Strain of marine oil-degrading bacteria, compounds obtained by fermentation and their applications

Abstract

A strain of marine oil degrading bacteria, compounds obtained by fermentation and their uses are published. The name is Alcanivorax dieselolei T6-6, with preservation Number: CGMCC NO: 9033. It belongs to -proteobacteria, oceanospirillales, alkane degrading bacteria branch, alcanivorax category and diesel alcanivorax species. The said bacteria can be used to degrade and remove petroleum hydrocarbon, control environmental pollution, produce surfactants, reduce surface tension of water, and for other purposes; a compound can be isolated from secondary metabolites after fermentation, one of which is dieselolei T6-6, 2-amino-6-(N-2-carbonyl-4-tridecyl-cyclobutane) hexanoic acid, and it is a kind of lysine ester. The compound has emulsifying activity on different organics, antibacterial effects against Gram-positive bacteria and fungus, and a better cytotoxic activity.

Claims

1. A compound, wherein, the compound is produced by a fermentation of bacterium Alcanivorax dieselolei T6-6, accession number: CGMCC NO: 9033; wherein a structural formula of the compound is: ##STR00005## wherein when n=1, a molecular formula of the compound is C.sub.18H.sub.34N.sub.2O.sub.3, a molecular weight is 326, the compound is named as 2-amino-6-(N-2-carbonyl-4-nonyl-cyclobutane) hexanoic acid; wherein when n=2, a molecular formula of the compound is C.sub.20H.sub.38N.sub.2O.sub.3, a molecular weight is 354, the compound is named as 2-amino-6-(N-2-carbonyl-4-undecyl-cyclobutane) hexanoic acid; wherein when n=3, a molecular formula of the compound is C.sub.22H.sub.42N.sub.2O.sub.3, a molecular weight is 382, the compound is named as 2-Amino-6-(N-2-carbonyl-4-tridecyl-cyclobutane) hexanoic acid or lysine aliphatic dieselolei T6-6; wherein when n=4, a molecular formula of the compound is C.sub.24H.sub.46N.sub.2O.sub.3, a molecular weight is 410, the compound is named as 2-amino-6-(N-2-carbonyl-4-pentadecyl-cyclobutane) hexanoic acid.

2. The compound of claim 1, wherein the compound is lysine aliphatic dieselolei T6-6 which has the structural formula: ##STR00006##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is Phylogenetic tree based on 16s rDNA nucleotide sequence of T6-6

(2) FIG. 2A and FIG. 2B are alkane biodegradation rate GC-MS spectras of strain Alcanivorax dieselolei T6-6. 2A is a C16 biodegradation rate of GC-MS specta. 2B is a C14 biodegradation rate of GC-MS specta.

(3) FIG. 3 is Relationship between cell growth and biosurfactant production during the growth of strain T6-6.

(4) FIG. 4 is TLC analysis of biosurfactants from strain T6-6 (4A) Iodine vapor as developer and (4B) ninhydrin as developer.

(5) FIG. 5A is HPLC (210 nm peak) analysis of the compound dieselolein T6-6; FIG. 5B is HPLC (210 nm peak) of methanol used to dissolve the compound dieselolein T6-6 in the control (no have biosurfactant in this sample).

(6) FIG. 6 is molecular mass spectra of the compound of dieselolein T6-6

(7) FIG. 7 is .sup.1H-NMR spectra of purified compound from dieselolein T6-6

(8) FIG. 8 is .sup.13C-NMR spectra of purified compound from dieselolein T6-6

(9) FIG. 9 is .sup.1H-1HCOSY spectra of purified compound from dieselolein T6-6

(10) FIG. 10 is HMBC spectra of purified compound from dieselolein T6-6

(11) FIG. 11 is HMQC spectra of purified compound from dieselolein T6-6

(12) FIG. 12 is Comparison of emulsifying activity of lysine lipid produced by T6-6 and synthetic surfactants Tween 20 and Triton X-100

(13) FIG. 13 is Effects of pH on the surface activity of the biosurfactant of T6-6.

(14) FIG. 14 is Effects of temperature on the surface activity of the biosurfactant of T6-6.

(15) FIG. 15 is a process of crude extract.

DETAILED DESCRIPTION OF THE INVENTION

(16) The following content is the detailed description of present embodiment. The example of said embodiment is shown in figures; furthermore, the same or similar label all along indicates the same or similar elements or the elements with same or similar functions. Following embodiment that is described with reference to figures is exemplary, and is intended for the explanation of this invention and shall not be interpreted as limiting the invention. Where there is without indication of specific technology or conditions in embodiment, the operation shall be handled in accordance with the technical or conditions described in the literature of this field, or with the product manual. If the used reagent or equipment is not marked with manufacturer, they can be purchased from the market.

Embodiment 1: Isolation and Culture of Strains

(17) Isolation method: Take 50 cm.sup.3 deep sea water (collected from southwest Indian Ocean at the 20.sup.th voyage) into a 250 cm.sup.3 conical flask, which is kept at 121 C. for 20 minutes, add 0.21 g pre-autoclavable mixture of crude oil and diesel fuel at a mass ratio of 1:1; add pre-autoclavable NH.sub.4NO.sub.3 (at final concentration of 1 g/dm.sup.3), KH2PO4 solution (pH=7.5; at final concentration of 1 g/dm.sup.3) and FeSO.sub.4 solution (at final concentration of 0.4 g/dm.sup.3 and being treated by 0.22 m membrane filtration sterilization); take 50 cm.sup.3 above deep sea water into above flask, which is kept at 28 C. and dark cultured in a shaker at 160 r/min; after being cultured for 6 days, black oil contamination disappears, and then take and transfer 2 cm.sup.3 of above solution to the same medium, after transferring for 5 times, the enrichment is gradient diluted and applied on a 2216 L flat (2216 L medium: NaAc: 1 g, yeast powder: 2 g, tryptone: 10 g, sodium lemon: 0.5 g, being dissolved in 1 dm.sup.3 aged seawater, adjust pH to 7.5. Add 1.5% AGAR powder to solid plate) to isolate single bacterium.

(18) Finally, a strain isolated from a fungal strain, the strain was named as: T6-6, i.e. the identification dieselolei T6-6 Alcanivorax strain

(19) (A) Form

(20) The method: inoculate the strain onto a 2216 L plate, which is cultured in darkness at the temperature of 20 C. for ten days; measure the diameter of the bacterial colony and record color of the bacterial colony. The Alcanivorax dieselolei T6-6 is Gram-negative bacteria and is positive in oxidase and catalase and capable of reducing nitrate but not nitrite. When the strain grows on the 2216 L plate, the bacterial colony formed is small and milk white, has translucent edges, smooth surface, central bumps and regular exterior, has no halos and is rod-shaped under oil lenses.

(21) (B) Colony PCR and 16s rDNA Sequences

(22) The PCR (polymerase chain reaction) method of the bacterial colony: DNA extraction of the Alcanivorax dieselolei T6-6 and amplification of 16S rDNA are carried out according to a document (Liu C, Shao Z. Alcanivorax dieselolei sp. nov., a novel alkane-degrading bacterium isolated from sea water and deep-sea sediment [J]. International Journal of Systematic and Evolutionary Microbiology, 2006, 55:11811186). PCR amplification of 16S rDNA is performed with the aid of a primer SEQ ID NO.2-3.

(23) TABLE-US-00003 SEQIDNO:1: 16SF: 5-AGAGTTTGATCCTGGCTCAT-3 i.e.,SEQIDNO:2; 16SR: 5-ACGGCTACCTTGTTACGACT-3 i.e.,SEQIDNO:3;

(24) The target fragment size is about 1500 bp. The primer used for PCR is synthesized by TaKaRa. The PCR reaction system (5.0 mm.sup.3 in total volume): 10*buffer 5.0 mm.sup.3, dNTP (0.2 mmol/dm) 4.0 mm.sup.3, 16SR (10 pmol/dm.sup.3) 1.0 mm.sup.3, 16SF (10 pmol/dm.sup.3) 1.0 mm.sup.3, Taq enzyme (2 U/mm.sup.3) 0.4 mm.sup.3, DNA template (50 ng1 g) 1.0 mm.sup.3 and 37.4 mm.sup.3 of deionized water. Conditions of PCR amplification: 95 C. for 5 minutes, 95 C. for 1 minute, 55 C. for 1 minute and 72 C. for 2 minutes for 30 cycles and 72 C. for 5 minutes. Products of PCR amplification are converted into E. coli DH5alpha competent cells and sequenced by Sangon Biotech (Shanghai) Co., Ltd after being purified and connected with pMD19-T. The sequencing result is analyzed by the NCBI (http://www.ncbi.nlm.nih.gov/BLAST), sequences of sibling species in the amplification sequence and the GenBank are compared via software DNAMA (version 5.1) and the phylogenetic tree is drawn by using MEGA3.1.

(25) Take oil and diesel oil mixture as the carbon source for enrichment culture; the strain of Alcanivorax dieselolei T6-6 is isolated from sediment samples of southwest Indian Ocean collected at the 20th voyage, and it has obvious diesel degradation ability; single bacterium validation showed that 4.2 g/dm.sup.3 diesel oil can be emulsified and mostly degraded after being cultured for 3 days; only a small part of emulsified oil droplets are left on the surface, which may be non-alkane component in diesel oil. The strain was determined, and has been preserved in Chinese Type Culture Collection since Apr. 9, 2014, with its Preservation Numbers: CGMCC NO: 9033.

(26) In this study, the amplified 16S rDNA on Genebank registration number as shown in FIG. 1 phylogenetic tree.

(27) The type analysis of the Alcanivorax dieselolei T6-6 and sequence analysis of 16S rDNA demonstrate that the Alcanivorax dieselolei T6-6 belongs to -proteobacteria, oceanospirillales, alkane degrading bacteria branch, Alcanivorax category and diesel Alcanivorax species. The 16S rDNA phylogenetic tree is shown as FIG. 1.

Embodiment 2: Test of Alkane Degradation Ability of Alcanivorax Dieselolei T6-6

(28) Test method of alkane degradation rate: Scrape Alcanivorax dieselolei T6-6 from the fresh flatin to a sterile EP tube added with MMC medium (formula: NaCl 24 g/l, MgSO.sub.4 7H.sub.2O: 7.0 g/l, NH.sub.4NO.sub.3: 1.0 g/l, KCl: 0.7 g/l, KH.sub.2PO.sub.4: 2.0 g/l Na.sub.2HPO.sub.4: 3.0 g/l, PH=7.4, 1 L distilled water, with appropriate amount of 0.22 um membrane filter sterilized trace elements mixture added after sterilization (CaCl.sub.2 2 mg/l; FeC.sub.1.6H.sub.2O, 50 mg/l; CuSO.sub.4, 0.5 mg/l; MnCl.sub.2.4H.sub.2O, 0.5 mg/l; ZnSO.sub.4.7H.sub.2O, 10 mg/l); after mixing, add a certain concentration of bacteria into a 150 ml conical flask added with 50 ml MMC medium (two parallel controls are provided for each alkane), add 1% alkane C.sub.12, C.sub.14, C.sub.16 and C.sub.18 into MMC liquid medium for culture observation. Alkane degradation rate (%)=(I-residual amount in the fermented liquid/blank control)100%; draw standard curve according to 2 ul, 1 ul, 0.5 ul, 0.25 ul and 0.125 ul internal chlorinated dodecane, and take the one added with C12, C14, C16 and C18 but without Alcanivorax dieselolei T6-6 as the blank control. Add 9 ml n-hexane into a fermentation bottle, shaking by table concentrator at 180 r/min for 30 minutes, standing for stratification, and take the sublayer as n-hexane phase. Take a 10 ml volumetric flask to make a constant volume of 10 ml, use part of sample for dilution to 10.sup.9, and this sample is used for GC-MS. At the same time, make corresponding standard curves of C12, C14, C16 and C18, calculate alkane degradation rate, and the results obtained are shown in Table 2.

(29) The Alcanivorax dieselolei T6-6 can utilize the straight-chain paraffins of C.sub.10-C.sub.36, with better degradation effect on the C.sub.18 and its following alkanes so that C.sub.12, C.sub.14, C.sub.16 and C.sub.18 are used for determining degradation rate of the Alcanivorax dieselolei T6-6. The GC-MS analysis result indicates that degradation rate of C.sub.16 is highest, at 92.3%, the degradation rates of the rest three are close, at about 50%. Details are shown in Table 2 and FIGS. 2A and 2B.

(30) TABLE-US-00004 TABLE 2 Strain Alcanivorax dieselolei T6-6 alkane degradation rate and cell growth table degradation rate alkane (%) OD.sub.600 C.sub.12alkane 57.46 2.5 1.62 0.06 C.sub.14alkane 50.28 0.6 1.55 0.05 C.sub.16alkane 92.03 0.5 2.69 0.02 C.sub.18alkane 46.62 0.9 1.53 0.05 indicates three experimental error
under the same experimental conditions, Alcanivorax dieselolei B-5 (AY683537) has alkane degradation too.

Embodiment 3: Test of Cell Surface Hydrophobicity of Alcanivorax dieselolei T6-6

(31) Wash the fermentation liquid of strain Alcanivorax dieselolei T6-6 using phosphate buffer (pH 7.0) at a volume of 1:1, centrifuge it at room temperature (12000 RPM, 3 minutes) and collect bacteria. Repeat washing for three times, and finally have the cells suspended in 4 ml phosphate buffer, adjust the ultraviolet-visible spectrophotometer A600=0.5 or so, with the specific value measured as A.sub.0. And then, divide the measured A.sub.0 samples into 6 groups, add 1 ml of hydrophobic material crude oil, is-xylene, n-hexadecane, octadecane, mineral oil and liquid paraffin respectively. Shake the samples for 2 minutes using a oscillator, and stratifies them after by-standing for 30 minutes. Insert a syringe needle into the lower layer to draw 2 ml liquid, and measure its A value with a spectrophotometer, and record the value as A1. The strength of cell surface hydrophobicity can be calculated with the formula: H %=(A0A1)/A0100. The higher the H value is, the bigger the cell surface hydrophobicity. A comparison was conducted with the cell surface hydrophobicity of a Myroides sp. SM1 that has been reported, and the results show that Alcanivorax dieselolei T6-6 has a good cellular surface hydrophobicity against most organic materials (except liquid paraffin), and has a higher bacterial hydrophobicity than Myroides sp. SM1. The results shown in Table 3.

(32) TABLE-US-00005 TABLE 3 Determination of the hydrophobicity of the surface of the strain Alcanivorax dieselolei T6-6 in the cell (%) Drain water content (hydrocarbon) Myroides sp. SM1 T6-6 Crude oil 85.48 68.88 1.2 the - xylene, 28.07 32.24 0.5 n-hexadecane 14.62 62.61 0.8 octadecane 0.13 78.52 1.5 mineral oil 3.78 72.45 1.2 liquid paraffin 5.23 7.66 0.8 NOTE: the data of Myroides sp. SM1 from Songklanakarin J. Sci Technol, 2007, 29 (3): . . . 769-779 indicates three experimental error

Embodiment 4: Analysis on Relationship Between Cell Growth of Strain Alcanivorax dieselolei T6-6 and Surface Tension

(33) Test method of liquid surface tension: Inoculate the Alcanivorax dieselolei T6-6 single bacterium into MSM medium on clean workbench (with 2% n-hexadecane as the sole carbon source), and culture it in constant temperature air bath shaker incubator (28 C., 200 rpm/min) until thalli obviously grow (about 3 to 5 days), during which take out 10 ml fermentation liquid every 24 hours, and measure its surface tension values using automatic interfacial tensiometer (Manufacturer: Chengde Precision Testing Machine Co., Ltd; Model: JZ-200-a automatic interface tensiometer). Put 10 ml fermented liquid of Alcanivorax dieselolei T6-6 into a measuring cup, the outer wall of which should be wiped dry, and then place the measuring cup on the operating platform of surface tension meter. Wash the suspension loop with pure water for several times in advance and rinse it with petroleum ether, acetone, then blow it to quickly volatilize acetone solution attached on its surface, finally place it on the operating platform to measurement. Before measurement, pay attention to the ambient temperature that is generally kept at 25 C. or so. (For refrigeration of supernatant and hydrophobic layer stratified after centrifugation, the surface tension should be measured when it gets to the room temperature. Thus, the result is comparability and credibility). When measuring liquid surface tension, pour 10 ml fermentation liquid of A. dieselolei T6-6 at temperature of 25 C. into a weighing cup, place the weighing cup in the center of the tray, press .box-tangle-solidup. to make the platinum ring deep into 5 mm to 7 mm in the liquid, press .circle-solid. to stop; if the peak needs to be kept, firstly press peak button, and then press .Math., the display value will increase gradually, and finally stay at the maximum; this maximum value is the actually measured value of surface tension of liquid, and then press .circle-solid. to stop, after the maximum value is recorded, press reset button. Conduct gradient dilution and isolation of HLB flat (NaCl 3%) for the enrichment liquid with surface tension less than 40 mN m.sup.1 and obvious oil drainage activity, to obtain pure culture and analyze the single bacterial surface activity.

(34) Hexadecane as the sole carbon source in the medium MSM (formula: (NH4)2SO4=1 g; KCl=0.11 g; NaCl=0.11 g; FeSO4.7H2O=2.810-5 g; anhydrous KH2PO4=0.34 g; K2HPO4.13H2O=0.44 g; MgSO4.7H2O=0.05 g; yeast extract=0.05 g; trace element solution 0.5 mL; hexadecane 2 mL, pH 7.4), 28 C., 150 rpm/min, cultured for 3-5 days.

(35) Trace element solution (%): ZnSO4.7H2O=0.029 g; CaCl2=0.024 g; CuSO4=0.025 g; MnSO4.H2O=0.017 g.

(36) For the fermented the strain Alcanivorax dieselolei T6-6, determine surface tension and OD.sub.600 value of the fermentation liquid every 12 hours in 120 hours. The results show that active substances in the fermentation products of the Alcanivorax dieselolei T6-6 are mainly generated in a logarithmic phase of strain growth and the surface tension is the lowest at 26.0 mNm.sup.1 in a stable period. The results are shown in FIG. 3.

(37) The strain A. dieselolei B-5 (AY683537) grows well in MSM medium with Hexadecane as sole carbon source and can reduce surface tension of the fermentation liquid to 31 mNm-1 or so. The method is the same as description above.

Embodiment 5: Isolation and Purification of Fermentation Products of Strain Alcanivorax dieselolei T6-6

(38) After culturing MSM liquid medium (with 2% n-hexadecane as the sole carbon source, other ingredients are shown in Example 2) of strain Alcanivorax dieselolei T6-6 for 5 days, the surface tension of fermentation liquid is about 27 mNm.sup.1. After centrifugation at 12000 rpm and 4 C. for 20 minutes, the fermentation liquid is divided into three layers from top to bottom: white hydrophobic layer, supernatant and cell sediment. Determination results of the surface tension of these three layers: hydrophobic layer is 27.2 mNm.sup.1; supernatant is 31.3 mNm.sup.1; cell sediment is 59.2 mNm.sup.1. It can be seen that surface active substances produced by strain Alcanivorax dieselolei T6-6 are mainly in hydrophobic layer and supernatant. Crude extract are shown as FIG. 15.

(39) Combine the organic phase (27.8 mNm-1) after extracting hydrophobic layer of Strain Alcanivorax dieselolei T6-6 and the organic phase (28.1 mNm-1) after sediment, freeze-drying and extraction of supernatant acid of Alcanivorax dieselolei T6-6, and make acidic ninhydrin coloration after running TLC plate. Compared with iodine coloration, most material is lipopeptide. Then use HPLC to detect polar size of active substance. HPLC results and TLC running results are used to determine eluting solvent passing through normal phase silica gel column and its eluting gradient. The subsequent isolation and purification process of target active substance is getting through the normal phase silica gel column first and then reversed phase silica gel column, and finally gel column to further purify and prepare isolated samples. The ratio for the eluent getting through normal phase column is 100%/o chloroform, followed by chloroform:methanol=50:1, chloroform:methanol=10:1, chloroform:methanol=4:1, and 100% methanol. The elution volume varies with amount of samples, and is always 2-3 column volumes. After getting through normal phase column for two times, non-basic lipopeptide substances can be removed. Use HPLC to detect size of polarity and range of absorption peak of lipopeptides components. Set conditions for getting through the reversed-phase column based on HPLC results. The reversed phase column adopts AKTA Purifier 10 connecting the reverse phase column. There are two detection wavelengths of 210 nm and 254 nm with continuous gradient elution (0-100% methanol at continuous gradient, at a flow rate of 10 ml/min). After continuously washing for 30 minutes with 100% methanol, wash the sample with pure acetonitrile for 40 minutes. According to elution order, there are 8 components; the seventh peak is 3.02 AU in height with duration of about 4.5 minutes, and the eluting gradient is 92%-93% methanol. The first peak of the first component is 4.5 AU in height with duration of about 2.5 minutes, and the eluting gradient is about 5% methanol. Surface tensions of the two components with higher peak are detected, and the results show that No. 1 component does not decrease its surface tension, while No. 7 component has a better surface activity. The surface tension of water decreases from 78.6 to 32.3 after adding 300 ul unconcentrated surfactant to 20 ml DDW; No. 7 component is purified by HPLC, and the results show the purity is so high that it is unnecessary to get through gel column. Layer expansion agents at different polarities are used to detect purity of final samples, with 100% chloroform, chloroform:methanol=3:1, chloroform:methanol=2:1, chloroform:methanol=1:1 and 100% methanol respectively used as expansion layer of expansion layer agent. Iodine coloration shows a single point, so the substances are dried to hit nuclear magnetism. During the whole process of isolation, chemical coloration and activity detection are considered as tracking indexes. The strains with high surface activity should be further isolated. Active substance produced by Alcanivorax dieselolei T6-6 after fermentation should get through positive column for two times; after collecting component sample, the expanded layer should be colored with iodine vapor and ninhydrin, and iodine coloration results of TLC layer expansion (FIG. 4A) and ninhydrin coloration results (FIG. 4B) are shown. Coloration results show that surfactant is so pure after getting through positive column for two times, and just few impurities with high polarity move to edge of layer expansion agent, so that No. 7 and No. 8 should be merged together. Combined surfactant should be detected by HPLC. The chromatogram of HPLC column for surfactant extracted from fermentation liquid of Alcanivorax dieselolei T6-6 are shown in FIG. 3. FIG. 5A is the figure of active substances merged by T6-6 running HPLC (wavelength of peak is 210 mm). and, FIG. 5B is the methanol control (wavelength of peak is 210 mm). In both figures, the solvent peak is found 5 minutes ago, and the peak of active substances is found at the point of 18 minutes in FIG. 5A.

Example 6: Structural Characterization of the Biosurfactant Produced by Hydrocarbon-Degrading Bacterium Alcanivorax dieselolei T6-6

(40) the molecular weight of biosurfactant was determined by FT-MS (obtained in the example 5)

(41) The results obtained by an FT-MS (Fourier transform ion cyclotron resonance mass spectrometer) indicate that molecular ion peaks are [M+H] of 383.32765, [M+Na] of 405.30854 and [M+Kcl] of 421.28506, while theoretical [M+H] and [M+Na] are 383.3268 and 405.3088 respectively, errors of an actual numerical value and a theoretical value are 2.2 ppm and 0.6 ppm respectively, and generally, experimental error smaller than 5 ppm is considered tolerable. Therefore, theoretical molecular weight of lipopeptide produced by the strain Alcanivorax dieselolei T6-6 is 382.32.

(42) According to comparison results of a high-resolution mass spectrum analyzer, the substance only contains C, H, O and N, and its molecular weight is 382.32 on the basis of results of mass spectrum 6. After comparison, only one molecular formula C.sub.22H.sub.42N.sub.2O.sub.3 is possible within an error range smaller than 5 ppm, which is completely identical with the molecular formula deduced by the NMR. Refer to Table 4 for the analysis result.

(43) TABLE-US-00006 TABLE 4 the FT-MS Analysis Table of strain Alcanivorax dieselolei T6- 6 produced biosurfactants (hexadecane as the sole carbon source) Lipopeptides Molecular Weight [M + H].sup.+ 383.32765 [M + Na].sup.+ 405.30854 [M + Kcl].sup.+ 421.28506

(44) (b) NMR Spectra Analysis

(45) The chemical formula C.sub.22H.sub.42N.sub.2O.sub.3 of the lipopeptide produced by the strain Alcanivorax dieselolei T6-6 is further proved by the analysis results of the NMR spectrum. The fatty acid of the strain Alcanivorax dieselolei T6-6 is a hexadecanoic acid with beta-hydroxy, and the amino acid is lysine. Amino of lysine and carboxyl of hexadecanoic acid are dehydrated and condensed to obtain amide bonds, and the imino group of lysine and beta-hydroxy of hexadecanoic acid are reacted to obtain beta-lactam four-membered ring containing nitrogen. At present, no rings formed by independent amino acid and fatty acid in well-known cyclic lipopeptide surfactant are reported.

(46) The substance has three polar groups including amido bonds, amino and carboxyl, with stronger polarity at the hydrophilic end. It can be seen from comparison that the lipopeptide is surfactant of novel structure.

(47) FIG. 7 is a 1H-NMR spectrum of the pure Compound dieselolei T6-6, FIG. 8 is a 13C-NMR spectrum of the pure compound dieselolei T6-6

(48) proton NMR spectral data of pure Compounds dieselolei T6-6:

(49) .sup.1H NMR (600 MHz, CD.sub.3OD-d.sub.4) .sub.H

(50) 3.68 (2H, m, H-8), 3.55 (1H, t, J=6.0, Hz, H-2), 3.35 (1H, m, H-6a), 3.10 (1H, m, H-6b), 3.01 (1H, dd, J=4.8, 14.7 Hz, H-9a), 2.54 (1H, dd, J=1.2, 14.7 Hz, H-9b), 1.91 (1H, m, H-10a), 1.47 (1H, m, H-10b), 1.91 (1H, m, H-3a), 1.84 (1H, m, H-3b), 1.63 (2H, m, H-5), 1.47 (2H, m, H-4), 1.38 (2H, m, H-11), 1.32 (16H, m, H-1219), 1.32 (2H, m, H-21), 1.31 (2H, m, H-20), 0.92 (3H, t, J=6.9 Hz, H-22)

(51) carbon NMR spectral data of pure Compounds dieselolei T6-6:

(52) .sup.13C NMR (151 MHz, CD.sub.3OD-d.sub.4), .sub.C

(53) 172.9 (C-1), 168.7 (C-7), 54.6 (C-2), 51.9 (C-8), 40.9 (C-9), 39.9 (C-6), 32.4 (C-10), 31.7 (C-20), 30.4 (C-3), 29.129.38 (C-1219) 27.4 (C-5), 25.1 (C-11), 22.5 (C-4), 22.3 (C-21), 13.0 (C-22); ESIMS m/z 383.32765 [M+Na].sup.+

(54) FIG. 9 is a 1H-1HCOSY spectrum of the pure Compound dieselolei T6-6. FIG. 10 is a HMBC spectrum of the pure Compound dieselolei T6-6. FIG. 11 is a 1H-1 HMQC spectrum of the pure Compound dieselolei T6-6.

(55) Combined with data of the FT-MS and the NMR detained in FIGS. 7 to 11, the new compound produced by fermenting the strain Alcanivorax dieselolei T6-6 is structured as following:

(56) ##STR00003##

(57) Common Chinese of new compound named: dieselolei T6-6, Chinese system named: 2-amino-6-(N-2-carbonyl-4-tridecyl-cyclobutane) hexanoic acid, English named: 2-amino-6-(2-oxo-4-tridecyl-azetidine-1-yl) hexanoic acid, as a lysine resin.

(58) When isolating by using the same method as above, subsequent isolation and purification process of the target active substance get through the normal phase silica gel column first and then the reversed phase silica gel column, and finally the gel column to prepare isolated samples after further purification. The ratio for getting through the normal phase column eluent is 100% chloroform, followed by chloroform:methanol=50:1, chloroform:methanol=10:1, chloroform:methanol=4:1, and 100% methanol. The elution volume varies with amount of samples, and is always 2-3 column volumes. After getting through the normal phase column for two times, non-basic lipopeptide substances can be removed. Use HPLC to detect the size of polarity and range of absorption peak of lipopeptides components. Set conditions for getting through the reversed-phase column based on HPLC results. The reverse phase column adopts AKTA Purifier 10 connecting the reverse phase column. There are two detection wavelengths of 210 nm and 254 nm with continuous gradient elution (0-100% methanol at continuous gradient, at a flow rate of 10 ml/min). Beginning with 0% methanol, substances with high polarity are eluted first, and then substances with poor polarity. Order of eluted materials are shown as the sequence of n=14 in the table below. After washing with 100% methanol for 30 minutes, the substances are washed with pure acetonitrile for 40 minutes. Structural formula of this compound is as follows:

(59) ##STR00004##

(60) TABLE-US-00007 TABLE 9 is elution conditions of the compounds Elution gradi- Molecular Molecular entrang- Compound Name Formula Weight n ing from 2-Amino--6-(N-2- C.sub.18H.sub.34N.sub.2O.sub.3 326 n = 1 70%-78% carbonyl-4-nonyl- methanol azetidine-1- yl)hexanoic acid 2-Amino--6-(N-2- C.sub.20H.sub.38N.sub.2O.sub.3 354 n = 2 80%-88% carbonyl-4-undecyl- methanol azetidine-1-yl) hexanoic acid 2-amino-6-(2-oxo- C.sub.22H.sub.42N.sub.2O.sub.3 382 n = 3 92%-93% 4-tridecyl-azetidine- methanol 1-yl)hexanoic acid 2-Amino--6-(N-2- C.sub.24H.sub.46N.sub.2O.sub.3 410 n = 4 More carbonyl-4-pentadecyl- than 94% azetidine-1-yl) of meth- hexanoic acid anol

Embodiment 7: Determination of Physicochemical Properties of Lipopeptide Surfactant Produced by Strain Alcanivorax dieselolei T6-6

(61) In order to understand physicochemical properties and functions of biosurfactants produced by strain Alcanivorax dieselolei T6-6 (also called dieselolei T6-6) after fermentation, we determined CMC value and MAD value of purified lysine ester (i.e. dieselolei T6-6), and also determined the size of oil expansion ring of lysine ester at different temperatures and pH values to verify the stability of biosurfactant, as well as its emulsification effect on different organisms.

(62) Determination method of biosurfactant CMC values: prepare different concentrations of water solution from pure biosurfactants to be determined, and measure its surface tension with surface tension meter. With the increase of surface activity (concentration), the surface tension of water solution gradually increases. The surface tension stops decreasing when the concentration increases to a certain level. The amount of surfactant corresponding to this critical surface tension is CMC value. The results are shown in Table 5.

(63) TABLE-US-00008 TABLE 5 is a comparison of CMC values of several biosurfactant with expanding oil test CMC biosurfactant (mg/l) lysine lipid produced by strains 32 Alcanivorax dieselolei T6-6 Rhamnolipid 50 Surfactin 45 SDS 23

(64) Determination method of biosurfactant MAD (Minimum Active Dose) values: prepare different concentrations of water solution from pure biosurfactants to be determined, and measure the oil expansion activity through oil expansion experiment. With the decrease of concentration, the oil expansion ability of water solution becomes weaker and weaker, and completely disappears when the concentration decreases to a certain level. The amount of surfactant at the time of oil expansion ability disappearing is the MAD value. The results are shown in Table 6.

(65) TABLE-US-00009 MAD values in Table 6 Table expanding oil experimental comparison of several surfactants MAD biosurfactant (g) lysine lipid produced by strains 0.105 0.05 Alcanivorax dieselolei T6-6 Tween 20 0.3 0.07 Tween 80 0.15 0.05 Triton X-100 0.3 0.08 SDS 6.0 0.12 indicates three experimental error

(66) In order to understand the emulsifying activity of lysine ester produced by strain Alcanivorax dieselolei T6-6, we determined the emulsifying stability value of lysine ester produced by strain Alcanivorax dieselolei T6-6 and the chemical surfactants Tween 20 and Triton X-100 against olive oil, n-hexane, dimethylbenzene, hexadecane and petroleum ether. Determination method of biosurfactant emulsifying property: add 5 ml commercially available diesel No. 0 and 5 ml surfactant solution (1%) into a test tube, treat it with ultrasonic generator at 800 W for 40 s, and measure the emulsion and oil phase at different times (0 to 24 hours). Emulsion stable value (Es %)=emulsified layer height (cm)/oil phase height (cm)100. Specific results are shown in FIG. 12. The emulsifying activity test result of lysine ester produced by strain Alcanivorax dieselolei T6-6 indicates that it has a higher emulsifying activity than chemical surfactant Tween 20 and Triton X-100. Lysine ester produced by strains Alcanivorax dieselolei T6-6 has a good emulsifying activity (831%) on olive oil, and can maintain 48 h at room temperature. Its emulsifying activity on four other organics is also more than 75%. Good emulsifying activity makes it greatly valuable in environmental pollution control.

(67) Activity test of lysine ester produced by strain Alcanivorax dieselolei T6-6 at different temperatures and pH values (oil expansion experiment) shows that it has a good thermal stability and pH tolerance, as shown in FIGS. 13 and 14. Determination method of biosurfactant oil expansion activity: Take a culture dish, add water, and add 0.1 ml hexadecane on the water surface to form oil film. Add the fermentation liquid from the shake flask to the center of oil film to make the central film squeezed to form a circle, the diameter of which positively correlates to the concentration and activity of the biosurfactant. Strains with cycle diameter more than 3 cm are retained for further study. Set pH value in the range of 2 to 10 to determine effect of pH on lysine ester produced by the strain Alcanivorax dieselolei T6-6. Oil expansion diameter varies from 3.6 to 3.9 for lysine ester produced by the strain Alcanivorax dieselolei T6-6 with small fluctuation. It has a good acid and base resistance, and the activity is the highest at pH=8. Considering the structure, it can be inferred that this material is alkaline. And also, when adding a few drops of ammonia expansion on the running TLC plate, the substance will expand more fully, which also proves that it is alkaline.

(68) Treat the pure lysine ester produced by strain Alcanivorax dieselolei T6-6 at temperatures of 20 C., 40 C., 60 C., 80 C. and 100 C. for one hour, and then determine its activity. The results show that the oil expansion diameter is 3.7 to 4.1 cm, without significant change in the activity. After the sample was treated at high temperature of 121 C. for 15 minutes, the oil expansion diameter slightly decreases to about 3.5 cm, indicating a slight decrease of activity. Good thermal stability makes it possible for the substance to be used in high temperature fields.

Embodiment 8: Test of Cytotoxic Activity and Antimicrobial Activity of Lysine Ester Produced by Strain Alcanivorax dieselolei T6-6

(69) Antibacterial activity test method: use filter paper method to detect antibacterial activity of biosurfactant produced by fermentation of strain Alcanivorax dieselolei T6-6. The indicator bacteria are Gram-negative bacteria, Gram-positive bacteria, endophytic pathogenic fungus in tea tree and yeasts. Specific steps: coat the well-shaken indicator bacteria on the culture plate, stick filter paper on each scribe region, and drip surfactant on filter paper. The control group is the methanol solvent of same amount. After bacteria are inverted cultured in constant temperature incubation at 28 C. for a certain period (different indicator bacteria have different growth cycles; bacteria grow quickly, but endophytic fungus in tea tree should grow 3-5 days), and observe the results to detect inhibition zone. If there is an inhibition zone, its size should be measured.

(70) As indicated by bacteriostasis experiments, the active substances produced by the Alcanivorax dieselolei T6-6 have better antimicrobial activity against six strains of gram-positive bacteria like bacillus, two strains of plant pathogenic fungi and one strain of yeast but no suppression effect against the gram-positive bacteria such as E. coli. DH5 alpha, aeromonas hydrophila and Campbell vibrio.

(71) The results are shown in Table 7.

(72) TABLE-US-00010 Inhibition zone Indicator bacteria genus name (mm) Lysinibacillus sphaericus 11.7 Ornithinibacillus bavariensis 14.2 Bacillus plakortidis 13.7 Bacillus thuringiensis 7.2 Bacillus rhizosphaerae 10.4 Bacillus safensis 10.9 Phomaexigua var. exigua 16.3 Pestalotiopsistheae 10.19 Rhodotorula mucilaginosa 12.1

(73) Cytotoxic activity test method: measure cytotoxic activity using a Cell Counting Kit-8 (CCK-8 kit for short and of Dojindo Laboratories, Japan), namely, a human CCK (cholecystokinin) octapeptide ELISA (enzyme-linked immunosorbent assay) kit. The human CCK octapeptide ELISA is a quick high-sensitivity detection kit based on WST-8 and widely applied to cell proliferation and cytotoxic activity.

(74) WST-8 is a compound similar to the MTT. With the presence of electronic coupling reagent, WST-8 can be reduced by some dehydrogenases in mitochondria and produce orange formazan. The more and faster the cell proliferation is, the deeper the color; the greater the cytotoxicity is, the lighter the color. For the same cells, the color depth has a linear relationship with the number of cells. Solvent DMSO is used in control group, and pure lysine ester produced by strain Alcanivorax dieselolei T6-6 dissolved in DMSO are used in experimental group. Cells are hepatoma cells 7402 and normal hepatocytes 7702 (available from Sino-US Joint Venture Biohermes Biomedical Technology Ltd). Medicinal concentration is 100 ug/ml, and operation is taken according to kit instruction, with each step repeated three times. After medication, OD.sub.600 is detected in cellular fluid after culturing for 48 hours. Cell toxicity results are shown in Table 8:

(75) TABLE-US-00011 The results in Table 8 Table Cytotoxicity Determination of cell culture fluid OD.sub.600 Samples of Hepatoma Normal liver different treatments cells 7402 cells 7702 DMSO 1.667 0.863 lysine lipid produced 0.121 0.282 by strains Alcanivorax dieselolei T6-6

(76) The medicine-to-cell suppression rate=[negative group (DMSO)experimental group (dosed)]/negative group (DMSO). Data in Table 8 are substituted into the formula to work out the suppression rate (92.74% for hepatoma carcinoma cells 7402 and 67.32% for normal hepatocyte cells 7702) of the active substances of lysine ester produced by the strain Alcanivorax dieselolei T6-6. Accordingly, the active substances of lysine ester generated by the strain Alcanivorax dieselolei T6-6 have better cytotoxic activity.

(77) Although the above has shown and described the embodiments of present invention, it is understood that the above embodiments are exemplary and are not to be construed as the limitation of this invention. General technician in the field can change, modify, substitute and vary above embodiments within the scope of inventory without departing from the principles and spirit of the invention.