CN102994418B - Methylotrophic bacillus for producing surfactins and iturin A compounds and application of methylotrophic bacillus - Google Patents

Methylotrophic bacillus for producing surfactins and iturin A compounds and application of methylotrophic bacillus Download PDF

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CN102994418B
CN102994418B CN201210431946.4A CN201210431946A CN102994418B CN 102994418 B CN102994418 B CN 102994418B CN 201210431946 A CN201210431946 A CN 201210431946A CN 102994418 B CN102994418 B CN 102994418B
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surfactin
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genus bacillus
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iraq subtilis
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CN102994418A (en
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漆淑华
张晓勇
彭江
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a methylotrophic bacillus for producing surfactins and iturin A compounds and an application of methylotrophic bacillus. The methylotrophic bacillus SCSGAB0092CCTCC (China Center For Type Culture Collection) NO:M2012339 can generate surfactins 1-7 and iturin A compounds 8-11, and also has the advantages of fouling resistance, bacterium resistance, fungus resistance and antiviral activity, thereby being capable of being used for preparing the surfactins 1-7 and the iturin A compounds 8-11 and applied to preparing anti-fouling materials, anti-bacterium materials, anti-fungus materials and antiviral materials, and having a wide industrial application prospect.

Description

Methylotrophy type genus bacillus and the application thereof of Surfactin and iraq subtilis actinomycin A compounds produced in one strain
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of methylotrophy type genus bacillus (Bacillus methylotrophicus) SCSGAB0092 that can produce Surfactin 1-7 and iraq subtilis actinomycin A compounds 8-11, and utilize this bacterium to prepare the method for Surfactin 1-7 and iraq subtilis actinomycin A compounds 8-11, also have this bacterium in the application of preparing in fouling resistance material, antibacterium material and antifungal substance.
Background technology:
As far back as nineteen fifty-seven, Delcambe separates in the soil-like of African Zaire with Devignat and has obtained a bacillus subtilis (Bacillus subtilis), it can produce a class antifungal substance-iraq subtilis actinomycin A (Iturin A), its structure be by beta-amino lipid acid and peptide with lactone bond in conjunction with the cyclic lipopeptide forming.Because iraq subtilis actinomycin A is amphipathic compound, therefore it has surface-active effect, and it also has very strong haemolysis and anti-mycotic activity simultaneously.Through the research discovery of decades, nearly all subtilis and partial solution bacillus amyloliquefaciens (Bacillus amyloliquefaciens) can both produce iraq subtilis actinomycin A (Ahimou and Jacques, 2000; Harris et al., 1999).
Structure and the iraq subtilis actinomycin A of Surfactin are similar, but Surfactin be by beta-hydroxy fatty acid and peptide with lactone bond in conjunction with the cyclic lipopeptide forming, its surface-active action is far longer than iraq subtilis actinomycin A.Research shows that Surfactin is a strongest type biological surfactant of finding up to now.As deriving from that organism can be again that biological institute degrades and active very strong Surfactin can be used on washing composition manufacture, oil recovery, sensitive emulsion and stablizes, the aspects such as the Fast Measurement of plant disease control, cytoclasis and microbe population.Because Surfactin does not have strict specificity and selectivity to the fragmentation of cell, therefore phytopathogen there is the killing action of wide spectrum.In addition, in surfactivity cellulose solution, add after a certain amount of iraq subtilis actinomycin A, killing of phytopathogen had to great enhancement (Wu Qing equality, 1999).Along with environmental consciousness is strengthened day by day, biodegradable tensio-active agent will more and more get more and more people's extensive concerning.
Surfactin
1 R 1=C 10H 21,R 2=H,R 3=H,R 4=CH(CH 3) 2or
R 1=C 9H 19,R 2=H,R 3=H,R 4=CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
2 R 1=C 11H 23,R 2=H,R 3=H,R 4=CH(CH 3) 2or
R 1=C 10H 21,R 2=H,R 3=H,R 4=CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
3 R 1=C 12H 25,R 2=CH 3,R 3=H,R 4＝CH(CH 3) 2or
R 1=C 11H 23,R 2=CH 3,R 3=H,R 4＝CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
4 R 1=C 12H 25,R 2=H,R 3=H,R 4=CH(CH 3) 2or
R 1=C 11H 23,R 2=H,R 3=H,R 4=CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
5 R 1=C 12H 25,R 2=CH 3,R 3=H,R 4＝CH(CH 3) 2or
R 1=C 11H 23,R 2=CH 3,R 3=H,R 4＝CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
6 R 1=C 12H 25,R 2=CH 3,R 3=H,R 4＝CH(CH 3) 2or
R 1=C 11H 23,R 2=CH 3,R 3=H,R 4＝CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
7 R 1=C 12H 25,R 2=CH 3,R 3=CH 3,R 4=CH(CH 3) 2or
R 1=C 11H 23,R 2=CH 3,R 3=CH 3,R 4=CH 2CH(CH 3) 2or CH(CH 3)CH 2CH 3
Formula (1)
7 compounds shown in formula (1) are in prior art, to disclose 7 Surfactins, are specifically disclosed in document: [1] TangJS, Gao H, Hong K, Yu Y, Jiang MM, Lin HP, Ye WC & Yao XS (2007) Complete assignments of 1h and 13c NMR spectral data of nine surfactin isomers.Magn Reson Chem 45:792-796.[2] TangJS, Zhao F, Gao H, Dai Y, Yao ZY, Hong K, Li J, Ye WC & Yao XS (2010) Characterization andOnline Detection of Surfactin Isomers Based on HPLC-MS nanalyses and Their Inhibitory Effectson the Overproduction of Nitric Oxide and the Release of TNF-α and IL-6 in LPS-InducedMacrophages.Marine Drugs 8:2605-2618.[3] Chen H, Wang L, Su CX, Gong GH, Wang P & YuZL (2008) Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis.Letters in Applied Microbiology 47:180-186) in.
Formula (2)
Iraq subtilis actinomycin A compounds 8-11, its structure as the formula (2), these compounds belong to compound of the prior art, be disclosed document: Hiradate S, Yoshida S, Sugie H, Yada H & Fujii Y (2002) Mulberryanthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2.Phytochemistry61:693-698).Compound 8-11 is respectively iraq subtilis actinomycin A compounds Iturin A in literary composition 2, Iturin A 4, Iturin A 6and IturinA 7.
Summary of the invention:
First object of the present invention is to provide one can produce Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, also there is methylotrophy type genus bacillus (Bacillus methylotrophicus) the SCSGAB0092CCTCC NO:M 2012339 of fouling resistance, antibacterium, antimycotic and antiviral activity, this bacterium is preserved in Chinese Typical Representative culture collection center on September 12nd, 2012, address: Wuhan, China Wuhan University, its deposit number: CCTCC NO:M 2012339.
Methylotrophy type genus bacillus of the present invention (Bacillus methylotrophicus) SCSGAB0092CCTCC NO:M2012339(sequence accession number JX315308) be that the inventor obtains from interior separation of tissue of squama seabed, marine animal South Sea cypress gorgonian (Melitodessquamata).The 16S rRNA gene order of this bacterial strain is as shown in SEQ ID NO.1, sequence in itself and Genbank database is carried out to BLAST compare of analysis, result shows: this bacterial strain with at first by Korean 2010 as novel species report a kind of can metabolism methyl alcohol, the bacterial isolates Bacillus methylotrophicusCBMB205(sequence accession number EU194897 of Promoting plant growth) (Madhaiyan et al., 2010) be in a minimum branch together, evolutionary distance is nearest.The 16SrRNA gene order of methylotrophy type genus bacillus (Bacillus methylotrophicus) SCSGAB0092CCTCC NO:M2012339 and the similarity of Bacillus methylotrophicus CBMB205 reach more than 99% (phylogenetic tree of this bacterial strain is shown in Fig. 1).Comprehensive physiological and biochemical property and colony morphology characteristic, be accredited as Bacillusmethylotrophicus called after: methylotrophy type genus bacillus (Bacillus methylotrophicus) SCSGAB0092, this bacterial strain is preserved in Chinese Typical Representative culture collection center on September 12nd, 2012, address: Wuhan, China Wuhan University, its deposit number: CCTCC NO:M 2012339.
Second object of the present invention is to provide the preparation method of Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, it is characterized in that, comprises the following steps:
(a) prepare the fermented liquid of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339;
(b) fermented liquid step (a) being obtained is extracted with ethyl acetate, and concentrates to obtain acetic acid ethyl ester extract;
(c) acetic acid ethyl ester extract purification on normal-phase silica gel column chromatography step (b) being obtained, using chloroform-methanol as eluent, carry out gradient elution from volume ratio 100:0 to 0:100, collecting chloroform-methanol volume ratio is the cut V that 98:2 elutes, and the cut VIII that elutes to 0:100 than 80:20 of collected volume;
Cut V, through reversed-phase silica gel column chromatography, taking methanol-water as eluent, is carried out gradient elution from volume ratio 30:70 to 100:0, collect cut V-4 that methanol-water volume ratio 100:0 elutes, then obtain Surfactin monomeric compound 1-7 through HPLC purifying;
Cut VIII is through gel filtration chromatography, taking chloroform-methanol as eluent, after remove portion impurity, use again reversed-phase silica gel column chromatography, taking methanol-water as eluent, carry out gradient elution from volume ratio 50:50 to 100:0, cut VIII-2 that collected volume elutes to 100:0 than 70:30, then obtain iraq subtilis actinomycin A compounds 8-11 through HPLC purifying.
The preferably preparation by the following method of fermented liquid that step (a) is described: methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 is inoculated in A1BFe substratum, at 200 revs/min, 30 DEG C of shaking culture obtain seed liquor in 2 days, and seed liquor is inoculated in A1BFe substratum, at 200 revs/min, 30 DEG C of shaking culture 108h, obtain fermenting culture, remove thalline and obtain fermented liquid, described A1BFe substratum is: starch 10g/L, yeast extract 4g/L, peptone 2g/L, Fe 2(SO 4) 34H 2o 40mg/L, KBr 100mg/L, thick sea salt 30g/L, surplus is water, pH 7.0.
Described concentrating can adopt conventional method, as concentrating under reduced pressure.
The 3rd object of the present invention is to provide methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 and preparing Surfactin 1,2,3,4,5,6 or 7, or application in iraq subtilis actinomycin A compounds 8,9,10 or 11.
The present invention finds through experiment, methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 is 20.12mm to the antibacterial circle diameter of streptococcus aureus, be 11.02mm to the antibacterial circle diameter of micrococcus luteus, show strong anti-microbial activity, because this bacterium can produce Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, these materials have strong antibacterium and fungi activity, and have antiviral activity.Also find through experiment, in the time of concentration 25 μ g/mL, the ethyl acetate extract of methylotrophy type genus bacillus SCSGAB0092 bacterial strain fermentation liquor can suppress adhering to of kentrogon and careless tongue worm larva completely, be that inhibiting rate is 100%, illustrate that this extract has anti-fouling activity, can be for the preparation of in antifouling paint, illustrate that thus methylotrophy type genus bacillus SCSGAB0092CCTCCNO:M 2012339 has anti-fouling activity.
Therefore the 4th object of the present invention is to provide methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M2012339 and preparing fouling resistance material, as the application in antifouling paint, or in the application of preparing in antibacterium material, as the application in anti-golden staphylococci and micrococcus luteus material, or in the application of preparing in antifungal substance, the application of withering in germ and wheat powdery mildew (disclose Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11 in document and there is this effect) material as resisting cotton blight.
Methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 of the present invention can produce Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, also there is fouling resistance, antibacterium, antimycotic and antiviral activity, therefore it can be for the preparation of Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, and prepare the application in fouling resistance material, antibacterium material, antifungal substance and antiviral substance, therefore there is wide prospects for commercial application.
Methylotrophy type genus bacillus of the present invention (Bacillus methylotrophicus) SCSGAB0092 is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 12nd, 2012, address: Wuhan, China Wuhan University, its deposit number: CCTCC NO:M 2012339.
Brief description of the drawings:
Fig. 1 is the phylogenetic tree of methylotrophy type genus bacillus (Bacillus methylotrophicus) SCSGAB0092CCTCC NO:M 2012339;
Fig. 2 is the efficient liquid phase chromatographic analysis comparison diagram of iturin and Surfactin two class mixtures and methylotrophy type fermentation of bacillus crude extract.
Fig. 3 is the H spectrogram of compound 1;
Fig. 4 is the C spectrogram of compound 2;
Fig. 5 is the H spectrogram of compound 2;
Fig. 6 is the H spectrogram of compound 3;
Fig. 7 is the H spectrogram of compound 4;
Fig. 8 is the H spectrogram of compound 5;
Fig. 9 is the H spectrogram of compound 6;
Figure 10 is the H spectrogram of compound 7;
Figure 11 is the C spectrogram of compound 8;
Figure 12 is the H spectrogram of compound 8;
Figure 13 is the H spectrogram of compound 9;
Figure 14 is the C spectrogram of compound 10;
Figure 15 is the H spectrogram of compound 10;
Figure 16 is the C spectrogram of compound 11;
Figure 17 is the H spectrogram of compound 11.
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the preparation of Surfactin 1-7 and iraq subtilis actinomycin A compounds 8-11
Choose a ring bacterial classification from methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 test tube slants in containing 150mL A1BFe substratum (starch 10g/L, yeast extract 4g/L, peptone 2g/L, Fe 2(SO 4) 34H 2o 40mg/L, KBr 100mg/L, thick sea salt 30g/L, surplus is water, pH 7.0) 500mL triangular flask in, in 30 DEG C of shaking tables with 200 revs/min, shaking culture 2 days, obtains the seed liquor of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339.
The 40LA1BFe substratum configuring is divided and is filled in 500mL triangular flask, every bottle approximately fills 150mL, at 121 DEG C of high pressure steam sterilizations after 25 minutes, every bottle adds the about 7.5mL seed liquor of cultured methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 in advance, cultivate 108h with the speed oscillation of 200 revs/min in 30 DEG C of shaking tables after, stop cultivating, obtain the fermenting culture of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339.Each bottle of fermenting culture that stops the methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 cultivating merged, then use speed (20-30 DEG C) centrifugal 15min at ambient temperature of 4000 revs/min, the supernatant liquor obtaining is the fermented liquid of removing after thalline in batches.By equal-volume ethyl acetate extraction for this fermented liquid, extraction liquid concentrating under reduced pressure obtains ethyl acetate extract 10g again.
Purification on normal-phase silica gel for ethyl acetate extract (100-200 order) dry method is mixed after sample, pack glass chromatography column (containing the about 300g of purification on normal-phase silica gel 100-200 order) into, carry out Column at Normal Temperature chromatography, using chloroform-methanol as eluent, carry out gradient elution from volume ratio 100:0 to 0:100, according to thin-layer chromatography (GF 254silica-gel plate) situation is concentrated each cut 8 the large cuts (I-VIII) of merging into, the part that wherein volume ratio 98:2 elutes is merged into the 5th large cut (V), and the part that volume ratio 8:2 elutes to 0:100 is merged into the 8th large cut (VIII).
After being mixed to sample with dry method, the 5th large cut (V) pack C into 18reverse phase silica gel post, taking methanol-water as eluent, carry out gradient elution from volume ratio 30:70 to 100:0, the cut each this step being eluted according to efficient liquid phase chromatographic analysis result merges, obtain 5 cuts (V-1 is to V-5), wherein volume ratio 100:0 elutes part and merges into cut V-4(677.1mg), this cut is Surfactin (surfactin) crude mixture, and the efficient liquid phase chromatographic analysis result of this crude product is as shown in Figure 2.This Surfactin crude mixture is further separated by high performance liquid phase half preparative chromatography (chromatographic column model: PhenomenexGimini 5 μ C18110A), and separation condition is 82% acetonitrile/water (0.01%CF 3cOOH), detection wavelength is 210nm, and flow velocity 3mL/min prepares respectively to separate obtaining Surfactin class monomeric compound 1-7 at retention time 16.5min, 20.5min, 26.0min, 27.0min, 28.5min, 40.5min and 45.0min.
By above-claimed cpd 1-7 is carried out to mass spectroscopy mensuration, record respectively its [M+H] +molecular weight is 994.6398,1008.67,1036.6887,1022.6718,1036.6887,1036.6905 and 1050.7054, shows that the molecular formula of compound 1-7 is respectively C 50h 87n 7o 13, C 51h 89n 7o 13, C 53h 93n 7o 13, C 52h 91n 7o 13, C 53h 93n 7o 13, C 53h 93n 7o 13and C 54h 95n 7o 13.Further control compounds 1-7's 1h NMR and 13c NMR spectrogram (500MHz, DMSO-d 6) (as shown in Fig. 3-10), find that the NMR data of compound 1-7 are closely similar, infer that thus it is same class compound.At this compounds 1in H NMR spectrum, δ h7-NH reactive hydrogen signal and the δ of 7.0-8.5 h3.94.8 7-NHCH-methyne signal shows to exist 7 amino-acid residues, δ h5.0-5.1 methyne signal and δ hthe methylene signals of the long chain alkane of 1.1-1.4 shows to exist beta-hydroxy fatty acid chain, in addition, and at δ h1.3-3.0and δ halso there is the β-H of a large amount of amino-acid residues in 0.7-0.9 place, γ-H and δ-H characteristic signal.At it 13in C NMR spectrum, δ cthere is 10 carbonyl signals, δ in 169-180 place cthere is 7-NH-CH methyne signal, δ in 49-60 place cthere is an oxidized methyne signal, δ in 69-73 place cthere is a large amount of methyl, methylene radical and methyne signal in 1045 places.These mass spectrums and NMR data and document (reference: [1] Tang JS, Gao H, Hong K, Yu Y, JiangMM, Lin HP, Ye WC & Yao XS (2007) Complete assignments of 1h and 13c NMR spectral data ofnine surfactin isomers.Magn Reson Chem 45:792-796.[2] Tang JS, Zhao F, Gao H, Dai Y, Yao ZY, Hong K, Li J, Ye WC & Yao XS (2010) Characterization and Online Detection of SurfactinIsomers Based on HPLC-MS nanalyses and Their Inhibitory Effects on the Overproduction ofNitric Oxide and the Release of TNF-α and IL-6in LPS-Induced Macrophages.Marine Drugs 8:2605-2618.[3] Chen H, Wang L, Su CX, Gong GH, Wang P & Yu ZL (2008) Isolation andcharacterization of lipopeptide antibiotics produced by Bacillus subtilis.Letters in AppliedMicrobiology 47:180-186) in the correlation spectrum data of surfactivity chlorins compound basically identical, therefore infer the structure of compound 1-7 as shown in formula (1), be Surfactin class monomeric compound 1-7.
The 8th large cut (VIII), after gel filtration chromatography (gel is sephedex LH-20, the chloroform-methanol that moving phase is 1:1 for diameter 18mm, column length 1600mm) remove portion impurity, then used to C 18reverse phase silica gel post carries out chromatographic separation, taking methanol-water as eluent, carry out gradient elution from volume ratio 50:50 to 100:0, the cut each this step being eluted according to efficient liquid phase chromatographic analysis result merges, obtain 2 cuts (VIII-1and VIII-2), the part that wherein volume ratio 70:30 elutes to 100:0 is merged into cut VIII-2(2424.1mg), this cut is iturin (Iturin) crude mixture, and the efficient liquid phase chromatographic analysis result of this crude product as shown in Figure 2.By this iturin crude mixture further by high performance liquid phase half preparative chromatography (chromatographic column model: YMC-Pack, ODS S-5 μ 250 × 10mm i.d.) separate, separation condition is 70% methanol/water, detection wavelength is 210nm, flow velocity 3mL/min, prepares respectively to separate obtaining iraq subtilis actinomycin A compounds 8-11 at retention time 22.3min, 30.2min, 53.4min and 60.4min.
By above-claimed cpd 8-11 is carried out to mass spectroscopy mensuration, record its [M+H] +molecular weight is respectively 1043.5449,1057.5631,1071.5773 and 1071.5754, shows that the molecular formula of compound 8-11 is respectively C 48h 74n 12o 14, C 49h 76n 12o 14, C 50h 78n 12o 14and C 50h 78n 12o 14.Further control compounds 8-11's 1h NMR and 13c NMR spectrogram (500MHz, DMSO-d 6) (as shown in Figure 11-17), find that the NMR data of compound 8-11 are closely similar, infer that thus it is same class compound.At this compounds 1in H NMR spectrum, δ h6.8-8.7-NH reactive hydrogen signal and δ h8-NHCH-methyne the signal of 4.0-4.6 shows to exist 8 amino-acid residues, δ hthe methyne signal of 6.6-7.1 shows to exist the phenyl ring of a para-orientation, δ hthe methylene signals of the long chain alkane at 1.1-1.4 place and δ hthe terminal methyl group signal at 0.8-1.0 place shows to exist beta-amino fatty acid chain, in addition, and at δ halso there is β-H and γ-H characteristic signal of a large amount of amino-acid residues in 1.3-3.0 place.At it 13in C NMR spectrum, δ cthere is 12 carbonyl signals, δ in 170-180 place cthere is the phenyl ring signal of 1 para-orientation, δ in 115-156 place cthere is 8-NH-CH methyne signal, δ in 50-65 place cthere is a large amount of methyl, methylene radical and methyne signal in 10-45 place.Further contrast again it 1hNMR and 13characteristic signal in C NMR spectrum, find and document (reference: Hiradate S, Yoshida S, Sugie H, YadaH & Fujii Y (2002) Mulberry anthracnose antagonists (iturins) produced by Bacillusamyloliquefaciens RC-2.Phytochemistry 61:693-698) middle iraq subtilis actinomycin A compounds Iturin A 2, IturinA 4, Iturin A 6with Iturin A 7correlation spectrum data basically identical, therefore infer the structure of compound 8-11 as shown in formula (2).
Embodiment 2: the antibacterial experiment of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339
Taking streptococcus aureus and micrococcus luteus etc. as indicator
Aseptic 10ml seawater is joined on the test tube slant of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339, scrape hypothallus with transfering loop, then bacterium liquid is transferred to containing in the sterile test tube of granulated glass sphere and with using 4 layers of lens wiping paper of sterilizing to filter after vibrator vibration evenly, be prepared into thallus suspension liquid, and adjust spore concentration 2 × 10 7individual/ml left and right.
Adjust the spore suspension of concentration through 10 5doubly after dilution, get appropriate diluent and be coated on the flat board containing 20mL seawater LBA substratum (the consisting of every liter and contain of substratum: bacteriology peptone 10g, yeast extract paste 5g, NaCl 5g, agar powder 20g, surplus is seawater).Cultivate after 7d for 30 DEG C, select the punch tool for lawn (diameter is 0.6mm) of diameter 0.4-0.5mm that lawn is punched together with agar, preparation is containing the agar block of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 lawns, to be ready for use on determination of activity.
10ml sterilized water is joined respectively on the test tube slant that is connected to streptococcus aureus, micrococcus luteus, scrape hypothallus with transfering loop, then pour in the sterile test tube containing granulated glass sphere, with the lens wiping paper filtration with 4 layers of sterilizing after vibrator vibration evenly, be prepared into respectively streptococcus aureus, micrococcus luteus suspension, and adjust cell concentration 1 × 10 8individual/ml left and right.
The thallus suspension liquid (streptococcus aureus or micrococcus luteus suspension) of getting lmL be added to containing 100ml LBA substratum (the consisting of every liter and contain of substratum: bacteriology peptone 10g, yeast extract paste 5g, NaCl 5g, agar powder 20g, surplus is water; After medium sterilization, be cooled to 55 DEG C of left and right) triangular flask in, make containing 10 6the nutrient solution of individual/mL cell concentration, is added to 15mL nutrient solution on agar (10ml, 2% the agar) flat board of in advance getting well and solidifying with transfer pipet, is the double-deck agar plate containing indicator after nutrient solution solidifies completely.
The agar block inoculating needle picking containing methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 lawns of accomplishing fluently with punch tool is also put on the double-deck agar plate containing indicator gently, it is enough large that distance between two agar blocks is wanted, in order to avoid inhibition zone is overlapped, with containing the agar block of lawn not in contrast.Make three flat boards, each 6 of dull and stereotyped upper placement contains methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 lawn agar blocks simultaneously.By flat board, in 37 DEG C of cultivation 12h, there is inhibition zone clearly in agar block edge now.
Measure the antibacterial circle diameter containing methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 agar blocks with vernier callipers, calculate the mean value of antibacterial circle diameter, calculating methylotrophy type genus bacillus SCSGAB0092CCTCCNO:M 2012339 is 20.12mm to the antibacterial circle diameter of streptococcus aureus, be 11.02mm to the antibacterial circle diameter of micrococcus luteus, shown strong anti-microbial activity.
Embodiment 3: the fouling resistance experiment of the ethyl acetate extract of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 bacterial strain fermentation liquors
Adult barnacle, taking Chaetoceros gracilis as food, is cultivated the cypris larva stage by the nauplius larva of barnacle in 0.22 μ m filtering sea, every milliliter of 2 larvas, 28 DEG C of culture temperature, with produce the Venus stage young carry out activity test; The ripe multicell grass tongue worm collecting is placed in the seawater with 0.22 μ m membrane filtration, with light stimulation, collect the larva that ripe multicell grass tongue worm produces through light stimulation, larva is placed in the watch-glass filling with the seawater of 0.22 μ m membrane filtration, does activity test with the larva that careless tongue worm produces through illumination.Adopt 24 hole polystyrene plates to measure anti-kentrogon and the anti-careless tongue worm larva attachment activity of the ethyl acetate extract of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 bacterial strain fermentation liquors.Ethyl acetate extract is the ethyl acetate extract of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 bacterial strain fermentation liquors of embodiment 1.This ethyl acetate extract is dissolved in to DMSO, is diluted to different concentration solution (25 – 200 μ g/mL) as test fluid with sterile filtration seawater.In each hole, add 1mL test fluid and 20 ± 2 ripe kentrogons (or 20 ± 2 careless tongue worm larvas), each concentration is established 5 and is repeated sample, and sterile filtration seawater does blank.24 porocyte culture plates are placed in to 30 DEG C of incubators and place, anti-kentrogon and careless tongue worm larva adhere to experiment and added up under the microscope the larva number adhering to after 24 hours and 3 hours, calculate the inhibiting rate that anti-larva adheres to.
Experimental result shows, in the time of concentration 25 μ g/mL, the ethyl acetate extract of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 bacterial strain fermentation liquors can suppress adhering to of kentrogon and careless tongue worm larva completely, be that inhibiting rate is 100%, the ethyl acetate extract that methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M 2012339 bacterial strain fermentation liquors are described has anti-fouling activity, can be for the preparation of in antifouling paint.

Claims (8)

1. methylotrophy type genus bacillus (Bacillus methylotrophicus) SCSGAB0092, its deposit number is: CCTCCNO:M2012339.
2. a preparation method of Surfactin 1-7, iraq subtilis actinomycin A compounds 8-11, is characterized in that, comprises the following steps: as the formula (1), the structural formula of iraq subtilis actinomycin A compounds 8-11 as the formula (2) for the structural formula of Surfactin 1-7
Surfactin
Formula (1)
Surfactin 1:R 1=C 10h 21, R 2=H, R 3=H, R 4=CH (CH 3) 2or R 1=C 9h 19, R2=H, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 2:R 1=C 11h 23, R 2=H, R 3=H, R 4=CH (CH 3) 2or R 1=C 10h 21, R 2=H, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 3:R 1=C 12h 25, R 2=CH 3, R 3=H, R 4=CH (CH 3) 2or R 1=C 11h 23, R 2=CH 3, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 4:R 1=C 12h 25, R 2=H, R 3=H, R 4=CH (CH 3) 2or R 1=C 11h 23, R 2=H, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 5:R 1=C 12h 25, R 2=CH 3, R 3=H, R 4=CH (CH 3) 2or R 1=C 11h 23, R 2=CH 3, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 6:R 1=C 12h 25, R 2=CH 3, R 3=H, R 4=CH (CH 3) 2or R 1=C 11h 23, R 2=CH 3, R 3=H, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Surfactin 7:R 1=C 12h 25, R 2=CH 3, R 3=CH 3, R 4=CH (CH 3) 2or R 1=C 11h 23, R 2=CH 3, R 3=CH 3, R 4=CH 2cH (CH 3) 2or CH (CH 3) CH 2cH 3;
Iraq subtilis actinomycin A
Formula (2)
Iraq subtilis actinomycin A compounds 8:R=(CH 2) 10cH 3;
Iraq subtilis actinomycin A compounds 9:R=(CH 2) 9cH (CH 3) 2;
Iraq subtilis actinomycin A compounds 10:R=(CH 2) 10cH (CH 3) 2;
Iraq subtilis actinomycin A compounds 11:R=(CH 2) 12cH 3;
(a) prepare the fermented liquid of methylotrophy type genus bacillus SCSGAB0092CCTCC NO:M2012339 claimed in claim 1;
(b) fermented liquid step (a) being obtained is extracted with ethyl acetate, and concentrates to obtain acetic acid ethyl ester extract;
(c) acetic acid ethyl ester extract purification on normal-phase silica gel column chromatography step (b) being obtained, using chloroform-methanol as eluent, carry out gradient elution from volume ratio 100:0 to 0:100, collecting chloroform-methanol volume ratio is the cut V that 98:2 elutes, and the cut VIII that elutes to 0:100 than 80:20 of collected volume;
Cut V, through reversed-phase silica gel column chromatography, taking methanol-water as eluent, is carried out gradient elution from volume ratio 30:70 to 100:0, collect cut V-4 that methanol-water volume ratio 100:0 elutes, then obtain Surfactin monomeric compound 1-7 through HPLC purifying;
Cut VIII is through gel filtration chromatography, taking chloroform-methanol as eluent, after remove portion impurity, use again reversed-phase silica gel column chromatography, taking methanol-water as eluent, carry out gradient elution from volume ratio 50:50 to 100:0, cut VIII-2 that collected volume elutes to 100:0 than 70:30, then obtain iraq subtilis actinomycin A compounds 8-11 through HPLC purifying.
3. preparation method according to claim 2, it is characterized in that, the described fermented liquid of step (a) is to prepare by the following method: methylotrophy type genus bacillus SCSGAB0092 CCTCC NO:M2012339 is inoculated in A1BFe substratum, at 200 revs/min, 30 DEG C of shaking culture obtain seed liquor for 2 days, seed liquor is inoculated in A1BFe substratum, at 200 revs/min, 30 DEG C of shaking culture 108h, obtain fermenting culture, remove thalline and obtain fermented liquid, described A1BFe substratum is: starch 10g/L, yeast extract 4g/L, peptone 2g/L, Fe 2(SO 4) 34H 2o40mg/L, KBr100mg/L, thick sea salt 30g/L, surplus is water, pH7.0.
4. preparation method according to claim 2, is characterized in that, described simmer down to concentrating under reduced pressure.
5. methylotrophy type genus bacillus SCSGAB0092 CCTCC NO:M2012339 claimed in claim 1 is at Surfactin 1,2,3,4,5,6 or 7 as the formula (1) of preparation, or application in iraq subtilis actinomycin A compounds 8,9,10 or 11 as the formula (2).
6. the application of methylotrophy type genus bacillus SCSGAB0092 CCTCC NO:M2012339 claimed in claim 1 in the medicine of preparing anti-Staphylococcus aureus or micrococcus luteus.
7. methylotrophy type genus bacillus SCSGAB0092 CCTCC NO:M2012339 claimed in claim 1 suppresses the application in fouling resistance preparation that kentrogon or careless tongue worm larva adhere in preparation.
8. application according to claim 7, is characterized in that, the material that described inhibition kentrogon or careless tongue worm larva adhere to is the antifouling paint that inhibition kentrogon or careless tongue worm larva adhere to.
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