WO2013097424A1 - 海洋链霉菌、吡喃型倍半萜类化合物及其制备方法和用途 - Google Patents

海洋链霉菌、吡喃型倍半萜类化合物及其制备方法和用途 Download PDF

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WO2013097424A1
WO2013097424A1 PCT/CN2012/077068 CN2012077068W WO2013097424A1 WO 2013097424 A1 WO2013097424 A1 WO 2013097424A1 CN 2012077068 W CN2012077068 W CN 2012077068W WO 2013097424 A1 WO2013097424 A1 WO 2013097424A1
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pro
cyclo
compound
streptomyces
scsio
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French (fr)
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龙丽娟
田新朋
李洁
罗雄明
齐振雄
尹团
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中国科学院南海海洋研究所
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Publication of WO2013097424A1 publication Critical patent/WO2013097424A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/32—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the invention belongs to the field of industrial microorganisms, and particularly relates to a py(R)-Pro-(D)-Ile capable of producing a new unique scented pyranosesquiterpene compound and a strong active cyclic dipeptide compound Cyclo(D)-Pro-(D)-Ile , Cyclo(D)-Pro-(D)-Leu and Cyclo(D)-trans-4-OH-Pro-(D)-Phe of Streptomyces treptomyces spJ SCSIO 01689, and the use of the bacterium to prepare pyran Pyranosesquiterpene compounds and strong active cyclic dipeptides Cyclo(D)-Pro-(D)-Ile, Cyclo(D)-Pro-(D)-Leu, Cyclo(D)-trans-4 A method of -OH-Pro-(D)-Phe, and a pyranosesquiterpene compound and use thereof.
  • Background technique :
  • Vibrio cmguilla m is a worldwide aquaculture animal pathogen that can infect salmon, rainbow trout, clams, sweetfish, squid, squid, turbot, gingiva, yellow croaker, etc. (Gao Dongmei, Li Jian, Wang Study on the immune effect of Vibrio anguilla inactivated vaccine on gingiva.
  • Vibrio anguillarum is an important disease in aquaculture.
  • Cyclic peptide compounds Cyclo(D)-Pra-(D)-Ile (compound 2), Cyclo(D)-Pro-(D)-Leu (compound 3) and Cyclo(D)-trans4-OH-Pro-( D)-Phe (Compound 4) is a known compound having a structural formula represented by the formula (II) and disclosed in the following document 1, Journal of Natural Products, 2003, 66 (10): 1299-1301. 2. Journal of Natural Products 1995, 58 (2): 201-208. 3. Anal Bioanal Chem 2010, 396: 1773 - 1779. Cyclo(D)-Pro-(D)-Ile(compound 2), Cyclo(D)-Pro-(disclosed in Fdhila F et al,.
  • a first object of the present invention is to provide Streptomyces sp. SCSIO 01689, which was deposited with the China Center for Type Culture Collection (CCTCC) on July 18, 2011, at Wuhan University, Wuhan, China. No.: CCTCC NO: M 2011257c
  • the Streptomyces sp. SCSIO 01689 of the present invention is isolated from seabed sediments of Sanya Bay ((109°32', ⁇ 18°1 ⁇ ) in the northern South China Sea, with a water depth of 45 meters. Streptomyces sp.
  • the 16S rDNA of SCSIO 01689 was sequenced and its sequence is shown in SEQ ID N0.1. BLAST analysis of the 16S rDNA nucleotide sequence showed that the strain had a similarity with Streptomyces sanyensis of 98.3%, indicating that the strain was SCSIO.
  • 01689 is a genus of Streptomyces. As shown in Fig. 1, the phylogenetic relationship between the strain and the Streptomyces species is clearly revealed by the proximity method, indicating that the bacterium belongs to the genus Streptomyces.
  • the bacterium SCSIO 01689 belongs to Gram-positive, aerobic actinomycetes, white branches of air and differentiate into curly spore chains; spore oval (Fig. 2), 1.5-2.0 ⁇ long, smooth surface. It can hydrolyze starch, Tween 20, 40, 60; gelatin liquefaction, milk coagulation and sputum positive; cellulose decomposition, H 2 S production, Tween 80 hydrolysis, oxidase and nitrate reduction reaction negative. Contact enzyme reaction was positive, melanin production and urease reaction were negative.
  • D-cellobiose D-galactose, D-glucose, D-maltose, D-mannose, D-raffinose, D-ribose, D-sucrose, D-sorbose and xylitol, fructose And D-xylose is the only carbon source and energy source to grow, but cannot use D-arabinose, D-trehalose, inositol, D-mannitol, L-rhamnose, dulcitol and D-lactose.
  • the tolerance ranges for pH, salt concentration and temperature are pH 6.0-9.5, 0-5% and 10-45 °C, respectively.
  • the cell wall contains L-DAP.
  • Phospholipid components are PG, DPG, PE, PI, PIM and Unknown phospholipid PL.
  • the advantages are MK-9 (H 6 ) and MK-9 (H 8 ).
  • the main fatty acids are i-C16:0 and ai-C15:0.
  • the molar content of G+C was 71.8 ( ⁇ 0.3)%.
  • strain Sanyensis GIM 4.003 T has different growth salt concentration, pH, temperature tolerance range, etc., and its unique carbon source utilization, hydrolysis starch, gelatin liquefaction, milk coagulation and deuteration, and antibacterial activity Difference, and genomic hybridization showed a hybridization value of 19% to the most similar strain Streptomyces sanyensis GIMN4.003 T ; well below 70% of the intra-species difference criterion ( Stackebrandt, E. & Ebers, J. Taxonomic parameters revisited: tarnished g Microbiol Today.
  • the Streptomyces sp. SCSIO 01689 of the present invention has fat degradation and protein degradation activity, and is capable of producing lipase and proteolytic enzyme, so the Streptomyces sp. SCSIO 01689 of the present invention can be applied to prepare lipase or protein. Hydrolase, added to aquatic foodstuffs and disease control materials.
  • Streptomyces sp. SCSIO 01689 is capable of producing a novel pyranosesquiterpene compound and Cyclo(D)-Pro-(D)- represented by formula (I).
  • Ile Compound 2
  • Cyclo(D)-Pro-(D)-Leu Compound 3
  • Cyclo(D)-trans-4-OH-Pro-(D)-Phe Compound 4
  • Streptomyces sp. SCSIO 01689 of the present invention is used for preparing a pyranosesquiterpene compound represented by the formula (I), Cyclo(D)-Pro-(D)-Ile, Cyclo. Application of (D)-Pro-(D)-Leu or Cyclo(D)-tranS"4-OH-Pro-(D)-Phe.
  • a second object of the present invention is to provide a novel pyranosesquiterpene compound having the formula (I) as shown in the formula -
  • a third object of the present invention is to provide a pyranosesquiterpene compound represented by formula (I), Cyclo(D)-Pro-(D)-Ile, Cyclo(D)-Pro a method for producing -(D)-Leu or Cyclo(D)-trans-4-OH-Pro-(D)-Phe, characterized in that the pyranosesquiterpene compound, Cyclo ( D)-Pro-(D)-Ile, Cyclo(D)-Pro-(D)-Leu or Cyclo(D)-tranS"4-OH-Pro-(D)-Phe is from Streptomyces sp. Separated from the fermentation culture of SCSIO 01689.
  • the pyranosesquiterpene compound, Cyclo(D)-Pro-(D)-Ile, Cyclo(D) are isolated and prepared from the fermentation culture of Streptomyces spJ SCSIO 01689 by the following method. )-Pro-(D)-Leu or Cyclo(D)-trans-4-OH-Pro-(D)-Phe, the specific steps are as follows:
  • the preparation of the a) step of the fermentation of Streptomyces sp. SCSIO 01689 is preferably prepared by the following method: The activated Streptomyces iStreptomyces sp. SCSIO 01689 is inserted into the seed medium at 28 ° C, 180 rpm The seed liquid was prepared by culturing for 3 days, and the seed liquid was introduced into the fermentation medium at a seeding rate of 5%, and cultured at 28 ° C, 180 rpm for 8 days with shaking to prepare a fermentation culture, and the seed culture was carried out.
  • the formula of the base and fermentation medium is: per liter of medium: 5 g of starch, 5 g of soy flour, 2 g of peptone, 20 g of glucose, 2 g of yeast extract, K 2 HP0 4 0.5 g, MgS0 4 7 H 2 0 0.5g, 30 g of crude sea salt, the balance is water, pH 7.4. Streptomyces iStreptom yces sp.
  • step b) prepared by the method) Pyroosesquiterpene compound, cyclodipeptide compound Cyclo(D)-Pro-(D)-Ile, Cyclo in fermentation culture of SCSIO 01689
  • the content of (D)-Pro-(D)-Leu and Cyclo(D)-trans-4-OH-Pro-(D)-Phe is relatively high.
  • the purification of step b) described above is preferably a column separation.
  • the pyranosesquiterpene compound of the present invention has strong anti-E. coli activity, and its MIC value is 1.58 g/ml, so that pyranosesquiterpene compounds can be applied.
  • anti-E. coli drugs For the preparation of anti-E. coli drugs.
  • the pyranosesquiterpene compound of the present invention has strong anti-halficidal activity, and its LC 5 Q value for Artemia larvae is 3.25 ⁇ / ⁇ 1, so the pyran type is halved. Pyranosesquiterpene compounds can be used to prepare anti-Artemia drugs.
  • the pyranosesquiterpene compound of the present invention has a structure and a unique flavoring compound contained in cooked chicken: pyran-type sesquiterpenoid (8Z)-8-tetradecen-5)lide, (8E) -8-tetrade cen-5-olide and (6E)-6-tetradecen-5-olide (Reference: J. Agric. Food Chem.
  • Pyranosesquiter pene compounds can be used as precursor compounds for the above-mentioned flavoring agents, and are applied to pyridinium sesquiterpenes (8Z 8-tetra decen-5-olide, (8E)-8-tetradecen-5- Preparation of olide or (6E)-6-tetradecen-5-olide flavoring agent.
  • Cyclo(D)-Pro-(D)-Ile, Cyclo(D)-Pro-(D)-Leu and Cyclo(D)-trans-4-OH-Pro-(D)-Phe have antihalogen Insect activity, LC 50 values were 55.67 g/ml, 87.63 g/ml and 13.45 g/ml, respectively. Therefore, Cyclo(D)-Pro-(D)-ne, Cyclo(D)-Pro-(D)-Leu and Cyclo(D)-trans-4-OH-Pro-(D)-Phe can be used to prepare anti-resistance Artemia drugs.
  • the Streptomyces sp. SCSIO 01689 of the present invention is capable of producing a novel Pyranosesquiterpene compound having good activity against Escherichia coli and anti-Artemia, and having a better resistance to Vibrio anguillarum Cyclo(D)-Pro-(D)-Ile, Cyclo(D)-Pr o-(D)-Leu and Cyclo(D)-trans4-OH-Pro-(D)-Phe with active and anti-halficidal activity , thus providing new Cyclo(D)-Pro-(D)-Ile, Cyclo(D)-Pro-(D)-Leu and Cyclo(D)-trans4-OH-Pro-(D)-Phe way.
  • Pyranosesquiterpene compounds can be used in the preparation of anti-E. coli drugs, anti-halogen drugs and precursor compounds as taste agents for the preparation of flavoring agents.
  • Streptomyces iStreptomyces sp. SCSIO 01689 of the present invention was deposited with the China Center for Type Culture Collection (CCTCC) on July 18, 2011, Address: Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 2011257c.
  • Figure 1 is a phylogenetic tree of Streptomyces sp. SCSIO 01689 based on the neighboring method reconstructed by the 16s rDNA sequence;
  • Figure 2 is a scanning electron micrograph of the spore morphology of Streptomyces sp. SCSIO 01689. detailed description:
  • Streptomyces sp. Extraction of genomic DNA involved in the identification of SCSIO 01689, PCR amplification of 16S rDNA, sequence alignment and establishment of phylogenetic tree, and physiology, chemical and morphological identification, etc., all references [Tian, XP, Zhi, XY, Qiu, ⁇ . Q., Zhang, YQ, Tang, SK, Xu, L. H" Zhang, S” Li, WJ Sciscionella marina gen. nov., sp. no v., a marine actinomycete Isolated from a sediment in the northern South China Sea. Int J Syst Evol Microbiol, 2009, 59(Pt 2): 222-228].
  • the Streptomyces sp. SCSIO 01689 of the present invention is from Sanya Bay in the northern part of the South China Sea.
  • the separation medium is 50% R2A medium, containing 0.5 g of peptone per liter, 0.5 g of acid hydrolyzed casein, yeast Extract 0.5 g, glucose 0.5g, soluble starch 0.5g, KH 2 P0 4 0.3g; MgS0 4 7H 2 0 0.05g, agar 15.0 g, natural seawater 500ml, distilled water 500ml, pH 7.2. Separation culture conditions: 28° C, 14 days. The strain SCSIO 01689c was isolated and purified from seafloor sediments.
  • the genomic DNA of the strain SCSIO 01689 was extracted according to the method in the reference or the conventional method, and the 16S rDNA of the strain SCSIO 01689 was routinely PCR-amplified and sequenced, and the sequence thereof is shown in SEQ ID N0.1.
  • BLAST analysis of the 16S rDNA nucleotide sequence showed that the similarity of this strain to Streptomyces sanyensis was 98.3%, indicating that the strain SCSIO 01689 is a Streptomyces genus.
  • the phylogenetic relationship between the strain and the Streptomyces species was clearly revealed by the proximity method, indicating that the strain belongs to the genus Streptomyces.
  • the strain SCSIO 01689 belongs to Gram-positive, aerobic actinomycetes, white-branched and differentiated into curly spore chains; spore oval (Fig. 2), 1.5-2.0 ⁇ m long, smooth surface.
  • Contact enzyme reaction was positive, melanin production and urease reaction were negative.
  • D-cellobiose D-galactose, D-glucose, D-maltose, D-mannose, D-raffinose, D-ribose, D-sucrose, D-sorbose and xylitol, fructose And D-xylose is the only carbon source and energy source to grow, and cannot be grown using D-arabinose, D-trehalose, inositol, D-mannitol, L-rhamnose, dulcitol and D-lactose.
  • the tolerances for pH, salt concentration and temperature were pH 6.0-9.5, 0-5% and 10-45 °C, respectively.
  • the cell wall contains L-DAP.
  • the phospholipid components are PG, DPG, PE, PI, PIM and unknown phospholipid PL.
  • the advantages are MK-9 (H 6 ) and MK-9 (3 ⁇ 4).
  • the main fatty acids are i-C16:0 and ai-C15:0.
  • the G+C molar content was 71.8 ( ⁇ 0.3)%. According to the above analysis of morphology, physiology, chemistry, etc., it has different growth salt concentration, pH, temperature tolerance range, etc., and its unique carbon source utilization, hydrolysis of starch, gelatin liquefaction, milk coagulation and deuteration, and antibacterial The activity and other aspects are significantly different from the known Streptomyces S.
  • Each liter of seed medium is prepared by: 5 g of starch, 5 g of soy flour, 2 g of peptone, 20 g of glucose, 2 g of yeast extract, K 2 HP0 4 0.5 g, MgS0 4 7H 2 0 0.5 g, 30 g of crude sea salt was added to 1 liter of water at pH 7.4. Sterilized at 121 °C for 30 min;
  • Fermentation medium The formula is the same as the seed medium. Sterilize at 121 °C for 30 min.
  • Seed culture A single colony of activated Streptomyces (&re/7to yces sp.) SCSIO 01689 on the culture dish plate was separately connected to 18 bottles, each bottle containing 250 mL of seed culture medium in a 250 mL conical flask. , 28 °C, 180 r min" 1 , cultured for 3 days, 900 mL of seed solution was prepared.
  • Fermentation culture The seed solution was added to 18 L of fermentation medium (in a 500 mL conical flask, each bottle containing 150 ml of fermentation medium, a total of 120 bottles) at a 5% inoculum (volume percentage). °C, ⁇ ⁇ - 1 , cultured for 8 days with shaking, to obtain 18 L of fermentation culture.
  • the fermentation broth of the 18 L volume of the fermentation culture was extracted three times with 2 volumes of ethyl acetate, and the ethyl acetate extract was combined.
  • the ethyl acetate layer was concentrated under reduced pressure to give 13.5 g of the extract.
  • the extract was subjected to column chromatography on silica gel (200-300 mesh) at room temperature, and eluted with a gradient of 100: 0-0: 100 by using chloroform/acetone as an eluent to collect a chloroform/acetone volume ratio of 95:5 and The fractions eluted by a gradient of 60:40 were recorded as fractions Fr.l and Fr.2, respectively.
  • Fr.l chloroform/acetone volume fraction eluted at 95:5
  • LH-20 gel column chromatography eluted with chloroform/methanol volume ratio of 1:1 as mobile phase
  • high pressure liquid eluted with chloroform/methanol volume ratio of 1:1 as mobile phase
  • the high performance liquid chromatograph was semi-prepared Waters 600 (250mmX 10mm, 5 ⁇ , YMC), the mobile phase was methanol/water with a volume ratio of 85:15, and the flow rate was 3ml/min to obtain compound 1 (retention time was 32.4 points).
  • Fraction Fr.2 (fraction eluted at a chloroform/acetone volume ratio of 60:40) was subjected to LH-20 gel column chromatography, eluted with a chloroform/methanol volume ratio of 1:1 as a mobile phase, and then subjected to high pressure liquid chromatography.
  • H-15 is related to C-13/C-14/C-16
  • H-13 is related to C-11/C-12/C-14/C-15
  • H-9 is related to Corresponding to C-7/C-8/C-10/C-11
  • ⁇ ⁇ 4 is related to C-2/C-3/C-5/C-6
  • ⁇ -7 is related to C-5/C-6/C-8/C-9
  • mass spectrometry data the structure is determined to be a new pyr Pyraosesquiterpene compound having the structure shown in formula (I):
  • the data for Compound 2-4 are shown in Table 2-3, in conjunction with the reference (Journal of Natural Products, 2003, 66 (10): 1299-1301. Journal of Natural Products 1995, 58(2): 201-208.
  • Anal Bioanal Chem 2010, 396: 1773 - 1779) the structure of the identified compound 2-4 is shown in the formula (II), and the compound 2-4 is a known compound, that is, Cyclo(D)-Pro-(D)-Ile (compound) 2), Cyclo(D)-Pro-(D-Leu (Compound 3), Cyclo(D)-trans-4-OH-Pro-(D)-Phe (Compound 4).
  • the iH-NMR data was measured at 500 MHz, the coupling constant (Hz) in parentheses, the 13 C-NMR data at 125 MHz, and the nuclear magnetic data measured in deuterated methanol.
  • the 3 ⁇ 4-NMR data was measured at 125 MHz and the nuclear magnetic data was measured in deuterated methanol.
  • Streptomyces SCSIO 01689 was inoculated into various media for testing hydrolase activity, and cultured at 28 degrees for 3-7 days. The presence or absence of the hydrolyzed circle around the colonies on the different hydrolase test media was observed. The ability of hydrolase.
  • the medium and test methods for various hydrolase assays are as follows:
  • Gelatin liquefaction test medium component peptone 5g; glucose 20g; gelatin 200g (boiled in a boiling water bath); pH 7.2; 8 pounds sterilized for 30 minutes.
  • the tube was slanted for later use; it was cultured at 28 degrees for 3-7 days after inoculation, and the liquefaction was observed after placing the refrigerator at 4 degrees for 30 minutes. If it is liquid, it will liquefy. If it is still solid, it will not be liquefied.
  • Proteolytic enzyme test medium KH 2 P0 4 lg, 2 HP0 4 0.5g, skim milk (Difco 232100) 15g, 15 agar powder; 1000ml distilled water, sterilized at 121 °C for 15 min, cooled to 55-60 degrees, poured Tablet spare.
  • the inoculated bacteria were cultured on a plate at 28 °C for 3-7 days to observe the occurrence of the transparent circle. The size of the transparent circle determines the activity of the enzyme, and no transparent circle proves that there is no protease.
  • Starch hydrolysis test medium soluble starch 10g, K 2 HP0 4 0.3g, MgCO 3 lg, NaCl 0.5g, K 03 lg, agar 20g, water 1000ml; pH 7.2-7.4, 121 °C sterilization for 15 min, cooling To 55-60 degrees, pour the plate for use; preparation of iodine solution: 1 g of iodine tablets, 2 g of potassium iodide dissolved in 300 ml of distilled water. The strains were inoculated separately on the starch agar plate by the point connection method. When the strains grew well, the iodine solution was added to the colonies. If amylase is produced, a transparent circle is formed, and the size of the circle indicates the activity of the amylase; if no amylase is produced, the iodine solution is blue in the surrounding part of the colony.
  • Esterase hydrolysis test medium (NH 4 ) 2 S04 2.64g, KH 2 P0 4 2.38g, 2 HP0 4 5.65g, MgS0 4 7H 2 0 l .Og , CuS0 4 5H 2 0 0.0064g, FeS0 4 7H 2 0 0.001 lg, MnCl 2 4H 2 0 0.0079g, ZnS0 4 7H 2 0 0.0015g, agar 20g, respectively, add Tween 20, 40 or 80 10g; water 1000ml, pH 7.2-7.4. 15 lbs for 30 minutes, preparation Tablet spare. The strain was inoculated on a plate by spotting and cultured at 28 degrees for 3-7 days.
  • Streptomyces sp. SCSIO 01689 has a significant hydrolyzed ring for Tween 20 and Tween 40.
  • the hydrolyzed ring diameter is 1.5-2 cm; the protease transparent circle is 1.0-1.3 cm; and gelatin does not produce liquefaction, and There is no amylase hydrolysis circle.
  • Streptomyces sp. SCSIO 01689 has lipid degradation and protein degradation activities, and is capable of producing lipase and proteolytic enzyme properties.
  • the samples were initially tested at 100 ⁇ 8 / piece of each sample and found to have good activity.
  • the MIC value was then tested by the dilution factor method (paper diameter was 6 mm, 6-10 mm was weak activity, 10-15 mm was medium, and 15 mm or more was strong).
  • the screening results are shown in Table 4.
  • Table 5 Compound 1-4 anti-E. coli activity test table
  • the new pyran sesquiterpenoids (Compound 1) have a good inhibitory activity against Escherichia coli, while Cyclo(D)-Pro-(D)-Ile (Compound 2), Cyclo (D)-Pro-(D)-Leu (Compound 3), Cyclo(D)-trans-4-OH-Pro-(D)-Phe (Compound 4) for Escherichia coli with MIC values greater than 100 ⁇ / ⁇ , the inhibitory activity is weak.
  • Artemia larval biological lethal method According to the modified method of Solis, a 96-well cell culture plate was added, and 200 artificial saline solution containing 10-15 Artemia was added per well to prepare a test plate.
  • Compound 1, Compound 2, Compound 3 or Compound 4 was formulated into a 20 mg/ml sample solution with DMSO, followed by dilution.
  • the blank control group and each concentration of the sample group were set up with three parallel holes, the blank control group was added with 5 ⁇ L of sample solvent (DMSO), and the sample group was added with 5 different concentrations of sample solution, and the final concentrations were 500, 50, 5 g/ respectively. mL. After 24 hours of incubation at room temperature, the number of dead individuals in the larvae of Artemia was counted under binocular dissection.
  • Table 6 Compound 1-4 anti-Artemia activity test table
  • the new pyran sesquiterpenoids (Compound 1), Cyclo(D)-Pro-(D)-Ile (Compound 2), Cyclo(D)-Pro-(D)- Both Leu (Compound 3) and Cyclo(D)-trans-4-OH-Pro-(D)-Phe (Compound 4) have good lethal Artemia activity, especially new pyran sesquiterpenoids.
  • Compound 1 its LC 5 . It is 3.25 g/mL and has excellent anti-halficidal activity and can be used to prepare anti-half-based drugs.
  • TGGTCGGTGT ACAGGTGGTG CATGGCTGTC GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA 1020 GTCCCGCAAC GAGCGCAACC CTTGTTCTGT GTTGCCAGCG TGCCCTTCGG GGTGACGGGG 1080 ACTCACAGGA GACTGCCGGG GTCAACTCGG AGGAAGGTGG GGACGACGTC AAGTCATCAT 1140 GCCCCTTATG TCTTGGGCTG CACACGTGCT ACAATGGCCG GTACAATGAG CTGCGATGCC 1200 GCGAGGTGGA GCGAATCTCA AAAAGCCGGT CTCAGTTCGG ATTGGGGTCT GCAACTCGAC 1260 CCCATGAAGT CGGAGTCGCT AGTAATCGCA GATCAGCACT GCTGCGGTGA ATACGTTCCC 1320 GGGCCTTGTA CACACCGCCC GTCACGTCAC GAAAGTCGGT AACACCCGAA GCCGGTGGCC 1380 CAACCCCTTG CGGGAGGAAT CG

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Abstract

提供了一种海洋链霉菌、吡喃型倍半萜类化合物及其制备方法和用途。链霉菌（Streptomyces sp.） SCSIO 01689于2011年7月18日保藏于中国典型培养物保藏中心（CCTCC），保藏编号为CCTCC NO： M 2011257。该链霉菌能够产生新的抗大肠杆菌活性和抗卤虫活性的吡喃型倍半萜类化合物，和具有抗鳗弧菌活性和抗卤虫活性的Cyclo(D)-Pro-(D)-Ile、Cyclo(D)-Pro-(D)-Leu和Cyclo(D)-trans-4-OH-Pro-(D)-Phe，因此为制备Cyclo(D)-Pro-(D)-Ile、Cyclo(D)-Pro-(D)-Leu和Cyclo(D)-trans-4-OH-Pro-(D)-Phe提供了新的途径。该吡喃型倍半萜类化合物可以用于制备抗大肠杆菌药物、抗卤虫药物，还可作为呈味剂的前体化合物应用于呈味剂的制备。

Description

海洋链霉菌、 吡喃型倍半萜类化合物及其制备方法和用途 技术领域：
本发明属于工业微生物领域，具体涉及一种能够产生新的具有独特香味的吡 喃型倍半萜类 （Pyranosesquiterpene ) 化合物和强活性环二肽类化合物 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans-4-OH-Pro-(D)-Phe 的链霉菌 treptomyces spJ SCSIO 01689新种， 以及利用该菌制备吡喃型倍半 萜类 ( Pyranosesquiterpene )化合物和强活性环二肽类化合物 Cyclo(D)-Pro-(D)-Ile， Cyclo(D)-Pro-(D)-Leu, Cyclo(D)-trans-4-OH-Pro-(D)-Phe 的方法， 还有吡喃型倍 半萜类 （Pyranosesquiterpene) 化合物及其用途。 背景技术：
鳗弧菌 ( Vibrio cmguilla m) 是世界范围内流行的水产养殖动物病原菌， 可感染鲑鱼、 虹鳟、 鳗鲡、 香鱼、 鲈鱼、 鳕鱼、 大菱鲆、 牙鲆、 黄鱼等 （高 冬梅，李健，王群.鳗弧菌灭活疫苗对牙鲆免疫效果的研究.海洋水产研究， 2004， 25(l):486-492) o 部分感染鳗弧菌的鱼体表最初出现局部褪色， 鳍条、 鳍基部 及鳃骨下部充血发红， ***红肿， 继而肌肉组织有弥撒性或点状出血， 体表 发黑， 鳍部出现溃烂； 解剖检验时有明显的黄色粘稠腹水， 肠粘膜组织腐烂 脱落， 部分鱼肝脏坏死 （肖慧,李军，徐怀恕，等. 鲈鱼苗烂鳃、 烂尾病病原的 研究. 青岛海洋大学报， 1999，29(1):89)。 鳗弧菌是水产养殖中的重要病害。
环肽类化合物 Cyclo(D)-Pra-(D)-Ile (化合物 2)、 Cyclo(D)-Pro-(D)-Leu (化合物 3)和 Cyclo(D)-trans4-OH-Pro-(D)-Phe (化合物 4)为已知化合物， 其结构式如式 ( II )所示， 公开于以下文献 1、 Journal of Natural Products, 2003, 66 ( 10)： 1299-1301。 2、 Journal of Natural Products 1995， 58 (2)： 201 -208。 3、 Anal Bioanal Chem 2010, 396:1773― 1779。并在 Fdhila F et al,. Journal of Natural Products, 2003, 66 ( 10 ) ： 1299-1301 中 公 开 Cyclo(D)-Pro-(D)-Ile(化 合 物 2) 、 Cyclo(D)-Pro-(D)-Leu (化合物 3)和 Cyclo(D)-tranS"4-OH-Pro-(D)-Phe (化合物 4)对 鳗弧菌具有很好杀菌活性。
Figure imgf000004_0001
式 （11 )。 发明内容：
本发明的第一个目的是提供一种链霉菌 Streptomyces sp. ) SCSIO 01689, 该菌于 2011年 7月 18日保藏于中国典型培养物保藏中心（CCTCC), 地址： 中 国武汉市武汉大学， 保藏编号为： CCTCC NO: M 2011257c
本发明的链霉菌 (Streptomyces sp. SCSIO 01689是从我国南海北部三亚湾 ((Ε109°32', Ν18°1Γ)水深 45米的海底沉积物中分离获得的。 常规 PCR扩增链霉 菌 Streptomyces sp. ) SCSIO 01689的 16S rDNA并测序，其序列如 SEQ ID N0.1 所示。 对 16S rDNA 核苷酸序列进行 BLAST 分析， 结果显示， 该菌株与 Streptomyces sanyensis的相似度为 98.3%， 说明该菌株 SCSIO 01689为链霉菌菌 属。如图 1所示，通过邻接法清晰地揭示了该菌株与链霉菌属物种的***进化关 系， 表明该菌属于链霉菌属的一种。
形态学特征和生理生化分析：
该菌 SCSIO 01689属于革兰氏阳性， 好氧放线菌， 气丝白色分支并分化成 卷曲的孢子链； 孢子椭圆形 （图 2), 长 1.5-2.0μηι， 表面光滑。 能水解淀粉、 吐 温 20, 40， 60； 明胶液化、 牛奶凝固和胴化阳性； 纤维素分解， H2S 产生， 吐 温 80水解， 氧化酶和硝酸盐还原反应阴性。 接触酶反应阳性， 黑色素产生和脲 酶反应阴性。 可以利用 D-纤维二糖， D-半乳糖， D-葡萄糖， D-麦芽糖， D-甘露 糖， D-棉子糖， D-核糖， D-蔗糖， D-山梨糖和木糖醇， 果糖和 D-木糖为唯一碳 源和能源生长， 而不能利用 D-***糖， D-海藻糖， 肌醇， D-甘露醇， L-鼠李 糖， 卫矛醇和 D-乳糖生长。 pH、 盐浓度和温度的耐受范围分别为 pH 6.0-9.5， 0 -5%和 10-45 °C。 细胞壁含有 L-DAP。 磷脂组分为 PG， DPG, PE， PI, PIM和 未知磷脂 PL。优势醌为 MK-9(H6)和 MK-9(H8)。主要脂肪酸为 i-C16:0和 ai-C15: 0。 G+C摩尔含量为 71.8(士0.3) %。 根据以上形态学、 生理学、 化学等分析， 其 与已知最接近的菌株
Figure imgf000005_0001
sanyensis GIM 4.003T, 在生长盐浓度、 pH, 耐受温度范围等有不同， 且其唯一碳源的利用、 及水解淀粉、 明胶液化、 牛奶凝 固和胴化， 以及抗菌活性等方面有较大的差异， 且基因组杂交显示其与最相似菌 株 Streptomyces sanyensis GIMN4.003T的杂交值为 19%;远低于种内差异标准的 70% ( Stackebrandt, E. & Ebers, J. Taxonomic parameters revisited: tarnished g old standards. Microbiol Today. (2006).33, 152-155.)； 因此， 综合分析多项分类 数据， 鉴定此菌株为链霉菌属 Streptomyces 的一个新种， 将此菌命名为： 链 霉菌 iStreptomyces sp. ) SCSIO 01689, 该菌于 2011年 7月 18日保藏于中国典 型培养物保藏中心 （CCTCC) , 地址： 中国武汉市武汉大学， 保藏编号为： CCT CC NO: M 2011257。
本发明的链霉菌 (Streptomyces sp. SCSIO 01689具有脂肪降解和蛋白降解 的活性， 能够产生脂肪酶和蛋白水解酶， 因此本发明的链霉菌（Streptomyces sp. ) SCSIO 01689可以应用于制备脂肪酶或蛋白水解酶，添加于水产食料和病害防治 原料中。
本发明人发现链霉菌 (Streptomyces sp. ) SCSIO 01689能够产生一种新的如 式 （ I ) 所示的吡喃型倍半萜类 （ Pyranosesquiterpene ) 化合物和 Cyclo(D)-Pro-(D)-Ile(化合物 2) ， Cyclo(D)-Pro-(D)-Leu(化 合物 3) ， Cyclo(D)-trans-4-OH-Pro-(D)-Phe (化合物 4)。
因此本发明的链霉菌 (Streptomyces sp. ) SCSIO 01689用于制备如式 （ I ) 所示的吡喃型倍半萜类 （Pyranosesquiterpene ) 化合物、 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu或 Cyclo(D)-tranS"4-OH-Pro-(D)-Phe的应用。
本发明的第二个目的是提供一个新的吡喃型倍半萜类 （Pyranosesquiterpene) 化 合物， 其结构式如式 （ I ) 所示-
Figure imgf000005_0002
式 （ I )。
本发明的第三个目的是提供一种如式 （ I ) 所示的吡喃型倍半萜类 ( Pyranosesquiterpene ) 化合物、 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu或 Cyclo(D)-trans-4-OH-Pro-(D)-Phe 的制备方法， 其特征在于， 所述的吡喃型倍半 萜类（Pyranosesquiterpene)化合物、 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu 或 Cyclo(D)-tranS"4-OH-Pro-(D)- Phe是从链霉菌 Streptomyces sp. ) SCSIO 01689 的发酵培养物中制备分离得到的。
优选， 是通过以下方法从链霉菌 (Streptomyces spJ SCSIO 01689的发酵培 养物中制备分离得到吡喃型倍半萜类 （Pyranosesquiterpene ) 化合物、 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu或 Cyclo(D)-trans-4-OH-Pro-(D)-Phe， 具体步骤如下：
a) 制备链霉菌（Streptomyces sp. ) SCSIO 01689的发酵培养物， 将该发酵培 养物的发酵液用乙酸乙酯萃取， 乙酸乙酯经蒸馏浓缩后得到浸膏； b) 将浸膏经硅胶柱层析，用氯仿 /丙酮作为洗脱剂，从体积比 100: 0-0： 100 进行梯度洗脱， 收集氯仿 /丙酮体积比 95:5和体积比 60: 40梯度洗脱下 来的馏分分别为 Fr. l和 Fr.2， 镏分 Fr.l经过凝胶柱层析得到粗品， 经纯 化， 得到吡喃型倍半萜类（Pyranosesquiterpene)化合物； 馏分 Fr.2经过 凝胶柱层析得到粗品， 经纯化， 得到 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans4-OH-Pro-(D)-Phe。
所述的 a) 步骤的制备链霉菌 Streptomyces sp. ) SCSIO 01689的发酵培养 物优选通过以下方法制备： 将活化的链霉菌 iStreptomyces sp. ) SCSIO 01689接 入种子培养基中， 28°C， 180 rpm， 培养 3天制得种子液， 将种子液以 5%的接 种量接入到发酵培养基中， 28 °C， 180 rpm, 振荡培养 8 天， 而制得发酵培养 物， 所述的种子培养基和发酵培养基的配方都为每升培养基中含有： 淀粉 5 g， 大豆粉 5 g, 蛋白胨 2 g， 葡萄糖 20 g， 酵母膏 2 g， K2HP04 0.5g， MgS04 7 H20 0.5g， 粗海盐 30 g, 余量为水， pH 7.4。 经本方法制备的链霉菌 iStreptom yces sp. ) SCSIO 01689的发酵培养物中吡喃型倍半萜类 （Pyranosesquiterpene ) 化合物、环二肽类化合物 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D) -trans-4-OH-Pro-(D)-Phe的含量比较高。 所述的步骤 b) 的纯化优选都为色谱柱分离。
经实验发现， 本发明的吡喃型倍半萜类 （Pyranosesquiterpene)化合物具有 较强的抗大肠杆菌活性， 其 MIC 值为 1.58 g/ml， 因此吡喃型倍半萜类 ( Pyranosesquiterpene)化合物可以应用于制备抗大肠杆菌药物。
经实验发现， 本发明的吡喃型倍半萜类（Pyranosesquiterpene)化合物具有 较强的抗卤虫活性， 其对卤虫幼体的 LC5Q值为 3.25μ§/πι1， 因此吡喃型倍半萜类 ( Pyranosesquiterpene)化合物可以应用于制备抗卤虫药物。
本发明的吡喃型倍半萜类（Pyranosesquiterpene)化合物， 其结构与煮熟的 鸡肉中含有的独特香味素： 吡喃型倍半萜 (8Z)-8-tetradecen-5 )lide, (8E)-8-tetrade cen-5-olide和 (6E)-6-tetradecen-5-olide (参考文献： J. Agric. Food Chem. 2011, 59, 11752-11763 ) 的结构类似， 因此本发明的吡喃型倍半萜类（ Pyranosesquiter pene)化合物可作为上述呈味剂的前体化合物， 应用于吡喃型倍半萜 (8Z 8-tetra decen-5-olide, (8E)-8-tetradecen-5-olide或 (6E)-6-tetradecen-5-olide呈味剂的制备。
经实验发现 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans-4- OH-Pro-(D)-Phe具有抗卤虫活性， 其 LC50值分别为 55.67 g/ml、 87.63 g/ml和 1 3.45 g/ml。 因此 Cyclo(D)-Pro-(D)-ne、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans-4- OH-Pro-(D)-Phe可以应用于制备抗卤虫药物。
本发明的链霉菌 Streptomyces sp. ) SCSIO 01689能够产生新的具有较好的 抗大肠杆菌活性和抗卤虫活性的吡喃型倍半萜类（Pyranosesquiterpene)化合物， 和具有较好的抗鳗弧菌活性和抗卤虫活性的 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pr o-(D)-Leu和 Cyclo(D)-trans4-OH-Pro-(D)-Phe，因此为制备 Cyclo(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans4-OH-Pro-(D)-Phe提供了新的途径。吡喃 型倍半萜类（Pyranosesquiterpene)化合物可以用于制备抗大肠杆菌药物、 抗卤 虫药物和作为呈味剂的前体化合物应用于呈味剂的制备。
本发明的链霉菌 iStreptomyces sp. ) SCSIO 01689于 2011年 7月 18日保藏 于中国典型培养物保藏中心（CCTCC), 地址： 中国武汉市武汉大学， 保藏编号 为 CCTCC NO: M 2011257c 附图说明: 图 1是链霉菌 Streptomyces sp. ) SCSIO 01689基于 16s rDNA序列的邻接法重建 的与之亲缘关系最近的种之间关系的***发育树；
图 2是链霉菌 (Streptomyces sp. ) SCSIO 01689的孢子形态扫描电镜图。 具体实施方式：
以下实施例是对本发明的进一步说明， 而不是对本发明的限制。
实施例 1 :
一、链霉菌 "treptomyces sp. ) SCSIO 01689的分离和鉴定
链霉菌 Streptomyces sp. ) SCSIO 01689鉴定中涉及的基因组 DNA的提取， 16S rDNA的 PCR扩增， 序列比对和***进化树的建立方法以及生理学、 化学 和形态学鉴定等， 均参考文献 [Tian, X. P., Zhi, X. Y.， Qiu, Υ. Q.， Zhang, Y. Q.， Tang, S. K., Xu, L. H" Zhang, S" Li, W. J. Sciscionella marina gen. nov.， sp. no v., a marine actinomycete isolated from a sediment in the northern South China Sea. Int J Syst Evol Microbiol, 2009， 59(Pt 2): 222-228]。
本发明的链霉菌 Streptomyces sp. ) SCSIO 01689是从我国南海北部三亚湾
((Ε109°32', Ν18°1 Γ)水深 45米的海底沉积物中分离获得的。 分离培养基为 50% 的 R2A培养基， 每升含有蛋白胨 0.5 g, 酸水解干酪素 0.5 g， 酵母浸膏 0.5 g， 葡萄糖 0.5g, 可溶性淀粉 0.5g, KH2P04 0.3g; MgS04 7H20 0.05g, 琼脂 15.0 g， 天然海水 500ml, 蒸馏水 500ml, pH 7.2。 分离培养条件为： 28°C， 14天。 由此 从海底沉积物分离纯化得到菌株 SCSIO 01689c
按照参考文献中的方法或者常规方法提取该菌株 SCSIO 01689 的基因组 DNA, 常规 PCR扩增菌株 SCSIO 01689的 16S rDNA并测序， 其序列如 SEQ ID N0.1所示。 对 16S rDNA核苷酸序列进行 BLAST分析， 结果显示， 该菌株与 Streptomyces sanyensis的相似度为 98.3%， 说明该菌株 SCSIO 01689为链霉菌菌 属。如图 1所示，通过邻接法清晰地揭示了该菌株与链霉菌属物种的***进化关 系， 表明该菌属于链霉菌属的一种。
形态学特征和生理生化分析：
该菌株 SCSIO 01689属于革兰氏阳性，好氧放线菌，气丝白色分支并分化成 卷曲的孢子链； 孢子椭圆形 （图 2)， 长 1.5-2.0μιη， 表面光滑。 能水解淀粉、 吐 温 20, 40, 60； 明胶液化、 牛奶凝固和胴化阳性； 纤维素分解， ¾S 产生， 吐 温 80水解， 氧化酶和硝酸盐还原反应阴性。 接触酶反应阳性， 黑色素产生和脲 酶反应阴性。 可以利用 D-纤维二糖， D-半乳糖， D-葡萄糖， D-麦芽糖， D-甘露 糖， D-棉子糖， D-核糖， D-蔗糖， D-山梨糖和木糖醇， 果糖和 D-木糖为唯一碳 源和能源生长， 而不能利用 D-***糖， D-海藻糖， 肌醇， D-甘露醇， L-鼠李 糖，卫矛醇和 D-乳糖生长。 pH、盐浓度和温度的耐受范围分别为 pH 6.0-9.5 , 0-5% 和 10-45 °C。 细胞壁含有 L-DAP。 磷脂组分为 PG， DPG, PE， PI, PIM和未知 磷脂 PL。 优势醌为 MK-9(H6)和 MK-9(¾)。 主要脂肪酸为 i-C16:0和 ai-C15:0。 G+C摩尔含量为 71.8(±0.3) %。根据以上形态学、 生理学、化学等分析， 其在生 长盐浓度、 pH， 耐受温度范围等有不同， 且其唯一碳源的利用、 及水解淀粉、 明胶液化、 牛奶凝固和胴化， 以及抗菌活性等方面与已知链霉菌 S. sanyensis GIM 4.0031 "有较大的差异， 且基因组杂交显示其与最相似菌株 S. sanyensis GIMN4.003T的杂交值为 19%; 远低于种内差异标准的 70% ( Stackebrandt, E. & Ebers, J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today.(2006).33, 152-155.)； 因此， 综合分析多项分类数据， 鉴定此菌株为链霉 菌属 (Streptomyces~) 的一个新种， 并将此菌命名为： 链霉菌 (Streptomyces sp. SCSIO 01689 , 该菌于 2011 年 7 月 18 日保藏于中国典型培养物保藏中心 ( CCTCC) , 地址： 中国武汉市武汉大学， 保藏编号为 CCTCC NO: M 2011257c 实施例 2:
一、 化合物的分离、 制备和解析
1、 培养基
A、 每升种子培养基的配制方法为： 将淀粉 5 g, 大豆粉 5 g， 蛋白胨 2 g， 葡萄糖 20 g， 酵母膏 2 g, K2HP04 0.5g, MgS04 7H20 0.5g, 粗海盐 30 g加 入到 1升水中， pH 7.4。 121 °C， 灭菌 30 min;
B、 发酵培养基： 配方同种子培养基。 121 °C , 灭菌 30 min。
2、 发酵
2.1、种子培养：将培养皿平板上活化的链霉菌（&re/7to yces sp. ) SCSIO 01689 的单菌落分别接入 18瓶， 每瓶含有 50 mL种子培养基的 250 mL的锥形培养瓶 中， 28 °C， 180 r min"1 , 培养 3天， 制得种子液 900 mL。 2.2、 发酵培养： 将种子液以 5%的接种量（体积百分比）接入到 18L发酵培 养基（置于 500mL的锥形培养瓶， 每瓶含有 150ml发酵培养基， 共 120瓶） 中， 28 °C, Ιδθ ηηΐη-1 , 振荡培养 8天， 得到 18L发酵培养物。
3、 萃取
将 18L体积的发酵培养物的发酵液用 2倍体积的乙酸乙酯进行萃取 3次，合 并乙酸乙酯萃取液， 乙酸乙酯层减压蒸馏浓缩得浸膏 13.5g。
4、 化合物的分离和制备
将浸膏经硅胶（200~300目）常温柱层析， 用氯仿 /丙酮作为洗脱剂， 从体积 比 100： 0-0： 100进行梯度洗脱， 收集氯仿 /丙酮体积比 95:5和体积比 60： 40 梯度洗脱下来的熘分， 分别记为馏分 Fr.l和 Fr.2。
熘分 Fr.l (氯仿 /丙酮体积比 95:5洗脱下来的熘分） 经 LH-20凝胶柱层析， 以氯仿 /甲醇体积比 1 :1作为流动相洗脱，再经高压液相色谱制备， 高效液相色谱 仪为半制备型 Waters 600(250mmX 10mm, 5μιη, YMC), 流动相为体积比 85: 15 的甲醇 /水， 流速为 3ml/分， 得到化合物 1 (保留时间为 32.4分)。
馏分 Fr.2 (氯仿 /丙酮体积比 60:40洗脱下来的馏分） 经 LH-20凝胶柱层析， 以氯仿 /甲醇体积比 1 : 1作为流动相洗脱，再经高压液相色谱制备，高效液相色谱 仪为半制备型 Waters 600(250mm X 10mm, 5μιη, YMC)],当流动相为体积比为 15 : 85的甲醇 /水时， 流速为 3ml/分， 得到化合物 2 (保留时间为 27.6分)； 当流动相 为体积比为 25 : 75 的甲醇 /水时， 流速为 3ml/分， 得到化合物 3 (保留时间为 24.4min )和化合物 4 (保留时间为 31.9min )。
5、 化合物的解析
通过结构分析， 化合物 1、 2、 3和 4的鉴定结果如下：
化合物 1 : 无色油状（甲醇）， UV (甲醇 )：215 , 223.0 inn. 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD)， 见表 1。 ESIMS mJz 259.1 [M+Na] +， 237.2 [M + H]+ , 473.4 [2M + H]+。 从氢谱和碳谱上能看到化合物 1含有 1个甲基 [δΗ 2.16 (t, 7=7.0 Hz, H-16) 6C 14.4 (q, C-16)]， 8个次甲基 [δΗ 2.85 (d, 7=6.5 Hz, H-3), 6c 30.3 (t, C-3); δΗ 2.29 (dt, 7=7.0 6.5 Hz, H-7), 6C 33.0 (t, C-7); δΗ 2.30 (dt, J= 6.5 5.5 Hz, H-8), 6C 38.4 (t， C-8); 6H 2.28 (dt, 7=11.0 6.0 Hz, H-l l), 8C 30.7 (t, C-l l); δΗ 2.18 (m, H-12), 8C 28.6 (t, C-12); δΗ 2.18 (m, H-13)„ 5C 26.3 (t, C-13); 6H 2.18 (m， H-14)„ 5C 26.2 (t, C-14); δΗ 2.22 (m, H-15)„ 6C 23.7 (t， C-15)], 5个亚甲基 [SH 6.52 (dd, J-11.0 6.5 Hz, H-4), 5C 126.6 (d, C-4); δΗ 6.00 (t, 7=11.0 6.0 Hz, H-5), 5c 129.4 (d, C-5); δΗ 4.10 (dd, 7=7.0 6.0 Hz, H-6), 5C 73.4 (d， C-6); δΗ 5.64 (dt, 7=15.0 5,5 Hz, H-9), 6c 137.4 (d, C-9); 6H 5.45 (dt, J=15.0 11.0 Hz, H-10),5C 132.4 (d, C-10)] 和 1个 季碳 [Sc 170.1 (s， C-2)]。 根据 HMBC相关谱可看到， H-15与 C-13/C-14/ C-16相 关， H-13与 C-11/C-12/C-14/C-15相关， H-9与 C-7/C-8/C-10/C-11相关， 可得出 具有一个单萜结构； Η·4与 C-2/C-3/C-5/C-6相关， 推测该化合物可能含有吡喃 环， Η-7与 C-5/C-6/C-8/C-9相关， 推测该吡喃环与单萜相连接； 结合质谱数据， 确定该结构为一个新的吡喃型倍半萜类 （Pyranosesquiterpene )化合物， 其结构 如式 （I)所示：
Figure imgf000011_0001
式 （I)
化合物 2-4的数据见表 2-3 ,结合参考文献（Journal of Natural Products, 2003, 66 ( 10)： 1299-1301。 Journal of Natural Products 1995， 58 ( 2)： 201 -208。 Anal Bioanal Chem 2010, 396: 1773― 1779), 鉴定化合物 2-4的结构如式 ( II )所示， 解析化合物 2-4为已知化合物， 即为 Cyclo(D)-Pro-(D)-Ile (化合物 2)， Cyclo(D)-Pro-(D -Leu (化合物 3), Cyclo(D)-trans-4-OH-Pro-(D)-Phe (化合物 4)。
Figure imgf000011_0002
表 1 : 化合物 1的氢谱和碳谱核磁数据归属
化合物 1
No.
H C
2 170.1 s
3 2.85 (d, 6.5) 30.3 t
4 6.52(dd, 11.0,6.5) 126.6 d
5 6.00(t, 11.0,6.0) 129.4 d
6 4.10(dd，7.0,6.0) 73.4 d
7 2.29 (t, 7.0,6.5) 33.01
8 2.30(dt, 6.5,5.5) 38.41
9 5.64(dt 15.0,5.5) 137.4 d
10 5.45(dt, 15.0,11-0) 132.4 d
11 2.28(dt,l 1.0,6.0) 30.71
12 2.18(m) 28.61
13 2.18(m) 26.3 t
14 2.18(m) 26.21
15 2.22(m) 23.71
16 2.16(t,7.0) 14.4 q
注： iH-NMR数据于 500MHz测定， 括号中为偶合常数 (Hz), 13C-NMR数据于 125MHz测定， 核磁数据在 氘代甲醇中测得。
表 2: 化合物 2·4的氢谱核磁数据归属
2 3 4
No.
H-NMR13 H- MR13 H-NMR13
1 6.20(s) 6.20 (s) 6.21(s)
2 3.75(t,4.5) 3.73(t,4.5) 3.78(4.5)
3 2.05(m),2.42(m) 2.24(m)， 2.40(m) 2.05(m),2.40(m)
4 1.93 (2H,m) 1.90 (2H,m) 1.91 (2H，m)
5 3.69(t,6.5),4.08(t,6.5) 3.69(t,6.5),4.09(t,6.5) 3.69(t,6.5),4.09(t,6.5)
6 3.48(d,5.0) 3.52(d,5.0) 3.50(d,6.0)
7 4.32(dd,l 1.5,5.0) 2.06(m) 1.98(m)
8 0.99(d,7.0) 1.88(2H, m)
9 7.22(d,7.0) 0.99(dJ.O) 0.93(t, 7.5)
10 7.35(dd,7.0J.5) 1.02(d, 7.0)
11 7.29(dd,7.0,7.5)
12 7.35(dd,7.0,7.5)
13 7.22(47.0)
注： 'H-NMR数据于 500MHz测定， 括号中为偶合常数 (Hz)， 核磁数据在氘代甲醇中测得。 表 3 : 化合物 2-4的碳谱核磁数据归属
2 3 4
No.
C-NMR13 C-NMR13 C-NMR13
1. 169.7 169.3 169.3
2. 63.5 63.6 62.9
3. 29.4 29.4 29.4
4. 22.1 22.0 22.0
5. 45.6 45.6 45.6
6. 165.2 165.2 165.2
7. 57.7 58.3 58.4
8. 68.3 33.1 24.5
9. 135.8 17.5 39.7
10. 129.1 19.0 11.3
11. 129.3 15.3
12. 127.6
13. 129.3
14. 129.1
¾-NMR数据于 125MHz测定， 核磁数据在氘代甲醇中测得。
实施例 3: 活性测试
1、 菌株酶解活性测试
将链霉菌 Streptomyces SCSIO 01689接种于测试水解酶活性的各种培 养基， 28度培养 3-7天，分别观察不同水解酶测试培养基上菌落周围水解圈有无 和大小，具体判断该菌株产对应水解酶的能力。各种水解酶测定的培养基及测试 方法如下：
明胶液化试验培养基组份： 蛋白胨 5g; 葡萄糖 20g; 明胶 200g (沸水浴加 热溶解)； pH 7.2; 8磅灭菌 30分钟制备。管斜面备用；接菌后 28度培养 3-7天， 放置 4度冰箱 30min后观察液化情况。 如果是液体， 则液化， 如果仍然是固体， 则无液化。
蛋白水解酶试验培养基： KH2P04 lg， 2HP04 0.5g, skim milk (Difco 232100) 15g, 15 琼脂粉； 1000ml蒸馏水， 121 °C灭菌 15 min， 冷却至 55-60度， 倒平 板备用。 接种细菌于平板上于 28度培养 3-7天观察透明圈产生情况， 透明圈大 小决定酶活情况， 无透明圈证明无蛋白酶。
淀粉水解试验培养基：可溶性淀粉 10g， K2HP04 0.3g， MgC03 lg, NaCl 0.5g， K 03 lg, 琼脂 20g, 水 1000ml; pH7.2-7.4, 121 °C灭菌 15 min, 冷却至 55-60 度， 倒平板备用； 碘液的制备： 碘片 1克， 碘化钾 2克溶于蒸馏水 300ml中. 采 用点接法将菌株分别接种于淀粉琼脂平板上，待菌种生长良好时，在菌落周围滴 加碘液检测。 如有淀粉酶产生， 形成透明圈， 圈的大小表示淀粉酶活性强弱； 如 不产生淀粉酶， 则菌落周围部位遇到碘液呈蓝色。
酯酶水解试验培养基： （NH4)2S04 2.64g, KH2P04 2.38g， 2HP04 5.65g, MgS047H20 l .Og , CuS045H20 0.0064g， FeS047H20 0.001 lg， MnCl2 4H20 0.0079g， ZnS047H20 0.0015g, 琼脂 20g， 分别添加吐温 20， 40或 80 10g; 水 1000ml, pH 7.2-7.4. 15磅灭菌 30分钟， 制备平板备用。 采用点接法将菌株接种 于平板上， 28度培养 3-7天。
实验结果： 链霉菌 Streptomyces sp. ) SCSIO 01689对吐温 20， 吐温 40有 明显的水解圈， 水解圈直径在 1.5-2cm;蛋白酶透明圈 1.0-1.3cm;而对明胶不产生 液化现象， 并没有淀粉酶水解圈。 由此可见， 链霉菌 (Streptomyces sp. ) SCSIO 01689具有脂类降解和蛋白降解的活性， 能够产生脂肪酶和蛋白水解酶的特性。
2、 化合物活性测试
2.1、 抗鳗弧菌活性测试
实验方法简述： 1.让鳗弧菌在常规培养条件生长备用； 2.把被测试样品 （化 合物 1、化合物 2、化合物 3和化合物 4)的用 DMSO作溶剂稀释至浓度为 20 mg/mL， 用枪头吸 5μ1加入到 6 mm直径的纸片上； 3.将培养好的鳗弧菌均匀的涂布于盛有 LB培养基 （胰蛋白胨 10g/L， 酵母提取物 5g/L， 氯化钠 10g/L， pH值 7.4， 1.2%琼 脂粉， 去离子水 1000ml ) 的 10 cm直径培养皿中于 37度培养； 4.将带有测试样品 的纸片放入培养皿中的平板上并编号， 24小时后观察。 5. 初筛后， 稀释 2、 4、 8、 16倍， 用同样的方法进行测试并计算 MIC值。
筛选结果
在每个样品 100μ8/片情况下初步测试样品，发现具有较好活性。然后采用稀 释倍数法测试 MIC值 （纸片直径为 6mm, 6-10mm为弱活性， 10-15mm为中等， 15mm以上为强活性） 。 筛选结果如表 4所示。
表 4: 化合物 1、 2、 3、 4对鳗弧菌 ( Vibrio anguiUarum) 的抗性分析表
Figure imgf000014_0001
化合物 2、 3、 4对 MIC值参见 Fdhila F et al,. Journal of Natural Products, 2003, 66 ( 10)： 1299-1301 从表 4可以看出 Cyclo(D)-Pro-(D)-Ile (化合物 2)， Cyclo(D)-Pro-(D)-Leu (化合 物 3)， Cyclo(D)-trans-4-OH-Pro-(D)-Phe (化合物 4)对鳗弧菌具有较好的抑制活性， 而新的吡喃型倍半萜化合物 （化合物 1 ) 对鳗弧菌， 其 MIC值大于 10(^g/mL， 抑制活性较弱。
2.2、 抗大肠杆菌活性测试
实验方法简述： 1.让大肠杆菌在常规培养条件下生长备用； 2.把被测试样品 (化合物 1、 化合物 2、 化合物 3和化合物 4) 的用 DMSO作溶剂稀释至浓度为 20 mg/mL,用枪头吸 5μ1加入到 6 mm直径的纸片上； 3.将培养好的大肠杆菌均勾的涂 布于盛有 LB培养基的 10 cm直径培养皿中于 37度培养； 4.将带有测试样品的纸片 放入培养皿中的平板上并编号， 24小时后观察。 5.将利富平用 DMSO作溶剂稀释 为 0.2 mg/mL， 做为阳性对照按照同样的方法进行处理。 6. 初筛后， 稀释 2、 4、 8、 16倍， 用同样的方法进行测试并计算 MIC值。
筛选结果：
在每个样品 ιοομΕ/片，利福平做标准品 1μ8/片的情况下，初步测试样品并与阳性 对照利福平进行比较。 然后采用稀释倍数法测试 MIC值 （纸片直径为 6nmi， 6-10mm为弱活性， 10-15mm为中等， 15mm以上为强活性） ， 筛选结果如表 5 所示：
表 5: 化合物 1-4抗大肠杆菌活性测试表
Figure imgf000015_0001
由表 5可以看出， 新的吡喃型倍半萜类化合物 （化合物 1 )对大肠杆菌具有 较好的抑制活性， 而 Cyclo(D)-Pro-(D)-Ile (化合物 2)， Cyclo(D)-Pro-(D)-Leu (化合 物 3)， Cyclo(D)-trans-4-OH-Pro-(D)-Phe (化合物 4)对大肠杆菌，其 MIC值大于 1 00μΒ/ηΛ, 抑制活性较弱。
2.3、 卤虫幼体致死试验
实验方法简述：
卤虫幼体的孵化： 取卤虫卵 100 mg置于 500 mL烧杯中， 加入人工海水 400 mL， 用一小充气泵缓缓充气， 室温孵化 24 h， 除去卵壳及未孵化的卵， 卤虫幼 体继续培养 24 h， 备用。
卤虫幼体生物致死法： 依照 Solis的改良法， 取 96孔细胞培养板， 每孔加 200 含 10-15个卤虫的人工海水液， 制成测试培养板。 用 DMSO将化合物 1、 化合物 2、 化合物 3或化合物 4配成 20mg/ml的样品液， 然后进行稀释。 空白对 照组和每个浓度的样品组各设三个平行孔， 空白对照组加 5 μL 样品溶剂 (DMSO), 样品组加 5 不同浓度的样品液， 终浓度分别为 500、 50、 5 g/mL。 室温培养 24 h后， 在双目解剖镜下检测计数卤虫幼体死亡个体数目。
卤虫幼体生物致死活性用校正死亡率表示， 按下列公式计算- 校正死亡率 =(对照组存活率一处理组存活率 )/对照组存活率 χ ΐοο%
将三个浓度及其对应的校正死亡率输入计算机， 计算 LC5o及其 95%置信区 间。
筛选结果如表 6所示：
表 6: 化合物 1·4抗卤虫活性测试表
Figure imgf000016_0001
从表 6 可以看出， 新的吡喃型倍半萜类化合物 （化合物 1 )、 Cyclo(D)-Pro-(D)-Ile(化合 物 2) ， Cyclo(D)-Pro-(D)-Leu(化 合物 3)和 Cyclo(D)-trans-4-OH-Pro-(D)-Phe (化合物 4)都具有较好的致死卤虫活性， 特别是 新的吡喃型倍半萜类化合物 （化合物 1 ), 其 LC5。为 3.25 g/mL， 抗卤虫活性非 常好， 可以用于制备抗卤虫药物。
序列表
<110>中国科学院南海海洋研究所
<120〉海洋链霉菌、 吡喃型倍半萜类化合物及其制备方法和用途
<160>1
<210>1
<211>1419
<212>DNA
<213>链霉菌 (Streptomyces sp. SCSIO 01689
<400>1
GGCTCTACCA TGCAGTCGAC GATGAACCTC CTCCGGGAGG GGAAGAGTGG CGAACGGGTG 60
AGTAACACGT GGGCAATCTG CCCTGCACTC TGGGATAACT CCGGGAAACC GGAGCTAATA 120
CCGGATACGA CTCTCCAGGG CATCCTGGGG GGTGGAAAGC TCCGGCGGTG CAGGATGAGC 180
CCGCGGCCTA TCAGCTGGTT GGTGGGGTGA CGGCCCACCA AGGCGACGAC GGGTAGCCGG 240
CCTGAGAGGG CGACCGGCCA CACTGGGACT GAGACACGGC CCAGACTCCT ACGGGAGGCA 300
GCAGTGGGGA ATATTGCACA ATGGGCGCAA GCCTGATGCA GCGACGCCGC GTGAGGGATG 360
ACGGCCTTCG GGTTGTAAAC CTCTTTCAGC AGGGAAGAAG CGGAAGTGAC GGTACCTGCA 420
GAAGAAGCAC CGGCTAACTA CGTGCCAGCA GCCGCGGTAA TACGTAGGGT GCGAGCGTTG 480
TCCGGAATTA TTGGGCGTAA AGAGCTCGTA GGCGGCCTGT CGCGTCGGAT GTGAAAGCCC 540
GGGGCTTAAC CCCGGGTCTG CATTCGATAC GGGCAGGCTA GAGTTCGGTA GGGGAGATCG 600
GAATTCCTGG TGTAGCGGTG AAATGCGCAG ATATCAGGAG GAACACCGGT GGCGAAGGCG 660
GATCTCTGGG CCGATACTGA CGCTGAGGAG CGAAAGCGTG GGGAGCGAAC AGGATTAGAT 720
ACCCTGGTAG TCCACGCCGT AAACGTTGGG CACTAGGTGT GGGCGGCATT CCACGTCGTC 780
CGTGCCGCAG CTAACGCATT AAGTGCCCCG CCTGGGGAGT ACGGCCGCAA GGCTAAAACT 840
CAAAGGAATT GACGGGGGCC CGCACAAGCG GCGGAGCATG TGGCTTAATT CGACGCAACG 900
CGAAGAACCT TACCAAGGCT TGACATACAC CGGAAACACG TGGAGACACG TGCCCCCTTG 960
TGGTCGGTGT ACAGGTGGTG CATGGCTGTC GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA 1020 GTCCCGCAAC GAGCGCAACC CTTGTTCTGT GTTGCCAGCG TGCCCTTCGG GGTGACGGGG 1080 ACTCACAGGA GACTGCCGGG GTCAACTCGG AGGAAGGTGG GGACGACGTC AAGTCATCAT 1140 GCCCCTTATG TCTTGGGCTG CACACGTGCT ACAATGGCCG GTACAATGAG CTGCGATGCC 1200 GCGAGGTGGA GCGAATCTCA AAAAGCCGGT CTCAGTTCGG ATTGGGGTCT GCAACTCGAC 1260 CCCATGAAGT CGGAGTCGCT AGTAATCGCA GATCAGCACT GCTGCGGTGA ATACGTTCCC 1320 GGGCCTTGTA CACACCGCCC GTCACGTCAC GAAAGTCGGT AACACCCGAA GCCGGTGGCC 1380 CAACCCCTTG CGGGAGGAAT CGTCGAAGGT GGACCGGGG 1419

Claims

¾禾' J ^ ^
1、 链霉菌（Stwpt n ces sp.)SCSIO 01689，保藏编号为： CCTCC NO: M 2011257»
2、 如式 （ I ) 所示的吡喃型倍半萜类 （Pyranosesquiterpene) 化合物：
Figure imgf000018_0001
式 （ I )。
3、 权利要求 1所述的链霉菌 Streptomyces sp. ) SCSIO 01689用于制备如权利 要求 2 所述的吡喃型倍半萜类 （Pyranosesquiterpene ) 化合物、 Cyclo(D)-Pro-(D)-ne 、 Cyclo(D)-Pro-(D)-Leu 或
Cyclo(D)-trans-4-OH-Pro-(D)-Phe的应用。
4、 一种权利要求 2所述的吡喃型倍半萜类 （Pyranosesquiterpene ) 化合物、 Cyclo(D)-Pro-(D)-Ee 、 Cyclo(D)-Pro-(D)-Leu 或
Cyclo(D)-trans-4-OH-Pro-(D)-Phe的制备方法， 其特征在于，所述的吡喃型倍 半 萜 类 （ Pyranosesquiterpene ) 化 合 物 、 Cyclo(D)-Pro-(D)-Ile 、 Cyclo(D)-Pro-(D)-Leu 或 Cyclo(D)-trans-4-OH-Pro-(D)-Phe 是从链霉菌 (Streptomyces sp. ) SCSIO 01689的发酵培养物中制备分离得到的。
5、 根据权利要求 4所述的制备方法， 其特征在于， 具体步骤如下：
a) 制备链霉菌 iStreptomyces sp.) SCSIO 01689的发酵培养物， 将该发酵培 养物的发酵液用乙酸乙酯萃取， 乙酸乙酯经蒸熘浓缩后得到浸膏； b) 将浸膏经硅胶柱层析， 用氯仿 /丙酮作为洗脱剂， 从体积比 100: 0-0： 100 进行梯度洗脱， 收集氯仿 /丙酮体积比 95:5和体积比 60: 40梯度洗脱下来的 馏分分别为 Fr.l和 Fr.2， 馏分 Fr.l经过凝胶柱层析得到粗品， 经纯化， 得到 权利要求 2所述的吡喃型倍半萜类（Pyranosesquiterpene)化合物； 熘分 Fr.2 经过凝胶柱层析得到粗品， 经纯化， 得到 Cycl0(D)-Pro-(D)-Ile、 Cyclo(D)-Pro-(D)-Leu和 Cyclo(D)-trans-4-OH-Pro-(D)-Phe。
6、 根据权利要求 5所述的制备方法， 其特征在于， 所述的 a) 步骤的制备链霉 菌 (Streptomyces sp. ) SCSIO 01689的发酵培养物是通过以下方法制备： 将 活化的链霉菌 （i^epto yc^sp.) SCSIO 01689接入种子培养基中， 28 °C， 180 rpm, 培养 3天制得种子液， 将种子液以 5%的接种量接入到发酵培养基 中， 28 °C， 180 rpm, 振荡培养 8天， 而制得发酵培养物， 所述的种子培养 基和发酵培养基的配方都为每升培养基中含有： 淀粉 5g， 大豆粉 5g， 蛋白 胨 2g， 葡萄糖 20g， 酵母膏 2g， K2HP040.5g, MgS047H200.5g， 粗海盐 30 g, 余量为水， pH7.4， 所述的步骤 b) 的纯化都为色谱柱分离。
、 权利要求 1所述的链霉菌 Streptomyces sp.) SCSIO 01689在制备脂肪酶或 蛋白水解酶中的应用。
、 权利要求 2所述的吡喃型倍半萜类（Pyranosesquiterpene)化合物在制备抗大 肠杆菌药物或制备抗卤虫药物或作为呈味剂的前体化合物在制备呈味剂中 的应用。
、 Cyclo(D)-Pro-(D)-Ee 、 Cyclo(D)-Pro-(D)-Leu 或 Cyclo(D)-trans-4-OH-Pro-(D)-Phe在制备抗卤虫药物中的应用。
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