CN106754408B - One plant of porous trichoderma strain and its method for preparing terpenoid - Google Patents

One plant of porous trichoderma strain and its method for preparing terpenoid Download PDF

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CN106754408B
CN106754408B CN201611116500.7A CN201611116500A CN106754408B CN 106754408 B CN106754408 B CN 106754408B CN 201611116500 A CN201611116500 A CN 201611116500A CN 106754408 B CN106754408 B CN 106754408B
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terpenoid
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porous trichoderma
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李二伟
张蕴之
刘杏忠
任晋玮
王文昭
裴云飞
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Institute of Microbiology of CAS
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Abstract

The invention discloses one plant of porous trichoderma (Tolypocladium inflatum) bacterial strain P086, on November 22nd, 2016, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.13191.

Description

One plant of porous trichoderma strain and its method for preparing terpenoid
Technical field
The present invention relates to technical field of microbial fermentation more particularly to terpene strain fermentation technical fields.
Background technique
The bioactive natural product separated from fungi belongs to terpenoid (terpestacin), terpenoid (terpestacin) carbon skeleton be a fifteen-membered ring and five-membered ring it is condensed made of bicyclic skeleton, wherein fifteen-membered ring Upper there are three trans- three to replace double bond, while having there are four chiral centre, including a quaternary carbon chiral centre, and five-membered ring For 1, the 2- double ketone structure that functional group is intensive, enol-type structure is presented in one of carbonyl.Its molecular structural formula are as follows:
Terpenoid (terpestacin) has mild antibacterial activity, and has good bioselection activity, because And it is widely paid close attention to as a kind of very promising drug.But the existing method production cycle for preparing terpenoid It is long, low efficiency.
Summary of the invention
The object of the present invention is to provide one plant of porous trichoderma (Tolypocladium inflatum) bacterial strain P086.
The separation of porous trichoderma strain
Porous trichoderma (Tolypocladium inflatum), be this laboratory separated from cordyceps sporophore and , belong to mycota double-core suberathem (Dikarya) Ascomycota (Ascomycota) Ascomycotina (Pezizomycotina) Excrement shell Gammaproteobacteria (Sordariomycetes) meat seat bacterium subclass (Hypocreomycetidae) Hypocreales (Hypocreales) worm Careless section (Ophiocordycipitaceae) Tolypocladium (Tolypocladium).
Porous trichoderma (Tolypocladium inflatum) bacterial strain P086 of the invention, on November 22nd, 2016, It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: Beijing's southern exposure The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.13191.
ITS sequence sequencing in the identification of porous trichoderma (Tolypocladium inflatum) bacterial strain P086 of the invention Used primer are as follows: ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ', PCR result is to see Fig. 1.
The present invention also provides utilize porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191 the method and terpenoid that) prepare terpenoid discharge the inhibiting effect and cell toxicant of NO to macrophage Property.
Terpene is prepared using porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) The method of compound (terpestacin), comprising the following steps:
(1) the porous trichoderma of activation (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) is connect Fermented and cultured is carried out in kind to solid medium.
(2) after step 1) fermentation, organic solvent is added to culture and extracts, obtains extracting solution, the extracting solution is subtracted Pressure distillation, obtains crude extract and sub- fraction.
Preferably, the solid medium is rice medium.
Preferably, the condition of the fermented and cultured is 18-28 DEG C, static gas wave refrigerator 20-60 days.
Preferably, the organic solvent is ethyl acetate, acetone, methanol, ethyl alcohol, chloroform, isopropanol and n-butanol etc..
Preferably, in the method, further include the crude extract and sub- fraction are carried out positive silica gel column layer chromatography or The isolated terpenoid of gel filtration chromatography.
Preferably, the chromatography, with petroleum ether-methylene chloride (ratio 100:0,100:1,100:2,100:3,20: 1,10:1,8:1,5:1,4:1,3:1,2:1,1:1,0:1v/v) or methylene chloride-methanol (ratio 100:0,100:1,100: 2,100:3,20:1,10:1,8:1,5:1,4:1,3:1,2:1,1:1,0:1v/v)) it is that eluent gradient elutes；Reversed silicagel column Chromatographic isolation, with methanol-water (ratio 5:95,10:90,15:85,20:80,25:75,30:70,35:65,40:60,45: 55,50:50,55:45,60:40,65:35,70:30,75:25,80:20,85:15,90:10,95:5,100:0v/v) it is flowing Phase gradient elution；Gel filtration chromatography then selects methanol for mobile phase elution.Sub- fraction is through positive, reversed-phase silica gel column chromatography, fraction It is dissolved in methanol solution after drying is concentrated under reduced pressure, is stored at room temperature, obtains each fraction sample.By preparative efficient liquid phase etc. Modern Natural Medicine Chemistry extracts separation means and obtains pure terpenoid.
It preferably, further include before solid fermentation by porous trichoderma (Tolypocladium in the step 1) Inflatum) bacterial strain P086 (CGMCC NO.13191) carries out preculture, the mycelium after taking preculture on solid medium It is transferred in fluid nutrient medium and carries out activation culture or be inoculated on several solid mediums to be activated；The condition of culture is equal Are as follows: it 25 DEG C, cultivates 7 days.
This patent strain growth is rapid, and it is very high to produce terpenoid efficiency；This patent bacterial strain is enterprising in PDB culture medium Row liquid fermentation 72 hours can output terpenoid, and 7 days can output when carrying out solid fermentation on rice medium Terpenoid；In addition, this patent bacterial strain output terpenoid total amount account for secondary metabolite crude extract 0.5% (10g is thick Extract is purified into 50mg terpenoid), yield is very high.
Detailed description of the invention
The porous trichoderma strain of Fig. 1 (bacterial strain name) qualification figure.
Fig. 2 terpenoid1H-NMR(CDCl3) spectrogram.
Fig. 3 terpenoid1H-NMR(Acetone-d6) spectrogram.
Fig. 4 terpenoid13C-NMR(Acetone-d6) spectrogram.
Specific embodiment
The present invention is described in further detail with embodiment below, but the contents of the present invention are not limited thereto.
Experimental method described in following embodiments is unless otherwise specified conventional method；The reagent and biological material Material, unless otherwise specified, commercially obtains.
Embodiment 1
Porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) obtains
(1) strain isolation
In the isolated porous trichoderma (Tolypocladium of a fungal strain endophyte from cordyceps sporophore Inflatum) bacterial strain P086 (CGMCC NO.13191).
Specific separation method is, firstly, carrying out surface sterilization (75% alcohol to cordyceps sporophore with ethanol for disinfection 30s, 5%NaClO 90s) and it is placed in drying in sterilizing filter paper with rinsed with sterile water, it is inoculated on PDA plate and trains after being cut into small pieces It supports and isolates and purifies bacterial strain
(2) porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) identification
Using Ai Delai Biotechnology Co., Ltd product fungal genomic DNA rapidly extracting kit, bacterial strain is carried out DNA is extracted, and utilizes primer I TS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-TCCTCCGCTTATTGATATGC- 3 ' carry out PCR to its DNA, send and are sequenced in Beijing Qing Kexin industry Bioisystech Co., Ltd, and ITS sequence is as shown in Figure 1.In beauty Sequence is carried out nucleic acid BLAST and bacterial strain is accredited as porous trichoderma by state's National Biotechnology Information Center (NCBI) (Tolypocladium inflatum)。
Porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) is in 2016 11 The moon 22, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: Beijing The institute 3 of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.13191.
Embodiment 2
Porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) fermentation prepares terpene Compound
(1) bacterial strain activation, fermented and cultured
By porous trichoderma (Tolypocladium inflatum) bacterial strain P086 (CGMCC NO.13191) from low temperature refrigerator, It is taken out under liquid nitrogen storage tank or other storage conditions, selects on mycelium inoculation to the inclined-plane or plate of PDA culture medium, trained in advance It supports；The condition of preculture are as follows: 25 DEG C are cultivated 3-7 days, and incubation time can be appropriately extended or shortened according to the growing state of bacterial strain.
Mycelium can be cut into about 0.5cm by growing to 3.0-5.0cm to bacterium colony2Fritter takes about 10-15 fungus block to be transferred to It is cultivated in PDB culture medium, obtains seed culture fluid；The condition of Liquid Culture are as follows: 25 DEG C are cultivated 3-7 days, shaking table speed 150-300rpm。
It takes 10 milliliters of seed culture fluids to be inoculated into rice medium, is inoculated with 40 bottles；Fermentation condition is 25 DEG C of cultures 30 It.
Aforesaid operations are sterile working, and each culture medium need to pass through high pressure steam sterilization.
Wherein, the composition of PDA culture medium are as follows: potato 200g, glucose 20g, agar powder 16g, water are settled to 1L.
Wherein, PDB fluid nutrient medium forms are as follows: potato 200g, glucose 20g, water are settled to 1L.
Wherein, the preparation method of rice medium are as follows: 80g rice and 100ml water, high pressure are added in 500 milliliters of wide-mouth bottles Steam sterilizing.
Fermentation strain need to inspect periodically growth conditions, prevent pollution microbes.
(2) the extraction separation of compound
A organic solvent extracts
After solid fermentation, 300 milliliters of ethyl acetate are added separately in each fermentation flask, in room temperature ultrasonic cleaning Machine ultrasound 30min, then soak at room temperature extracts, and 24 hours primary, repeats to extract 3 times, and the decompression of combined ethyl acetate extracting solution is steamed Drying is evaporated, the secondary metabolite crude extract about 10g of porous trichoderma (Tolypocladium inflatum) bacterial strain P086 is obtained.
B compound isolates and purifies
Sub- fraction in the crude extract and experimentation is subjected to silica gel column layer chromatography or gel filtration chromatography separation.Its In, positive silicagel column with petroleum ether-methylene chloride (ratio 100:0,100:1,100:2,100:3,20:1,10:1,8:1,5: 1,4:1,3:1,2:1,1:1,0:1v/v) or methylene chloride-methanol (ratio 100:0,100:1,100:2,100:3,20:1, 10:1,8:1,5:1,4:1,3:1,2:1,1:1,0:1v/v)) it is that eluent gradient elutes；Reversed silica gel column chromatography separation, with first Alcohol-water (ratio 5:95,10:90,15:85,20:80,25:75,30:70,35:65,40:60,45:55,50:50,55:45, 60:40,65:35,70:30,75:25,80:20,85:15,90:10,95:5,100:0v/v) it is that eluent gradient elutes；Gel Column chromatography then selects methanol for mobile phase elution.Sub- fraction is molten after fraction reduced pressure is dry through positive, reversed-phase silica gel column chromatography Solution is stored at room temperature in methanol solution, obtains each fraction sample.By the modern Natural Medicine Chemistry such as preparative efficient liquid phase It extracts separation means and obtains pure terpenoid 50mg, account for crude extract total amount: 0.5% (=50mg/10g).
C compound structure confirmation
Terpenoid is white solid；Specific rotation angle value(0.1mg/ml, MeOH)；HRESIMS m/z 403.2869[M+H]+,827.5475[2M+Na]+；Under different deuterated reagents hydrogen spectrum (1H-NMR), carbon spectrum (13C-NMR) as follows: according to According to the physicochemical property of compound, hydrogen modal data, and with bibliography numeric ratio to (Johann Chan, Timothy F.Jamison, Synthesis of (-)-terpestacin via Catalytic, Stereoselective Fragment Coupling:Siccanol is terpestacin, not 11-epi-terpestacin, J.Am.Chem.Soc., 2003, 125 (38), pp 11514-11515), determine the structure of the compound with it is identical.
Terpenoid1H-NMR(CDCl3) spectrogram such as Fig. 2, spectrum analysis:
1H NMR(500MHz,CDCl3): δ 5.94 (s, 1H), 5.40 (m, 1H), 5.24 (dd, J=23.6,11.7Hz, 1H), 5.14 (dd, J=4.2Hz, 1H), 4.06 (dd, J=9.6,3.6Hz, 1H), 3.89 (dd, J=10.4,7.0Hz, 1H), 3.82 (dd, J=10.4,5.4Hz, 1H), 2.71 (dd, J=11.4,2.0Hz, 1H), 2.67 (m, 1H), 2.43 (d, J= 17.3Hz, 1H), 2.39 (dd, J=13.6,10.5Hz, 1H), 2.22-2.32 (m, 2H), 2.06-2.16 (m, 2H), 2.02 (m, 1H), 1.93 (m, 1H), 1.75-1.83 (m, 3H), 1.75-1.66 (m, 3H), 1.64 (d, J=3.4Hz, 3H), 1.57 (s, 3H), 1.30 (d, J=7.1Hz, 3H), 0.99 (s, 3H).
Terpenoid1H-NMR(Acetone-d6) spectrogram such as Fig. 3, spectrum analysis:
1H NMR (500MHz, Acetone-d6): δ 5.33 (m, 2H) 5.20 (d, J=8.6Hz, 1H) 3.99 (dd, J= 9.4,4.5Hz, 1H) 3.84 (dd, J=10.2,6.7Hz, 1H) 3.73 (dd, J=10.2,6.2Hz, 1H) 3.39 (s, 1H) 2.81 (s, 1H) 2.76 (dd, J=11.4,2.2Hz, 1H) 2.64 (m, 1H) 2.45 (d, J=17.1Hz, 1H) 2.22-2.35 (m, 2H) 2.07-2.17 (m, 2H) 1.94 (sextuplete, 1H) 1.81 (dd, J=22.8,9.5Hz, 1H) 1.74 (m, 2H), spectrogram See annex 3.
Terpenoid13C-NMR(Acetone-d6) spectrogram such as Fig. 4, spectrum analysis:
13CNMR(500MHz,Acetone-d6): δ 207.7qC, 150.0qC, 148.4qC, 138.0qC, 137.9qC, 133.7qC, 128.9CH, 124.8CH, 123.4CH, 76.3CH, 65.9CH2, 50.4CH, 49.5qC, 41.1CH2, 40.1CH2, 38.5CH 35.7CH2, 31.3CH2, 29.8CH2, 24.6CH2, 16.9CH3, 16.0CH, 15.5CH3, 14.9CH3, 10.6CH3。
Embodiment 3
The biological activity determination and Study of cytotoxicity for inhibiting macrophage release NO of terpenoid
By mouse monokaryon macrophage RAW 264.7 with 1640 culture medium of RPMI (containing 10% fetal calf serum (FBS), 100U/ml Benzylpenicillin sodium salt, 100 μ g/ml streptomysins) in CO2Constant incubator (contain 5%CO2, 37 DEG C) in be incubated for growth.
Its cell concentration is diluted to by logarithmic growth phase mouse macrophage RAW 264.7 with 1640 culture medium of RPMI 5×105Mouse macrophage after dilution is carefully inoculated in 96 porocyte culture plates by cells/ml, and it is outstanding that 200 μ l cells are added in every hole Supernatant liquid is replaced in CO21h is cultivated in constant incubator.
It prepares the terpenoid sample of various concentration and 0.4 μ l (DMSO final concentration 0.2%) is taken to be separately added into 96 orifice plates In, LPS, which is added, in every hole makes its final concentration of 1 μ g/ml, in CO2It is cultivated for 24 hours in constant incubator.LPS group is arranged (i.e. in this step It is added without tested sample) and blank control group (DMSO), 3 repetitions of every sample setting.
100 μ l of culture solution supernatant is drawn into ELISA Plate, isometric Griess reagent is added, reacts 10min at room temperature The light absorption value at 550nm is measured afterwards.Griess reagent A: w (N- naphthalene ethylamine hydrochloride)=0.1% is soluble in water；Griess reagent B:w (P-aminobenzene-sulfonamide)=1% is dissolved in φ (H3PO4In)=5%, preceding isometric mix reagent A and B is used.
It is respectively the NaNO of 0,1,2,5,10,50,100 μm of ol/L with concentration2Standard curve is drawn, according to NaNO2Standard is bent NO in line computation cell culture supernatant2 -Concentration and to NO release inhibiting rate.It is molten that MTT is added in the every hole of above-mentioned culture plate Liquid (200 μ g/mL of final concentration), is placed in CO2Continue after cultivating 4h in incubator, discard supernatant, blot residual liquid, DMSO is added 150 μ L, shaking 10min make the first a ceremonial jade-ladle, used in libation generated crystallization after completely dissolution, measure extinction at 570nm using 630nm as reference wavelength Value, evaluates the cytotoxicity of sample.
Above-mentioned 1640 culture medium prescription of used RPMI comes from document Moore, G.E.Gerner, R.E.andFranklin, H.A. (1967) A.M.A., 199,519, with before heat inactivation 30min, 56 DEG C.
The A liquid of used Griess reagent: p-aminobenzene sulfonic acid 0.5g, spirit of vinegar (10% or so) 150mL；B liquid: α naphthalene Amine 0.1g, distilled water 20mL, spirit of vinegar (10% or so) 150mL.
NO in inhibition mouse macrophage has been carried out to terpenoid using the above method and has discharged active test, selection Hydrocortisone (Hydrocortisone) is used as positive control drug, is not only influencing normal cell normally value-added concentration model In enclosing, what is showed just has real meaning to the inhibitory activity of macrophage release NO.
Experiment measures terpenoid and truly has the bioactivity for inhibiting macrophage release NO, half-inhibitory concentration IC50 =10.36 ± 1.28 μM；In addition, terpenoid also has certain cytotoxicity, the cytotoxicity of sample is 85 ± 6.27 μ M。
Sequence table
<110>Institute of Microorganism, Academia Sinica
<120>one plants of porous trichoderma strains and its method for preparing terpenoid
<160> 1
<210> 1
<211> 529
<212> DNA
<213>porous trichoderma (Tolypocladium inflatum)
<400> 1
ctgcggaggg atcattaccg agttatcaac tcccaaaccc ctgtgaacat acccaacgtt 60
gcttcggcgg gaccgccccg gcgcctcggc gtcccggaac caggcgcccg ccggaggacc 120
caaactcttg tttaaccata gtggcatatt ctgagtctca caagaaaaat gaatcaaaac 180
tttcaacaac ggatctcttg gctctggcat cgatgaagaa cgcagcgaaa tgcgataagt 240
aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg cgcccgccag 300
tattctggcg ggcatgcctg ttcgagcgtc atttcaaccc tcaagcccca gcggcttggt 360
gttggggacc ggccccggcc gccccccaaa tgcagtggcg acctcgccgc agcctcccct 420
gcgtagtagc acaactcgca ccggagcgcg gagacggtca cgccgtaaaa cgcccaactt 480
ctcagagttg acctcggatc aggtaggaat acccgctgaa cttaagcat 529

Claims (8)

1. one plant of porous trichoderma (Tolypocladium inflatum) bacterial strain P086 CGMCC NO.13191.
2. a kind of method that porous trichoderma (Tolypocladium inflatum) bacterial strain P086 prepares terpenoid, feature It is:
(1) the porous trichoderma strain P086 of activation is inoculated on solid medium and carries out fermented and cultured；
(2) organic solvent is added to culture to extract, obtains extracting solution.
3. according to the method described in claim 2, it is characterized by: the solid medium is rice medium.
4. according to the method described in claim 2, temperature is 18-28 DEG C, static training it is characterized by: the fermented and cultured It supports 20-60 days.
5. according to the method described in claim 2, it is characterized by: the organic solvent be ethyl acetate, acetone, methanol, One of ethyl alcohol, chloroform, isopropanol, n-butanol.
6. according to the method described in claim 2, it is characterized by also including obtain crude extract and son evaporates after extracting solution is concentrated Point.
7. according to the method described in claim 6, it is characterized by: the crude extract and sub- fraction carry out positive silica gel column layer Chromatography or the isolated terpenoid of gel filtration chromatography.
8. according to the method described in claim 2, it is characterized by: the porous trichoderma strain P086 of the activation, refer to by Porous trichoderma strain P086 carries out preculture on solid medium, and the mycelium after taking preculture is transferred in fluid nutrient medium It carries out activation culture or is inoculated on several solid mediums to be activated；The condition of culture is equal are as follows: 25 DEG C, cultivates 3-7 days.
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