin, fengycin, iturin, bacillus and mycosubtilin isolated from Bacillus subtilis; lichenysin isolated from Bacillus licheniformis and pumilacidin isolated from Bacillus pumilus and the like [3-5 ]. Although many types of lipopeptide molecules have been discovered, due to their wide variety and complex structure, the mechanism of action of most lipopeptides is still unclear and their potential for use is still very limited.

China is a big aquaculture country, and diseases of aquatic animals caused by pathogenic bacteria are frequent due to continuous expansion of aquaculture scale and aggravation of aquaculture environment. In order to solve the problems of the increase of drug resistance of pathogenic bacteria and the damage of aquaculture food safety caused by the abuse of traditional antibiotics and chemical drugs, and the like, environment-friendly antibacterial active substances are to be deeply developed and widely applied. In recent years, with the implementation of the national marine strategy and the development of the blue economic area, the deep research of the marine biology and the marine biotechnology in China is promoted. Currently, a variety of microorganisms capable of producing lipopeptides have been isolated from marine organisms or marine sediments and the lipopeptides produced are biologically active. The lipopeptide molecules have important application prospects in the fields of development of novel antibiotics, environmental management, improvement of aquaculture safety in China and the like.

Reference documents:

1. marvellous, gorgeous, golden, Yu xing torch, Zhang Wei 2006, structure, function and biosynthesis of cyclic lipopeptide biosurfactant, microbiological notification 33: 122-.

2.Das P,Mukherjee S,Sen R.2008.Antimicrobial potential of alipopeptide biosurfactant derived from a marine Bacilluscirculans.J.Appl.Microbiol.104:1675-1684.

3.Vater J,Kablitz B,Wilde C,Franke P,Mehta N,Cameotra SS.2002.Matrix-assisted laser desorption ionization-time of flight mass spectrometry oflipopeptide biosurfactants in whole cells and culture filtrates of Bacillussubtilis C-1isolated from petroleum sludge.Appl.Environ.Microb.68:6210-6219.

4.Yakimov MM,Timmis KN,Wray V,Fredrickson HL.1995.Characterization ofa new lipopeptide surfactant produced by thermotolerant and halotolerantsubsurface Bacillus licheniformis Bas50.Appl.Environ.Microb.61:1706-1713.

5.Naruse N,Tenmyo O,Kobaru S,Kamei H,Miyaki T,Konishi M,OkiT.1990.Pumilacidin,a complex of new antiviral antibiotics-production,isolation,chemical-properties,structure and biological-activity.J.Antibiot.43:267-280.

Disclosure of Invention

The invention aims to provide a marine Bacillus sp.BS176 and application thereof in producing lipopeptide.

In order to achieve the purpose, the invention adopts the technical scheme that:

a marine Bacillus, the lipopeptide-producing marine bacterium is Bacillus sp.BS176, the strain is preserved in China general microbiological culture Collection center, the address is as follows: west road No.1 north chen, north yang district, beijing, 3 (institute for microbiology, chinese academy of sciences), with a collection date of 2016, 12 and 29 days, and a collection number: CGMCC No. 13515.

Use of a marine Bacillus sp.bs176 for the production of a lipopeptide.

Application of marine Bacillus in preparation of a medicament for inhibiting pathogenic bacteria.

A lipopeptide molecule comprises molecules CLP1 and CLP2, CLP1 has molecular formula of C₅₇H₁₀₁N₇O₁₃CLP2 with the molecular formula C₅₈H₁₀₃N₇O₁₃。

The polypeptide parts of the lipopeptide molecules CLP1 and CLP2 are both composed of glutamic acid (Glu) -leucine (Leu) -aspartic acid (Asp) -leucine (Leu) -isoleucine (Ile), the fatty acid chains of the lipopeptide molecules are β hydroxy fatty acids, and the difference is only that the fatty acid chains are different by one methylene group (CH)₂-)。

A method for preparing lipopeptide molecules comprises the steps of inoculating Bacillus marinus sp.BS176 into a 2216E liquid culture medium, and carrying out shake culture at 28 ℃ overnight; then taking the bacterial liquid to transfer to a fermentation medium, and carrying out shake culture at 28 ℃ for 2 days; and after the fermentation is finished, centrifugally collecting fermented supernatant, acidifying, freeze-drying and rotary evaporating the supernatant to obtain crude extracts (CLPs) of extracellular active substances of the marine Bacillus sp.BS176, and further purifying to obtain lipopeptide molecules CLP1 and CLP 2.

The fermentation medium is peptone 20g/L, yeast powder 10g/L, glucose 5g/L, seawater 1L, pH7.5.

And after the fermentation is finished, performing centrifugation at 8000rpm for 10 minutes to collect fermentation supernatant, and performing acidification, freeze drying, rotary evaporation and other treatments on the supernatant to obtain a crude extract of the extracellular active substance of Bacillus sp.BS176. Separating the crude extract with Sephadex LH-20 chromatographic column to obtain bioactive fraction. The fraction was further separated and purified by high performance liquid chromatography (HPLC, UV241 nm).

Two single substance fractions with the activity of inhibiting the movement ability of bacteria are obtained by HPLC coseparation, and the two active substances are analyzed by a primary mass spectrum (ESI-MS) and a secondary mass spectrum (MS/MS) to find that the molecular weights of the two active substances are 1114.73Da and 1128.75Da respectively, and the two active substances have the same amino acid composition form, namely a cyclic peptide structure consisting of Glu-Leu-Leu-Leu-Asp-Leu-Ile, and Glu and Asp are both modified by methoxyl. Further Nuclear Magnetic Resonance (NMR) analysis of the two active substances revealed that they present a fatty acid structure in addition to the amino acid chain structure, indicating that the active substances are lipopeptide molecules and belong to the family of homologues differing by only one methylene group (CH2-) in the part of the fatty acid chain (FIG. 1).

The application of lipopeptide molecules CLP1 and CLP2 in preparing medicines for inhibiting pathogenic bacteria is provided.

The pathogenic bacteria are gram-negative bacteria or gram-positive bacteria. The pathogenic bacteria are vibrio anguillarum, vibrio splendidus, vibrio vulnificus, pseudomonas aeruginosa, pseudomonas stutzeri, staphylococcus aureus, bacillus subtilis or bacillus BS176 and the like.

The invention has the advantages that: the Bacillus sp.BS176 is separated from the sea area of the western Pacific ocean, belongs to marine microorganisms, and lipopeptide produced by the Bacillus sp.BS176 has the activity of inhibiting the movement capability of aquaculture pathogenic bacteria and has the effect of promoting bacterial agglutination and settlement on various pathogenic bacteria. The bacillus and the lipopeptide generated by the bacillus have potential application values in the aspects of developing broad-spectrum antibacterial drugs, improving aquaculture environment and the like.

Drawings

Fig. 1 is a structural diagram of a Bacillus sp.bs176 lipopeptide molecule provided by an embodiment of the present invention. FIG. 2 is a diagram showing the activity of Bacillus sp.BS176 and its lipopeptide molecule in inhibiting the motility of Vibrio alginolyticus.

FIG. 3 is a diagram showing the activity of lipopeptide molecules of Bacillus sp.BS176 in promoting the aggregation of Vibrio alginolyticus.

Fig. 4 is a broad-spectrum diagram of the effect of Bacillus sp.bs176 lipopeptide molecules provided by the embodiments of the present invention.

Detailed Description

The invention will be further illustrated in the following experimental examples, but is not limited thereto. The lipopeptide-producing marine bacterium is Bacillus sp.BS176, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the address: west road No.1 north chen, north yang district, beijing, 3 (institute for microbiology, chinese academy of sciences), with a collection date of 2016, 12 and 29 days, and a collection number: CGMCC No. 13515. The marine Bacillus sp.BS176 is separated from the sea and mountain area of the western Pacific ocean, and the strain has the activity of inhibiting the movement capacity of pathogenic bacteria. Through analysis, two lipopeptides CLP1 and CLP2 in Bacillus sp.BS176 are key active molecules for inhibiting the movement capability of pathogenic bacteria; meanwhile, CLP1 and CLP2 also have the activity of promoting the agglutination and sedimentation of various pathogenic bacteria. The marine Bacillus sp.BS176 obtained by the method and the lipopeptide generated by the marine Bacillus sp.BS176 can be used for developing novel marine pathogenic bacteria prevention and control preparations.