CN101397579B - Method for preparing natamycin - Google Patents
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- CN101397579B CN101397579B CN2007101874347A CN200710187434A CN101397579B CN 101397579 B CN101397579 B CN 101397579B CN 2007101874347 A CN2007101874347 A CN 2007101874347A CN 200710187434 A CN200710187434 A CN 200710187434A CN 101397579 B CN101397579 B CN 101397579B
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Abstract
The invention relates to a method for preparing natamycin, which carries out fermentation to Streptomyces lydicus so as to obtain the natamycin. The method initiatively uses the fermentation of the Streptomyces lydicus for producing the natamycin, thus providing a new way for producing the natamycin. The natamycin produced by the method has high purity. The method can be widely applied to the production of the high-purity natamycin.
Description
Technical field
The present invention relates to a kind of method for preparing tennecetin.
Background technology
Tennecetin (Natamycin) is a kind of polyene macrolide antifungal microbiotic, also claim natamycin or pimaricin (Pimaricin), it is synthetic by the multi-enzyme system of 5 polyketide synthase enzymes genes encodings, can obligate suppress yeast and mould, stop the formation of flavacin in the filamentous fungus.Its actual using dosage is 10
-6The order of magnitude, have low dosage, high-level efficiency, the characteristics that the anti-microbial effect time is long, be a kind of efficient, safe natural biological food preservatives and antimicrobial additive, be extensive use of in more than 30 countries at present, comprise European Union, most of North America and the East European countries and some middle east, even the country that Switzerland pays much attention to food safety like this allows also to use tennecetin in bread and cheese product.China foodstuff additive council in 1996 estimates tennecetin and advises that approval uses, and in March, 1997, China Ministry of Health official approval tennecetin is as food preservatives, and its trade name is mould gram (Natamycin
TM).
Tennecetin has resistance to nearly all mould and yeast, and bacteriostatic action is stronger about 50 times than Sorbic Acid, and uses for several years running and be difficult for also causing that the target fungi forms resistance, but invalid to bacterium and virus.Therefore it has a wide range of applications in the food service industry based on fermentation using bacteria.As food preservatives, can be applicable to the mildew-resistant of jam, butter, orange juice, raw meat and salad sauce etc., prolong the quality guaranteed period of food, what prevent that yeast and mould from causing goes mouldy, reducing the food that causes because go bad reclaims, reduce production costs, and do not change flavours in food products, satisfy the requirement of human consumer whole food.Tennecetin also can be used as high-efficient antibacterial agent medically, and coccus is read in the oral cavity infect, fungoid cornea ulcer, keratitis, diseases such as skin and mucous membrane fungi infestation all have good result of treatment.Along with deepening continuously of research, the range of application of tennecetin is continuing to enlarge, and is bringing into play increasing effect.
Streptomyces lydicus (Sireptomyces lydicus DeBoer et al.) is to practise the saprophytic microbe occupy soil, studies with a long historyly, and known have many bacterial strains can produce various antibacterial substances in metabolic process.Its famous representative strain is S.lydicus NRRL2433, this bacterial strain is studied and is widely used at field of medicaments, can produce to have wide spectrum antibacterium and the lydimycin (Lydimycin) of mycobacterium effect and the streptolydigin (Streptolydigin) of resisting gram-positive bacterium and mycobacterium; The S.lydicus 1574 that the oxysuccinic acid Tubiserin (Malioxamycin) that produces anti-minority intestinal bacteria and Ke Shi pneumobacillus is arranged in addition, and the bacterial strain that produces the microbiotic Lydicamycin of resisting gram-positive bacterium and methicillin resistant staphylococcus aureus.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing tennecetin.
The method for preparing tennecetin provided by the present invention is that fermentation streptomyces lydicus (Streptomyceslydicus) obtains tennecetin.
Described streptomyces lydicus (Streptomyces lydicus) can be streptomyces lydicus (Streptomyceslydicus) A01 CGMCC No.1653 or streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654.
Streptomyces lydicus A01 CGMCC No.1653 is that separation screening obtains from the soil of Miyun County vegetables field, Beijing, and streptomyces lydicus A02 CGMCC No.1654 separation screening from the Natural Secondary Forests soil of outer suburbs, Beijing obtains.It has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 03 16th, 2006, preservation registration number is respectively CGMCC No.1653 and CGMCC No.1654.
Wherein, consisting of of the fermention medium of the described streptomyces lydicus that ferments (Streptomyces lydicus) A01 CGMCC No.1653: contain in every liter of substratum: analysis for soybean powder 2%, peptone 0.5%, Zulkovsky starch 0.5%, glucose 2%, corn steep liquor 0.25%, (NH
4)
2SO
40.25%, MgSO
4.7H
2O 0.025%, K
2HPO
40.002%, NaCl 0.4%, CaCO
30.2%, all the other are water; Naturally the initial pH value of the fermention medium of described streptomyces lydicus A01 CGMCCNo.1653 is about 5-6; Described percentage composition is the quality percentage composition.
The condition of described streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 of fermenting can be: under 28 ℃ condition, cultivate with the rotation radius of 13mm, the speed oscillation of 180rpm.
Ferment the consisting of of fermention medium of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654: contain the extracting solution that obtains by the 15g soybean grain in every liter of substratum, the 5g peptone, 10g sucrose, 10g starch, 2.5g ammonium sulfate, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate, all the other are water; The pH of the fermention medium of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is 7-8.
Wherein, the extracting solution that is obtained by the 15g soybean grain prepares as follows: the 15g soybean grain is added in the distilled water, boil 0.5-1h, the elimination solid substance obtains the extracting solution that is obtained by the 15g soybean grain.
The condition of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 of fermenting specifically can be: under 31 ℃ condition, cultivate with the rotation radius of 13mm, the speed oscillation of 240rpm.
The described method for preparing tennecetin also comprises the step of separation and purification tennecetin.
Wherein, the step of separation and purification tennecetin specifically can be: 1) collect the fermented liquid that the described streptomyces lydicus of fermentation (Streptomyces lydicus) obtains, precipitate with dehydrated alcohol, collect supernatant liquor;
2) described supernatant liquor is carried out the resin absorption chromatography successively, the silica gel adsorption chromatography separates with high performance liquid chromatography, obtains tennecetin.
When described streptomyces lydicus (Streptomyces lydicus) was streptomyces lydicus (Streptomyceslydicus) A01 CGMCC No.1653, separation purification method was specific as follows:
Described resin absorption chromatography adopts the D4006 macroporous resin, elution requirement is 50% methyl alcohol of the deionized water of using 1 times of column volume successively, 1.5 times of column volumes and 80% ethanol elution of 2.5 times of column volumes, and collecting discharge is 3.5 times to the 4.5 times elutriants between the column volume; Described percentage composition is a volumn concentration;
Described silica gel adsorption chromatography adopts 200～300 order silica gel, and elution requirement is to be that 1: 2: 1 propyl carbinol, the mixed solution of first alcohol and water carry out wash-out with volume ratio, and the collection discharge is 0.33 times to the 0.6 times elutriant between the column volume;
Described high performance liquid chromatography separates employing LC-9101 type cycles prepare high performance liquid chromatograph and JAIGEL-ODS-AP type SP-120-15 preparative column, with volume ratio is that the mixed solution of 7: 3 first alcohol and water is a moving phase, separated secondary, retention time was the elution peak of 38.399min when collection separated for the second time.
When described streptomyces lydicus (Streptomyces lydicus) was streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654, separation purification method was specific as follows:
Described resin absorption chromatography adopts the X-5 macroporous resin, and elution requirement is 30% methyl alcohol of the deionized water of using 2 times of column volumes successively, 2 times of column volumes and 70% ethanol elution of 2 times of column volumes, and collecting discharge is 4.8 times to the 5.6 times elutriants between the column volume; Described percentage composition is a volumn concentration;
Described silica gel adsorption chromatography adopts 100～200 order silica gel, and elution requirement is to be that the mixed solution of 8: 1: 1 ethanol, ammoniacal liquor and water carries out wash-out with its volume ratio, and the collection discharge is 0.23 times to the 1.2 times elutriant between the column volume;
Described high performance liquid chromatography separates employing LC-9101 type cycles prepare high performance liquid chromatograph and JAIGEL-ODS-AP type SP-120-15 preparative column, with volume ratio is that the mixed solution of 7: 3 first alcohol and water is a moving phase, separate three times, retention time was the elution peak of 39.766min when collection separated for the third time.
The present invention produces the method for tennecetin, uses the streptomyces lydicus natamycin fermentation preparation in a creative way, thereby provides new approach for producing tennecetin.The tennecetin purity that the inventive method separation and purification obtains is very high, reaches more than 99%.Therefore, the present invention will be used widely in the production of high purity tennecetin.
Description of drawings
Figure 1A is that the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 is to saccharomycetic restraining effect.
Figure 1B is the restraining effect of the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 to Botrytis cinerea.
Fig. 2 A is that the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is to saccharomycetic restraining effect.
Fig. 2 B is the restraining effect of the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 to Botrytis cinerea.
Fig. 3 is that Czech's eight solvent systems ply of papers of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result are analysed the result.
Fig. 4 is the uv absorption spectra of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active substance crude extract.
Fig. 5 A is that the HPLC of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result separates spectrogram for the first time.
Fig. 5 B is that the HPLC of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result separates spectrogram for the second time.
Fig. 6 A is that the HPLC of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result separates spectrogram for the first time.
Fig. 6 B is that the HPLC of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result separates spectrogram for the third time.
Fig. 7 A is the UV scanning collection of illustrative plates of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Fig. 7 B is the UV scanning collection of illustrative plates of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Fig. 8 A is the infrared absorption spectrum of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Fig. 8 B is the infrared absorption spectrum of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Fig. 9 A-a is the high resolution mass spectrum figure (negative ion) of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Fig. 9 A-b is the high resolution mass spectrum figure (positive ion) of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Fig. 9 B-a is the high resolution mass spectrum figure (negative ion) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Fig. 9 B-b is the high resolution mass spectrum figure (positive ion) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Figure 10 A is the proton nmr spectra (500MHz) of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Figure 10 B is the carbon-13 nmr spectra (500MHz) of streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 active result pure sample product.
Figure 11 A is the proton nmr spectra (500MHz) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Figure 11 B is the carbon-13 nmr spectra (500MHz) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 active result pure sample product.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Employed substratum is as follows among the embodiment:
1, Gause I substratum: contain K in every liter of substratum
2HPO
40.5g, NaCl 0.5g, KNO
31.0g, FeSO
47H
2O 0.01g, MgSO
47H
2O 0.5g, Zulkovsky starch 20g, agar 20g, all the other are water; The pH of described Gause I substratum is 7.2-7.4.
2, the seed culture medium of streptomyces lydicus A01 CGMCC No.1653: contain analysis for soybean powder 20g in every liter of substratum, peptone 5g, Zulkovsky starch 10g, glucose 20g, corn steep liquor 2.5g, (NH
4)
2SO
42.5g, K
2HPO
40.02g, NaCl 4g, CaCO
36g, all the other are water; The pH of the seed culture medium of described streptomyces lydicus A01 CGMCC No.1653 is 7.2.121 ℃ of sterilization 30min.
3, the fermention medium of streptomyces lydicus A01 CGMCC No.1653: contain analysis for soybean powder 2% in every liter of substratum, peptone 0.5%, Zulkovsky starch 0.5%, glucose 2%, corn steep liquor 0.25%, (NH
4)
2SO
40.25%, MgSO
4.7H
2O 0.025%, K
2HPO
40.002%, NaCl 0.4%, CaCO
30.2%, all the other are water; Naturally the initial pH value of this fermention medium is 5.6.Wherein, each percentage composition is the quality percentage composition.
4, the seed culture medium of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654: the extracting solution that obtains by the 15g soybean grain, the 5g peptone, 20g glucose, 10g starch, 2.5g ammonium sulfate, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, be made into the aqueous solution, after transferring pH7-8, add 1g lime carbonate, add water and be settled to 1000ml.
5, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 fermention medium:
By the extracting solution that the 15g soybean grain obtains, the 5g peptone, 10g sucrose, 10g starch, 2.5g ammonium sulfate, 0.25g sal epsom, the 0.2g potassium primary phosphate, 5g sodium-chlor is made into the aqueous solution, behind the accent pH7-8, adds 1g lime carbonate, adds water and is settled to 1000ml.
Wherein, the extracting solution that is obtained by the 15g soybean grain prepares as follows: the 15g soybean grain is added in an amount of distilled water, boil 0.5-1h, the elimination solid substance obtains the extracting solution that is obtained by the 15g soybean grain.
6, PDA substratum: contain potato 200g in every liter of substratum, sucrose 10-20g, agar 17-20g, all the other are water; The pH nature.
The separation of embodiment 1, streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653
Gather soil sample from Miyun County vegetables field, Beijing, get 10g add in the triangular flask of dress 100ml sterilized water and an amount of granulated glass sphere, put 100rpm vibration 20min on the shaking table, get 0.5ml and add in the 4.5ml sterilized water, dilute 10 successively again
2, 10
3, 10
4Doubly, respectively get above soil supension 0.1ml and be uniformly coated on respectively on the Gause I culture medium flat plate, blow in the Bechtop to slightly doing; 3 repetitions of every concentration are put in 28 ℃ of thermostat containers and were cultivated 5-10 days, and the single bacterium colony of picking actinomycetes dilutes the line separation and purification, obtains the pure culture bacterial strain.
Primary dcreening operation: the pure culture bacterial strain of acquisition carries out shake flask fermentation, and fermented liquid obtains aseptic ferment filtrate through the aseptic filtering with microporous membrane degerming of 0.45 μ m; With yeast saccharomyces cerevisiae (Saccharomyces cerevisiae ACCC20036), aspergillus niger (Aspergillus nigerACCC30005) and Botrytis cinerea (Botrytis cinerea ACCC30091) is indicator, utilizes cup-plate method or agar punch method to detect the bacteriostatic activity of fermented liquid;
The aseptic ferment filtrate of the bacterial strain that bacteriostatic activity is arranged that primary dcreening operation is obtained carries out ethanol sedimentation and removes impurity, supernatant liquor carries out UV scanning and obtains uv-spectrogram, and compare with the collection of illustrative plates of polyene antibiotics, filter out the bacterial strain of uv-spectrogram with polyenoid quasi-representative absorption peak;
The aseptic ferment filtrate of the bacterial strain that multiple sieve is obtained is removed and is carried out column chromatography for separation after impurity concentrates, and follows the tracks of the activity that detects each separated portion with indicator, main active component is prepared the type high performance liquid chromatography separates its pure sample product that obtain; Detect these pure sample product of evaluation, finishing screen is selected the bacterial strain that can produce tennecetin---streptomyces lydicus A01 CGMCC No.1653.
The Microbiological Characteristics of streptomyces lydicus A01 CGMCC No.1653 is identified:
(1) morphological specificity of thalline
Gram-positive; At GYM agar, JCM42
#Growth is after 7 days on the substratum such as agar, oatmeal agar, and substrate mycelium physically well develops, and no tabula does not rupture; The aerial hyphae well-grown, multi-branched; Fibrillae of spores is straight, gentle bent or crooked, the spore ellipse.
(2) cultural characters
Cultural characters on 6 kinds of solid mediums is as shown in table 1.
The cultural characters of table 1 streptomyces lydicus A01 CGMCC No.1653
| Substratum | Aerial hyphae | Substrate mycelium | But lysochrome |
| Gao Shi synthesizes No. 1 agar ISP4 agar GYM agar Bennett ' s agar JCM42 #Substratum oatmeal agar | The light gray lime is yellow greyish white slightly grey greyish white to the greyish white white of ecru | The yellowish-brown orange of little Huang is brown yellowish-brown shallow brown | There is not yellow yellowish shallow palm fibre |
(3) physio-biochemical characteristics
The physio-biochemical characteristics of streptomyces lydicus A01 CGMCC No.1653 are as shown in table 2.
The physio-biochemical characteristics of table 2 streptomyces lydicus A01 CGMCC No.1653
| Characteristic | The result | Characteristic | The result |
| I. growth and 45 ℃ of Mierocrystalline cellulose starch-splittings of other 3%NaCl 5%NaCl 7 |
+W-----++-W++ | II. utilization of carbon source sorbyl alcohol sorbose synanthrin fructose lactose semi-lactosi pectinose ribose rhamnosyl raffinose sucrose wood sugar inositol Alpha-Methyl-D-glucoside malonate | W--+++W--++-WW- |
Annotate: the weak positive findings of " W " expression; "+" expression positive findings; "-" expression negative findings.
(4) 16SrDNA sequence
The part 16SrDNA sequence of streptomyces lydicus A01 CGMCC No.1653 is shown in sequence in the sequence table 1.Show that with the result relatively of correlated series Blast among the GenBank it belongs to streptomyces; It is very high that its 16SrDNA sequence and known streptomyces lydicus bacterial strain NRRL2433 compare similarity, is 100%.
The result of comprehensive its morphological feature, cultural characters, physio-biochemical characteristics and 16SrDNA sequential analysis, it is accredited as streptomyces lydicus (Streptomyces lydicus), and this bacterial strain named is streptomyces lydicus (Streptomyces lydicus) A01.Streptomyces lydicus (Streptomyces lydicus) A01 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 03 16th, 2006, preservation registration number is CGMCC No.1653.
Streptomyces lydicus A01 CGMCC No.1653 and A02 CGMCC No.1654 can produce great deal of bioactive substances in substratum in metabolic process.This bio-active substance confrontation indicator strain yeast saccharomyces cerevisiae ACCC20036, aspergillus niger ACCC30005 and Botrytis cinerea ACCC30091 have had strong inhibitory effects; The ply of paper of its Czech's eight solvent systemss is analysed the consistent of collection of illustrative plates and polyene antibiotics; The peculiar typical absorption of its UV spectrum performance tetraenes microbiotic peak, this is consistent with the result that Czech eight solvent systems ply of papers are analysed; It is carried out finding to have 2 active ingredients after the initial gross separation, to carrying out UV spectrum (UV), infrared spectra (IR), mass spectrum (MS) and nuclear magnetic resonance spectrum (NMR) check and analysis behind the main active component purifying, the result shows that its molecular weight, molecular formula and chemical structure are all identical with tennecetin.The concrete production and the authentication method of tennecetin are as follows:
One, the preparation of tennecetin
1, utilize streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 shake flask fermentation to prepare tennecetin
Slant culture: streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 is inoculated on the Gause I slant medium 28 ℃ of spores of cultivating 7-10 days to its generation capacity;
Seed culture: scrape with aseptic platinum loop and to get its spore 3-5 ring and be inoculated in the 500ml triangular flask that the 100ml seed culture medium is housed, put on the temperature controllable shaking table, under 28 ℃ of conditions, with the rotation radius of 13mm, the rotating speed constant-temperature shaking culture 20-24h of 180rpm;
Fermentation culture: get above-mentioned seed culture fluid and be inoculated in the 500ml triangular flask that the 100ml fermention medium is housed, under 28 ℃ of conditions, with the rotation radius of 13mm, the rotating speed constant-temperature shaking culture 96h of 180rpm by the inoculum size of 3% (V/V).
This moment, bacterial strain produced the tennecetin of high density in fermented liquid.Fermented liquid obtains aseptic ferment filtrate through the aseptic filtering with microporous membrane degerming of 0.45 μ m, be indicator with yeast saccharomyces cerevisiae ACCC20036 and Botrytis cinerea ACCC30091 respectively, utilize the agar punch method to carry out activity and detect, the antibacterial circle diameter of fermented liquid reaches 31.0mm and 43.0mm (Figure 1A and Figure 1B) respectively.
2, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 shake flask fermentation prepares tennecetin
Slant culture: streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is inoculated on the Gause I slant medium 28 ℃ of spores of cultivating 7-10 days to its generation capacity;
Seed culture: scrape with aseptic platinum loop and to get 2-3 ring spore inoculating in the 250ml triangular flask that the 50ml seed culture medium is housed, under 28 ℃ of conditions, with the rotation radius of 13mm, the rotating speed constant-temperature shaking culture 24h-30h of 200rpm;
Fermentation culture: under aseptic condition, the inoculum size of seed culture fluid with 5% (V/V) is inoculated in the 500ml triangular flask that the 100ml fermention medium is housed, under 31 ℃ of conditions, with the rotation radius of 13mm, the rotating speed constant-temperature shaking culture 96h of 240rpm.
This moment, bacterial strain produced the tennecetin of high density in fermented liquid.Fermented liquid obtains aseptic ferment filtrate through the aseptic filtering with microporous membrane degerming of 0.45 μ m, measure its bacteriostatic activity as stated above, its antibacterial circle diameter to yeast saccharomyces cerevisiae ACCC20036 and Botrytis cinerea ACCC30091 reaches 32.0mm and 47.0mm (Fig. 2 A and Fig. 2 B) respectively.
Two, slightly the carrying and preliminary evaluation of tennecetin in the fermented liquid
(1) slightly the carrying of tennecetin in the fermented liquid
The fermented liquid of above-mentioned streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 and streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is used the dehydrated alcohol pre-treatment of 3 times of volumes respectively, 4 ℃ leave standstill 2h, with precipitation thalline, solid particles, solubility sticking jelly, nucleic acid and heteroproteins and mesostate etc., supernatant liquor with 2 metafiltration paper with the B vacuum filtration, filtrate through Rotary Evaporators at 45 ℃ of following concentrating under reduced pressure, concentrated solution is the active substance crude extract, and 4 ℃ of preservations are standby.
(2) preliminary evaluation of tennecetin in the fermented liquid
Adopt Czech's eight solvent systems ply of papers to analyse the chemical type of differentiating active substance, in 190nm～400nm scope, it is carried out full wavelength scanner simultaneously.
1, Czech's eight solvent systems ply of papers are analysed
Solvent systems is: the water saturated propyl carbinol of I.; II. the water saturated butanol solution that contains 2% (quality percentage composition) p-methyl benzenesulfonic acid; III. propyl carbinol: acetic acid: water (volume ratio)=2: 1: 1; IV. the water saturated propyl carbinol that contains 2% (quality percentage composition) hexahydropyridine; V. the 0.5mol/l pH that propyl carbinol is saturated is 7.0 phosphoric acid buffer; VI. the saturated water of propyl carbinol that contains 2% (quality percentage composition) p-methyl benzenesulfonic acid; VII. benzene: methyl alcohol (volume ratio)=4: 1, filter paper is handled with the phosphoric acid buffer of 0.5mol/l pH 7.0; VIII. methyl alcohol: 3% (quality percentage composition) the NaCl aqueous solution (volume ratio)=3: 1, filter paper is handled with 5% (quality percentage composition) aqueous sodium persulfate solution;
Adopt filter paper of Xinhua, be cut into the paper slip of long 16 * 1cm; Chromatography carries out in the test tube of 20cm * 3cm; The concrete operations step is as follows:
1. add each solvent systems developping agent 4ml respectively in every test tube, seal, allow the steam of developping agent be full of whole exhibition sheaf space with soft rubber ball;
2. get above-mentioned tennecetin crude extract sample, at an end of filter paper bar point sample several times, the edge point limit is with hot blast drying under automatic hand dryer at every turn with micropipet or kapillary, and applied sample amount is 15 μ l, and the diameter of point sample is advisable to be no more than 5mm;
3. after treating that sample on the filter paper blots, earlier it is suspended in vitro,, makes filter paper in the steam of developping agent behind the balance 0.5h, its point sample end is dipped in the developping agent up exhibition layer, solvent is directly contacted with sample with the airtight mouth of pipe of soft rubber ball; When treating developping agent near the forward position, taking-up filter paper bar is suspended to dry in the stink cupboard or with blowing hot wind solvent is tried one's best and volatilizees;
4. the square glass dish with customization prepares the PDA flat board under aseptic condition, and the spore suspension of getting an amount of cultured Botrytis cinerea ACCC30091 is uniformly coated on the flat board; Filter paper bar behind the chromatography is affixed on the flat board successively, and room temperature leaves standstill about 30min, and the active substance infiltration of corresponding site on the paper slip is diffused in the substratum; Take out paper slip with aseptic pincet, and its position on flat board of mark; Flat board is put 25 ℃ of constant incubators cultivation 48h carry out the biology development;
5. according to the biological activity result displayed, measure the exhibition layer distance of effective constituent under different pH, by following formula computation migration rate Rf value, the pH chromatogram draws:
The Rf=initial point arrives the distance of solvent front to the distance/initial point at inhibition zone center.
Ply of paper is analysed the result and is shown, the collection of illustrative plates basically identical of the active substance of 2 bacterial strains, and tracing not in solvent II and solvent VI does not move among the solvent VII, and promptly the Rf value is 0 (Fig. 3), and it is close that this and the ply of paper of polyene antibiotics in eight solvent systemss are analysed collection of illustrative plates.This active substance that shows its generation may belong to polyene antibiotics.Wherein in solvent II I, when the point sample amount is big, occur two antibacterial zones of big (Rf=0.333) little (Rf=0.754) once in a while, show that this active substance has two active ingredients at least.
2, UV scanning
The tennecetin crude extract sample that step () is obtained dilutes 40 times with deionized water, does blank with deionized water, adopts the UV-VIS of Hitachi 3010 ultraviolet-visible pectrophotometers to carry out full wavelength scanner in 190nm～400nm scope.
In the ultra-violet absorption spectrum of 2 bacterial strain crude extracts, though impurity peaks is more, but at 292nm, 305nm and 319nm place tangible three absorption peaks (Fig. 4) are arranged, repeatedly repeat this test and find that the peak value of the big more absorption peak of concentration of sample is high more, show that content of active substance is high more.These three typical absorption peaks are peculiar characteristic peaks of tetraenes microbiotic in the polyenoid class, and this is consistent with the measurement result that Czech eight solvent systems ply of papers are analysed.
According to above-mentioned analysis, determine that tentatively the active principle of 2 active substances that bacterial strain produced belongs to the tetraenes microbiotic.
Three, the separation and purification of tennecetin crude extract
Carry out progressively separation and purification by macroporous resin column chromatography, silica gel column chromatography and high performance liquid chromatograph HPLC.
1, macroporous resin adsorption column chromatography
For streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653, select 40cm * 2.6cm glass chromatography column for use, D4006 macroporous resin (Tianjin Chemical Plant of Nankai Univ.), macroporous resin is mixed well with appropriate amount of deionized water after by producer pre-treatment being described, slowly add and be equipped with in the chromatography column of 1/3 volumes of deionized water, simultaneously from the column bottom at the uniform velocity slowly to emit distilled water, make that liquid level remains at above the resin layer in the post.Be filled to about 3/4 post height, natural subsidence 6～10h, making the dress column volume after the balance is 150ml.
Tennecetin crude extract and resin are carried out dynamic adsorption by 2: 1 volume ratio; After absorption finishes, close the constant flow pump that is connected with post, chromatography column leaves standstill 2h, use 50% (volumn concentration) methyl alcohol of the deionized water of 1 times of column volume, 1.5 times of column volumes and 80% (volumn concentration) ethanol elution of 2.5 times of column volumes then successively, elution speed is 0.5ml/min, be in charge of the collection elutriant with automatic Fraction Collector, every pipe 15ml.With S. cervisiae ACCC20036 is indicator, utilizes the filter paper agar diffusion method to detect every pipe elutriant activity.
The result shows that active eluant concentrates on the 35th～45 pipe (being 3.5 times of elutriants between column volume to the 4.5 times column volume from discharge promptly).
For streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, select 40cm * 2.6cm glass chromatography column for use, X-5 macroporous resin (Tianjin Chemical Plant of Nankai Univ.).With above-mentioned same method dress post.
Get tennecetin crude extract and resin and carry out dynamic adsorption by 1: 1 volume ratio.Elution process is: the deionized water wash-out of 2 times of column volumes is removed partial pigment and a large amount of water-soluble impurity; 30% (volumn concentration) methanol-eluted fractions of 2 times of column volumes element that discolors; Use 70% (volumn concentration) ethanol elution activeconstituents of 2 times of column volumes at last.Be in charge of the collection elutriant, every pipe 15ml, the filter paper method is carried out determination of activity.
The result shows that active eluant concentrates on the 48th～56 pipe (i.e. elutriant between 4.8 times of column volume to 5.6 times column volumes).
Merge the activated elutriant of each bacterial strain respectively, supply next step separation behind 45 ℃ of following concentrating under reduced pressure.
2, silica gel adsorption column chromatography
For streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653, select 40cm * 2.6cm glass chromatography column for use, 200～300 order silica gel are that 1: 2: 1 propyl carbinol, the mixed solution of first alcohol and water are elutriant with volume ratio; Get about 150g silica gel and soak 3h with deionized water, the fine particle that inclines, the B vacuum filtration is removed moisture; Soak 12h with 6mol/L HCl again, be washed till neutrality with deionized water then, vacuum is drained; Spend the night with soaked in absolute ethyl alcohol, vacuum is drained; Face with the preceding 2h that under 120 ℃, activates, be dried to constant weight; Pack in the chromatography column elutriant of 1/3 column volume slowly adds the silica gel that mixes with elutriant then, stops to add when high to 3/4 post approximately, leaves standstill 6～10h, makes the slow sedimentation of silica gel.With the elutriant of 2～3 times of volumes flow velocity flushing cylinder, make it balance then with 1mL/min.Column volume after the balance is 150ml.The active wash-out concentrated solution of the macroporous resin 10ml upper prop of getting step 1 carries out dynamic adsorption, carries out wash-out with the elutriant of 2～3 times of column volumes by the flow velocity of 0.5ml/min, is in charge of the collection elutriant with automatic Fraction Collector, every pipe 5ml.With S. cervisiae ACCC20036 is indicator, utilizes the filter paper agar diffusion method to detect every pipe elutriant activity.
The result shows: the active ingredient in its elutriant concentrates on the 10th～18 pipe (i.e. elutriant between 0.33 times of column volume to 0.6 times column volume).
For streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, select 40cm * 2.6cm glass chromatography column for use, 100 orders～200 order silica gel are that the mixed solution of 8: 1: 1 ethanol, ammoniacal liquor and water is an elutriant with volume ratio; Use the same method and adorn post, go up sample absorption, wash-out and carry out the activity detection.
The result shows: the active ingredient in its elutriant concentrates on the 7th～36 pipe (i.e. elutriant between 0.23 times of column volume to 1.2 times column volume).
The active eluant that merges each bacterial strain respectively, 45 ℃ of vacuum decompressions concentrate, and concentrated solution is used for next step separation.
3, preparation HPLC separation and purification
Adopt LC-9101 type cycles prepare HPLC, JAIGEL-ODS-AP type SP-120-15 preparative column.
Get the active wash-out concentrated solution behind the silica gel column chromatography of step 2, filter automatic sampler sample introduction, each sample size 6ml with 0.45 μ m millipore filter; With methyl alcohol: water (volume ratio is 7: 3) is that moving phase is separated, and utilizes the UV detector to detect at wavelength 305nm place and forms separating spectrum automatically; Utilize run tank to collect each pairing elutriant in curve peak in the collection of illustrative plates respectively; With S. cervisiae ACCC20036 is indicator, utilizes the filter paper agar diffusion method to detect every pipe elutriant activity.For strains A 01 CGMCC No.1653, separate in succession 2 times with the flow rate pump of 2ml/min; For strains A 02 CGMCC No.1654, after carrying out separating the first time with the flow rate pump of 2ml/min, the flow rate pump with 3ml/min separates 2 times in succession again.
Experimental result shows, the HPLC first time of strains A 01 CGMCC No.1653 separates and detects 6 peaks altogether, is that the peak of 57.399min is main active peak (Fig. 5 A) in retention time wherein; The separation detection second time that it is carried out is that the peak of 38.399min is active peak (Fig. 5 B) to retention time, and its relative peak area per-cent is 99.503%, shows that sample purity reaches more than 99.503%.The HPLC separation first time of strains A 02 CGMCC No.1654 detects 30 peaks altogether, and wherein retention time is that the strong absorption peak of 57.866min is active peak (Fig. 6 A), and its relative peak area is 35.121%; To its separation detection to the 6 peak second time that carries out, wherein retention time is that the peak of 41.699min is active peak, and its relative peak area is 97.020%; Separation detection is that the peak of 39.766min is active peak (Fig. 6 B) to retention time for the third time, is 99.845% by its purity of calculated by peak area.
Final sample to 2 bacterial strains carries out vacuum concentration respectively, is white in color after the drying or cream-colored powder.Utilize Tianjin, island analysis mode HPLC to adopt following two kinds of methods that it is carried out the purity checking respectively.
1) sample that takes a morsel is dissolved in 70% methanol aqueous solution, is that moving phase is carried out gradient elution with methyl alcohol (A) and water (B).Chromatographic condition is: C
18Reversed-phase column, 30 ℃ of column temperatures, the UV detector detects wavelength 305nm, and SIL-10ADVP automatic sampler sample introduction 10 μ l are with the flow velocity wash-out 60min of 1ml/min.The gradient elution step is as follows:
| Time (branch) | A(％) | B(％) |
| 0～10 11～20 21～30 31～40 41～50 51～60 | 35 50 65 80 95 100 | 65 50 35 20 5 0 |
The result shows that elution curve is single peak, illustrates that it is an one-component, and purity reaches the requirement of determination of chemical structure.
2) sample that takes a morsel is dissolved in 70% methanol aqueous solution, is that moving phase is carried out gradient elution with acetonitrile (A) and water (B).Chromatographic condition is: C
18Reversed-phase column, 30 ℃ of column temperatures, the UV detector detects wavelength 305nm, and SIL-10ADVP automatic sampler sample introduction 10 μ l are with the flow velocity wash-out 60min of 1ml/min.The gradient elution step is as follows:
| Time (branch) | A(％) | B(％) |
| 0～15 16～30 31～45 46～60 | 35 50 70 100 | 65 50 30 0 |
The result shows that elution curve is single peak, illustrates that it is an one-component, and purity reaches the requirement of determination of chemical structure.
Two kinds of unanimities as a result that verification method obtains.
Four, the parsing of purification of samples chemical structure is identified
1, ultra-violet absorption spectrum (UV)
The sample denier of above-mentioned steps three purifying is dissolved in the ultrapure water, is that blank carry out full wavelength scanner with the ultrapure water with the UV-VIS of Hitachi 3010 ultraviolet-visible pectrophotometers in 190nm～400nm wavelength region, forms ultraviolet absorpting spectrum automatically.
By the UV scanning collection of illustrative plates as seen, the ultraviolet absorption peak of 2 bacterial strain active substances all shows typical tetraenes microbiotic spectral pattern, near the absorption peak that typical conjugation tetraene chromophoric group is promptly all arranged wavelength 281nm, 291nm, 305nm and 319nm, its medium wavelength 305nm place absorption value maximum, 281nm place absorption value minimum (Fig. 7 A and 7B) has verified that once more this active substance belongs to the tetraene microbiotic in the polyenoid class.
2, infrared absorption spectrum (IR)
Adopt the KBr pressed disc method, carry out 400cm with German BRUKER company's T ENSOR 27 Fourier infrared spectrographs
-1-4000cm
-1The zone interscan.
The infrared spectra of the tennecetin purification of samples that strains A 01 CGMCC No.1653 is produced shown in Fig. 8 A, v wherein
Max3593,3493,3280cm
-1For N-H in the molecule and-the stretching vibration charateristic avsorption band of OH; v
Max3017,2978,2940cm
-1For in the molecule-CH
3With-CH
2The stretching vibration charateristic avsorption band; v
Max1716cm
-1Strong absorption characteristic peak for C=O; v
Max1634,1570cm
-1Charateristic avsorption band for the C=C stretching vibration; In addition, at v
Max1401,1267,1190,1107,1060,1003,885,847,798cm
-1At the place absorption peak is arranged all.
The infrared spectra of the purification of samples of strains A 02 CGMCC No.1654 shown in Fig. 8 B, v wherein
Max3416.78cm
-1Charateristic avsorption band for-OH; v
Max3288.23cm
-1Stretching vibration charateristic avsorption band for N-H; v
Max2940.44 and 2980.27cm
-1Be-CH
3Charateristic avsorption band; v
Max3017.23cm
-1Be-CH
2Charateristic avsorption band; v
Max1715.38cm
-1Show the strong absorption peak of typical carbonyl; v
Max1571.44cm
-1The strong absorption peak of performance-C=C-; v
Max1634.40cm
-1The weak absorption peak of performance-C=C-.
3, high resolution mass spectrum
Adopt the German BRUKER ultrahigh resolution 9.4T of company mixed type level Four bar fourier tandom mass spectrometer (9.4TQ-FT-MS); Condition: capillary 4000, Dry Gas:4.0l/s, the source temperature: 180 ℃, scan range:300～2000, syringe pump:1.5ml/min, data analysis software is Bruker DaltonicsDataAnalysis 3.4.
The result shows, in the spectrogram that the tennecetin purification of samples that A01 CGMCC No.1653 is produced is analyzed, the positive ion detection mode detects molecular ion peak [M+Na]
+Compound (Fig. 9 A-b) for m/z=688.2938; The negative ion detection mode detects molecular ion peak [M-H]
-Compound (Fig. 9 A-a) for m/z=664.2976.
In the collection of illustrative plates that the tennecetin purification of samples that A02 CGMCC No.1654 is produced is analyzed, adopt the positive ion detection mode to detect adduct ion [M+Na]
+Be m/z688.2937 (Fig. 9 B-b); Adopt the negative ion detection mode to detect quasi-molecular ion [M-H]
+Compound (Fig. 9 B-a) for m/z664.2975; Adopt the positive and negative ion detection mode that the pure product of A02 antagonism product are carried out check and analysis, determine that 664.2975 are molecular ion peak.
In sum, analysis determines that the molecular formula of the main active component of 2 active substances that bacterial strain produced is C
33H
47NO
13, molecular weight is 665; By formula: degree of unsaturation (n)=1+Nc+ (Nn-Nh)/2 (Nc: carbonatoms; Nn: nitrogen-atoms number; Nh: number of hydrogen atoms) degree of unsaturation of calculating its molecular formula is 11, shows and contains a plurality of unsaturated link(age)s and ring etc. in its molecular structure.
4, nuclear magnetic resonance spectrum (NMR)
With deuterium generation-dimethyl formamide (d-DMF) is solvent, is interior mark with tetramethylsilane (TMS), measures under the room temperature.
Adopting Bruker AVANCE DRX-500 nuclear magnetic resonance spectrometer (German Bruker spectral instrument company), is solvent with deuterium generation-dimethyl formamide (d-DMF), and tetramethylsilane (TMS) is interior mark, carry out hydrogen compose (
1HNMR) and carbon spectrum (
13CNMR) mensuration; The former resonant frequency is 500.1325156MHz, sampling number 32768 times; Latter's resonant frequency 125.7577612MHz, sampling number are 65536 times.
Experimental result shows, the active result pure sample product that produced at A01 CGMCC No.1653
1In the H-NMR collection of illustrative plates (Figure 10 A),, make that the hydrogen on the carboxyl is neutralized owing to adopt DMF to make solvent, the peak of δ=12 disappears, increased by two place's solvent peaks of δ=3 and 8 simultaneously, the hydrogen on the conjugated polyene has been represented near one group of peak δ=6, and one group of peak about δ=5 is representation hydroxy then;
13In the C-NMR collection of illustrative plates (Figure 10 B), the peak of intensity maximum system is produced by solvent DMF, and δ=180 and 165 peak show the existence of carboxyl and ester group in the molecule, and one group of peak about δ=130 is then produced by conjugated polyene; When θ=135 °, CH and CH in the DEPT spectrogram
3The spectral line at peak up, CH
2The spectral line at peak has 5-CH down in the molecule
2
The nucleus magnetic resonance of the active result pure sample product that produced from A02 CGMCC No.1654
13As can be seen, the chemical shift (δ 165.217) of a carboxyl carbon atom is arranged in the molecule in the C collection of illustrative plates (Figure 11 B); The chemical shift of a carbonylic carbon atom (δ 178.603); The chemical shift (δ 66.102～70.326) of one group of carbon atom that links to each other with hydroxyl of the chemical shift of one group of conjugation tetraene carbon atom (δ 125.089～145.447); Sugar ring last five carbon atom resonance peaks (δ 71.194～97.894) methine carbon atom resonance peaks (δ 18.062, and δ 20.360).The nucleus magnetic resonance of 500 megahertzes
1As can be seen, ((δ 5.686～6.625ppm) in chemical shift CH=CH-) for the proton hydrogen that links to each other with four two keys on the polyenoid ring in the H collection of illustrative plates (Figure 11 A); (δ 4.187～4.741ppm) for the chemical displacement value of the proton hydrogen in five hydroxyls; (δ 1.274～2.439ppm) in the chemical shift of the proton hydrogen in five methylene radical and two methyl.
The above-mentioned experimental data of analysis-by-synthesis, the main active component of judging these two active results that bacterial strain produced is a tennecetin, its chemical structural formula is:
Sequence table
<160>1
<210>1
<211>1270
<212>DNA
＜213〉streptomyces lydicus (Streptomyces lydicus)
<400>1
gggtctaata?ccggatacga?cacggggtcg?catgacctcc?gtgtggaaag?ctccggcggt 60
gaaggatgag?cccgcggcct?atcagcttgt?tggtggggtg?atggcctacc?aaggcgacga 120
cgggtagccg?gcctgagagg?gcgaccggcc?acactgggac?tgagacacgg?cccagactcc 180
tacgggaggc?agcagtgggg?aatattgcac?aatgggcgaa?agcctgatgc?agcgacgccg 240
cgtgagggat?gacggccttc?gggttgtaaa?cctctttcag?cagggaagaa?gcgagagtga 300
cggtacctgc?agaagaagcg?ccggctaact?acgtgccagc?agccgcggta?atacgtaggg 360
cgcaagcgtt?gtccggaatt?attgggcgta?aagagctcgt?aggcggcttg?tcacgtcgga 420
tgtgaaagcc?cggggcttaa?ccccgggtct?gcattcgata?cgggcaggct?agagttcggt 480
aggggagatc?ggaattcctg?gtgtagcggt?gaaatgcgca?gatatcagga?ggaacaccgg 540
tggcgaaggc?ggatctctgg?gccgatactg?acgctgagga?gcgaaagcgt?ggggagcgaa 600
caggattaga?taccctggta?gtccacgccg?taaacgttgg?gaactaggtg?tgggcgacat 660
tccacgtcgt?ccgtgccgca?gctaacgcat?taagttcccc?gcctggggag?tacggccgca 720
aggctaaaac?tcaaaggaat?tgacgggggc?ccgcacaagc?agcggagcat?gtggcttaat 780
tcgacgcaac?gcgaagaacc?ttaccaaggc?ttgacataca?ccggaaaacc?ctggagacag 840
ggtccccctt?gtggtcggtg?tacaggtggt?gcatggctgt?cgtcagctcg?tgtcgtgaga 900
tgttgggtta?agtcccgcaa?cgagcgcaac?ccttgttctg?tgttgccagc?atgcccttcg 960
gggtgatggg?gactcacagg?agactgccgg?ggtcaactcg?gaggaaggtg?gggacgacgt 1020
caagtcatca?tgccccttat?gtcttgggct?gcacacgtgc?tacaatggcc?ggtacaatga 1080
gctgcgatac?cgcgaggtgg?agcgaatctc?aaaaagccgg?tctcagttcg?gattggggtc 1140
tgcaactcga?ccccatgaag?tcggagttgc?tagtaatcgc?agatcagcat?tgctgcggtg 1200
aatacgttcc?cgggccttgt?acacaccgcc?cgtcacgtca?cgaaagtcgg?taacacccga 1260
agccggtggc 1270
Claims (9)
1. a method for preparing tennecetin is that fermentation streptomyces lydicus (Streptomyces lydicus) obtains tennecetin; Described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653 or streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654.
2. method according to claim 1, it is characterized in that: the consisting of of the fermention medium of the described streptomyces lydicus that ferments (Streptomyces lydicus) A01 CGMCC No.1653: contain in every liter of substratum: analysis for soybean powder 2%, peptone 0.5%, Zulkovsky starch 0.5%, glucose 2%, corn steep liquor 0.25%, (NH
4)
2SO
40.25%, MgSO
4.7H
2O 0.025%, K
2HPO
40.002%, NaCl 0.4%, CaCO
30.2%, all the other are water; Described percentage composition is the quality percentage composition.
3. method according to claim 1 and 2, it is characterized in that: the condition of the described streptomyces lydicus that ferments (Streptomyces lydicus) A01 CGMCC No.1653 is: under 28 ℃ condition, cultivate with the rotation radius of 13mm, the speed oscillation of 180rpm.
4. method according to claim 1, it is characterized in that: the consisting of of the fermention medium of the described streptomyces lydicus that ferments (Streptomyces lydicus) A02 CGMCC No.1654: contain the extracting solution that obtains by the 15g soybean grain in every liter of substratum, the 5g peptone, 10g sucrose, 10g starch, 2.5g ammonium sulfate, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate, all the other are water; The pH of the fermention medium of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is 7-8.
5. according to claim 1 or 4 described methods, it is characterized in that: the condition of the described streptomyces lydicus that ferments (Streptomyces lydicus) A02 CGMCC No.1654 is: under 31 ℃ condition, cultivate with the rotation radius of 13mm, the speed oscillation of 240rpm.
6. method according to claim 1 is characterized in that: the step that also comprises the separation and purification tennecetin in the described method.
7. method according to claim 6 is characterized in that: described purification procedures comprises:
1) collects the fermented liquid that the described streptomyces lydicus of fermentation (Streptomyces lydicus) obtains, precipitate, collect supernatant liquor with dehydrated alcohol;
2) described supernatant liquor is carried out the resin absorption chromatography successively, the silica gel adsorption chromatography separates with high performance liquid chromatography, obtains tennecetin.
8. method according to claim 7 is characterized in that: described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A01 CGMCC No.1653;
Described resin absorption chromatography adopts the D4006 macroporous resin, elution requirement is 50% methyl alcohol of the deionized water of using 1 times of column volume successively, 1.5 times of column volumes and 80% ethanol elution of 2.5 times of column volumes, and collecting discharge is 3.5 times of elutriants between column volume to the 4.5 times column volume; Described percentage composition is a volumn concentration;
Described silica gel adsorption chromatography adopts 200～300 order silica gel, and elution requirement is to be that 1: 2: 1 propyl carbinol, the mixed solution of first alcohol and water carry out wash-out with volume ratio, and the collection discharge is 0.33 times to the 0.6 times elutriant between the column volume;
Described high performance liquid chromatography separates employing cycles prepare type high performance liquid chromatograph and JAIGEL-ODS-AP type SP-120-15 preparative column, with volume ratio is that the mixed solution of 7: 3 first alcohol and water is a moving phase, separated secondary, retention time was the elution peak of 38.399min when collection separated for the second time.
9. method according to claim 7 is characterized in that: described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654;
Described resin absorption chromatography adopts the X-5 macroporous resin, and elution requirement is 30% methyl alcohol of the deionized water of using 2 times of column volumes successively, 2 times of column volumes and 70% ethanol elution of 2 times of column volumes, and collecting discharge is 4.8 times to the 5.6 times elutriants between the column volume; Described percentage composition is a volumn concentration;
Described silica gel adsorption chromatography adopts 100～200 order silica gel, and elution requirement is to be that the mixed solution of 8: 1: 1 ethanol, ammoniacal liquor and water carries out wash-out with volume ratio, and the collection discharge is 0.23 times to the 1.2 times elutriant between the column volume;
Described high performance liquid chromatography separates employing cycles prepare type high performance liquid chromatograph and JAIGEL-ODS-AP type SP-120-15 preparative column, with volume ratio is that the mixed solution of 7: 3 first alcohol and water is a moving phase, separate three times, retention time was the elution peak of 39.766min when collection separated for the third time.
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