CN100448984C - Streptomyces vietnamensis - Google Patents
Streptomyces vietnamensis Download PDFInfo
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- CN100448984C CN100448984C CNB2006100341497A CN200610034149A CN100448984C CN 100448984 C CN100448984 C CN 100448984C CN B2006100341497 A CNB2006100341497 A CN B2006100341497A CN 200610034149 A CN200610034149 A CN 200610034149A CN 100448984 C CN100448984 C CN 100448984C
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Abstract
The present invention relates to microbiological technology, and is especially one new streptomycete strain capable of generating violet blue pigment. The new streptomycete strain is named as Streptomyces vietnamensis and has the preservation number of CCTCCM No. 205143. Streptomyces vietnamensis can produce great amount of soluble violet blue pigment in the culture medium comprising ISP2, ISP3, Gao's No 1 synthetic agar, etc. and the soluble violet blue pigment has high ultraviolet stability, high heat stability, relatively high heat resistance and great potential for developing new efficient non-toxic natural pigment.
Description
[affiliated technical field]
The present invention relates to a kind of new streptomycete, belong to microbial technology field.
[background technology]
In recent years, the development research of nontoxic, no teratogenesis disease, the strong production by biological natural pigment of stability more and more is subject to people's attention.Microbe-derived kind of pigment is many, and quantity is big, and is with low cost, and production technique is simple, have plant pigments and animal pigment incomparable advantage.The kind of pigment that utilizes the production of occurring in nature microbial fermentation is than horn of plenty, but the natural blue kind of pigment of producing both at home and abroad is less, and rare especially with the violet blue pigment of microbial fermentation production.The violet blue pigment has very high added value as one of 3 basic pigments, can be deployed into multiple color tones with natural pigment red, yellow strain, makes natural pigment abundanter.Therefore, obtain natural violet blue pigment by the microbial fermentation approach is the research focus always.
[summary of the invention]
The purpose of this invention is to provide a kind of novel species streptomycete---streptomyces vietnamensis that can produce the violet blue pigment.
The soil of gathering in the D φ O Xong pha forest rhizosphere soil of streptomyces vietnamensis system of the present invention by Vietnam obtains through separation and Culture.This bacterium culture condition is: (1) substratum: adopt synthetic No. 1 nutrient agar of Gao Shi.Concrete prescription is: Zulkovsky starch 20.0g, saltpetre (KNO
3) 1.0g, dipotassium hydrogen phosphate (K
2HPO
4) 0.5g, sal epsom (MgSO
47H
2O) 0.5g, sodium-chlor (NaCl) 0.5g, ferrous sulfate (FeSO
47H
2O) 0.01g, agar 20.0g, water 1000ml; (2) pH7.2～7.4; (3) culture temperature: 28 ℃.This bacterium called after streptomyces vietnamensis Streptomyces vietnamensis (viet.nam.en ' sis.N.L.adj.), be numbered GIMV4.0001.This bacterial classification is in specified depositary institution of State Intellectual Property Office China typical culture collection center and the preservation of external culture presevation unit Japan IAM DSMZ, preservation date is respectively on December 9th, 2005 and on February 16th, 2006, and preservation registration number is CCTCC No:M 205143 and IAM15340.
Streptomyces vietnamensis of the present invention (Streptomyces vietnamensis GIMV4.0001) has following character:
1. morphological specificity
As seen, spore is many under scanning electron microscope, and spore chain is straight, branch, and spore long column shape, there is one deck sheath on the surface.
2. the feature on various substratum
Cultural characteristic on different substratum such as the synthetic nutrient agar of yeastex malt extract agar (ISP2), oatmeal agar (ISP3) and Gao Shi sees Table 1.
The cultural characteristic of table 1 streptomyces vietnamensis on different substratum
| Feature | Yeast extract paste malt extract nutrient agar (ISP2) | Oat nutrient agar (ISP3) | Inorganic salt-Starch Agar substratum (ISP4) | Glycerine asparagine nutrient agar (ISP5) | Czapek agar medium | Gao Shi synthesizes No. 1 nutrient agar |
| Growing state | Good | Good | Generally | Generally | A little less than | Good |
| Produce the spore situation | Good | Good | Generally | Generally | A little less than | Good |
| The aerial hyphae color | White | White | White | White | White | White |
| The substrate mycelium color | Reddish-brown | Reddish-brown | Reddish-brown | Reddish-brown | The tangerine grey | Reddish-brown |
| The soluble pigment color | The violet blueness | The violet blueness | The violet blueness | The violet blueness | - | The violet blueness |
The violet blue pigment that streptomyces vietnamensis of the present invention is produced on substratum is soluble in basic solution and methyl alcohol, be slightly soluble in acidic solution (the strong more then solubleness of acidity is low more) and the big organic solvent of ethanol isopolarity, be insoluble to acetone, organic solvent that the ether isopolarity is little; This pigment is in the scope of pH≤5, and color is more stable; Pigment stability under UV-light is stronger, this pigment better heat stability, ability higher temperatures.
3. physiological and biochemical property
This bacterial strain amphimicrobian, Gram-positive, the catalase positive produces melanochrome and H
2S, milk peptonizes, and gelatine liquefication is fast, can hydrolyzed starch, can reduce nitrate.
4. utilization of carbon source
Can utilize melibiose, sorbose, sucrose, glucose, D-fructose, wood sugar, D-semi-lactosi, pectinose, rhamnosyl to be carbon source, but can not utilize D-N.F,USP MANNITOL.
5. other character
28 ℃ of optimum growth temperatures; Cell walls contains LL diamino pimelic acid, belongs to cell walls I type; Contain seminose, semi-lactosi and ribose in the cell hydrolyzate.Cell walls lipid acid contains TETRADECONIC ACID, palmitic acid, oleic acid, 16 carbon monoenoic acids, linolic acid and stearic acid.
There were significant differences for its morphological specificity of streptomyces vietnamensis of the present invention and other streptomycete, and the streptomycete that reaches 98%-99% with the gene order homology is compared, also different fully.Bikini streptomycete (Streptomyces bikiniensis) reaches 99.4% with streptomyces vietnamensis 16SrDNA sequence homology of the present invention, but morphological specificity is different fully, and bikini streptomycete fibrillae of spores is straight, gentle bent.Spore oval, Long Circle, smooth surface, no soluble pigment or mark amount yellow on the ISP substratum; Scorched earth (knowing many) streptomycete (Streptomyces showdoensis) reaches 99% with streptomyces vietnamensis 16SrDNA sequence homology of the present invention, but scorched earth (knowing many) streptomycete (Streptomyces showdoensis) does not produce soluble pigment on the ISP substratum; Streptomyces viridobrunneus and streptomyces vietnamensis 16SrDNA sequence homology of the present invention reach 99%, but Streptomyces viridobrunneus produces green soluble pigment; Other reach 98% streptomycete kind with streptomyces vietnamensis 16SrDNA sequence homology of the present invention and also have significantly different in some aspects with streptomyces vietnamensis, Streptomyces tanashiensis, Streptomyces cinereoruber, Streptomyces filamentosus andStreptomyces venezuelae chromogenesis (Shirling﹠amp not on the ISP substratum; Gottlieb, 1968a, 1968b, 1969); Streptomyces exfoliatus aerial hyphae pink (Shirling﹠amp; Gottlieb, 1968a); Streptomyces rubiginosohelvolus does not produce or produces light yellow soluble pigment (Shirling﹠amp on the ISP substratum; Gottlieb, 1968b); Streptomyces crystallinus produces light brown or chocolate soluble pigment (Williams, 1989).The streptomycete bacterial strain Microbiological Characteristics that streptomyces vietnamensis of the present invention and its homology are the highest relatively see Table 2, and with the part correlation bacterial strain according to the 16SrDNA sequence construct phylogenetic tree, see accompanying drawing 5.
The streptomycete bacterial strain Microbiological Characteristics that table 2 streptomyces vietnamensis and its homology are the highest compares
| Characteristic | Streptomyces vietnamensis S.vietnam GIMV4.0001 | Bikini streptomycete Streptomyces bikiniensis | Scorched earth (knowing many) streptomycete Streptomyces showdoensis | Green brown streptomycete Streptomyces viridobrunneus |
| Colony colour on the ISP2 substratum | Canescence | Grey | Yellow-gray | Grey |
| Spore shape | Long column shape | Olive shape | Ellipsoid or cylindricality | Ellipse or Long Circle |
| The spore chain form | Long and straight | Straight or little song | Long, straight or little song | Straight chain or song |
| The color of soluble pigment | The violet blueness | - | - | Green |
| Milk solidifies | + | W | W | w |
| Milk peptonizes | + | + | + | + |
| The starch hydrolysis | + | + | + | |
| Grow on the Mierocrystalline cellulose | - | ND | - | - |
| Whether produce H 2S | + | + | + | + |
| Whether produce melanochrome | + | + | + | ND |
| Melibiose | + | ND | ND | ND |
| Glucose | + | + | + | + |
| Sorbose | + | ND | - | ND |
| Sucrose | + | - | W | + |
| D-fructose | + | + | + | ND |
| Wood sugar | + | + | + | ND |
| The D-semi-lactosi | + | ND | + | ND |
| D-N.F,USP MANNITOL | - | - | - | - |
Annotate: in the table+expression is positive, and-expression is negative, and W represents a little less than the reaction that ND represents not measure.
[description of drawings]
Accompanying drawing 1: the 16SrDNA sequence of streptomyces vietnamensis bacterial strain of the present invention.
Accompanying drawing 2: the chromatogenous photo of streptomyces vietnamensis of the present invention.
Accompanying drawing 3: the electron scanning micrograph of streptomyces vietnamensis spore chain of the present invention, left figure: 2 μ m, amplify 10000 times; Right figure: 10 μ m, amplify 2000 times.
Accompanying drawing 4: the electron scanning micrograph of streptomyces vietnamensis spore of the present invention (amplifying 10000 times).
Accompanying drawing 5: streptomyces vietnamensis of the present invention and part correlation bacterial strain are according to the phylogenetic tree of 16SrDNA sequence construct.
[embodiment]
Embodiment 1: the isolation cultivation method of streptomyces vietnamensis
Herborization rhizosphere 0-30cm soil sample from Vietnam D φ o Xongpha virgin forest, get 10g soil and put into the triangular flask (band bead) that the 90mL sterilized water is housed, vibration is 30 minutes under the 180rpm rotating speed, leave standstill to get after 10 minutes and get 0.1mL after 10 times of the upper strata liquid dilutions and be uniformly coated on the synthetic No. 1 nutrient agar plate of Gao Shi, the upset flat board places 28 ℃ of incubators to cultivate several days, can obtain streptomyces vietnamensis CCTCC No:M 205143 of the present invention.
Gao Shi synthesizes No. 1 nutrient agar:
Zulkovsky starch 20.0g, saltpetre (KNO
3) 1.0g, dipotassium hydrogen phosphate (K
2HPO
4) 0.5g, sal epsom (MgSO
47H
2O) 0.5g, sodium-chlor (NaCl) 0.5g, ferrous sulfate (FeSO
47H
2O) 0.01g, agar 20.0g, water 1000ml, pH7.2～7.4.
Culture temperature is 28 ℃.
Carry out fermentation culture after 7 days with streptomyces vietnamensis of the present invention with synthetic No. 1 nutrient agar of Gao Shi, pH transfers to 9 with its fermented liquid, behind the centrifugal 5min of 8000rpm, get supernatant, damping fluid with pH9 dilutes 25 times with it again, recording violet blue pigment OD under 588nm is 0.5158, and promptly the look valency of pigment is 12.5 in this streptomyces vietnamensis fermented liquid.
Embodiment 2: the extracting method of streptomyces vietnamensis bacterial strain 16SrDNA
The present invention also provides extraction and the sequence measurement of the 16SrDNA of this streptomyces vietnamensis (Streptomyces vietnamensis GIMV4.0001) CCTCC JNo:M 205143, specifically carries out according to following operation:
1. agents useful for same
(1)5M NaCl
(2)TE1(10mM Tris，25mM EDTA)pH8.0
(3)TE2(10mM Tris，1mM EDTA)pH8.0
(4)10％SDS
(5) chloroform: primary isoamyl alcohol (24: 1)
(6) Virahol
(7)CTAB/NaCl
(8) Proteinase K mother liquor 20mg/ml
2.DNA extracting method
(1) mycelia is transferred in the mortar of the bacterium of going out, fully grinds.
(2) add 567ulTE and suspend, and add 30uL 10%SDS, 3uL 20mg/ml Proteinase K, mixing, 37 ℃ of incubations 1 hour.
(3) add 100ul 5mol/L NaCl, mixing.
(4) add 80ul CTAB/NaCl solution, mixing, 65 ℃ of incubations 20 minutes.
(5) use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, 10, centrifugal 10 minutes of 000rpm moves to clean centrifuge tube with supernatant liquor.
(6) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, get supernatant liquor and move in the clean pipe.
(7) add 0.6 times of volume Virahol, put upside down mixing, under the room temperature static 1 hour, deposit D NA.
Centrifugal 20 minutes of (8) 14,000rpm collect the DNA precipitation.
(9) the DNA precipitation adds 500uL 70% ethanol rinsing, natural air drying.
(10) add 50ul TE dissolving DNA ,-20 ℃ of preservations.
3.PCR amplification method
The universal primer that the pcr amplification of the 16SrDNA of streptomyces vietnamensis of the present invention is used: forward primer F27 (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and reverse primer 1522R (5 '-AAGGAGGTGAT CCAGCCGCA), the design of primer sequence is with reference to Edwards﹠amp; The article of Rogall (1989), primer is synthetic by Shanghai Ying Jun company.The used test kit that increases is bought the precious biotech firm in Dalian.
The pcr amplification system is:
The composition final concentration
Concentration response damping fluid 1 * T makes concentration
Each 0.2mmol/L of dNTP mixture
Taq archaeal dna polymerase 0.5～1.0U/50uL
Magnesium chloride (MgCl
2) 1.5mmol/L
Upstream primer 1umol/L
Downstream primer 1umol/L
Template 10
2～10
5Copy/50ul
Sterilized water complements to 50ul with reaction volume
The PCR response procedures
94℃ 5min
72℃ 2min
72℃ 7min
The sequencing of 16SrDNA entrusts the handsome bio tech ltd in Shanghai to carry out, and the accession number on the GenBank database is DQ311081, and test result is seen accompanying drawing 1.
Sequence table
＜110〉Guangdong Microbes Inst
＜120〉streptomyces vietnamensis
<140>200610034149.7
<141>2006-03-07
<160>1
<210>1
<211>1419
<212>DNA
＜213〉streptomyces vietnamensis (Streptomycex vietnamensis)
<400>1
GCAAGTCGAA CGATGAAGCC CCTCGGGGTG GATTAGTGGC GAACGGGTGA GTAACACGTG 60
GGCAATCTGC CCTTCACTCT GGGACAAGCC CTGGAAACGG GGTCTAATAC CGGATACGAC 120
CCGGGAAGGC ATCTTCCCGG GTGGAAAGCT CCGGCGGTGA AGGATGAGCC CGCGGCCTAT 180
CAGCTTGTTG GCGGGGTAAC GGCCCACCAA GGCGACGACG GGTAGCCGGC CTGAGAGGGC 240
GACCGGCCAC ACTGGGACTG AGACACGGCC CAGACTCCTA CGGGAGGCAG CAGTGGGGAA 300
TATTGCACAA TGGGCGCAAG CCTGATGCAG CGACGCCGCG TGAGGGATGA CGGCCTTCGG 360
GTTGTAAACC TCTTTCAGCA GGGAAGAAGC GCAAGTGACG GTACCTGCAG AAGAAGCGCC 420
GGCTAACTAC GTGCCAGCAG CCGCGGTAAT ACGTAGGGCG CAAGCGTTGT CCGGAATTAT 480
TGGGCGTAAA GAGCTCGTAG GCGGCTTGTC ACGTCGGGTG TGAAAGCCCG GGGCTTAACC 540
CCGGGTCTGC ATCCGATACG GGCAGGCTAG AGTGTGGTAG GGGAGATCGG AATTCCTGGT 600
GTAGCGGTGA AATGCGCAGA TATCAGGAGG AACACCGGTG GCGAAGGCGG ATCTCTGGGC 660
CATTACTGAC GCTGAGGAGC GAAAGCGTGG GGAGCGAACA GGATTAGATA CCCTGGTAGT 720
CCACGCCGTA AACGTTGGGA ACTAGGTGTT GGCGACATTC CACGTCGTCG GTGCCGCAGC 780
TAACGCATTA AGTTCCCCGC CTGGGGAGTA CGGCCGCAAG GCTAAAACTC AAAGGAATTG 840
ACGGGGGCCC GCACAAGCAG CGGAGCATGT GGCTTAATTC GACGCAACGC GAAGAACCTT 900
ACCAAGGCTT GACATATACC GGAAAGCATT AGAGATAGTG CCCCCCTTGT GGTCGGTATA 960
CAGGTGGTGC ATGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG 1020
AGCGCAACCC TTGTCCTGTG TTGCCAGCAT GCCCTTCGGG GTGATGGGGA CTCACAGGAG 1080
ACCGCCGGGG TCAACTCGGA GGAAGGTGGG GACGACGTCA AGTCATCATG CCCCTTATGT 1140
CTTGGGCTGC ACACGTGCTA CAATGGCCGG TACAAAGAGC TGCGATGCCG TGAGGCGGAG 1200
CGAATCTCAA AAAGCCGGTC TCAGTTCGGA TTGGGGTCTG CAACTCGACC CCATGAAGTC 1260
GGAGTTGCTA GTAATCGCAG ATCAGCATTG CTGCGGTGAA TACGTTCCCG GGCCTTGTAC 1320
ACACCGCCCG TCACGTCACG AAAGTCGGTA ACACCCGAAG CCGGTGGCCC AACCCCTTGT 1380
GGGAGGGAGC TGTCGAAGGT GGGACTGGCG ATTGGGACG 1419
Claims (1)
1, a kind of streptomycete, it is characterized in that called after streptomyces vietnamensis (5treptomyces vietnamensis GIMV4.0001), this bacterial classification is in China's typical culture collection center preservation, and preservation date is on December 9th, 2005, and preservation registration number is CCTCC NO:M 205143.
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| CN105255210B (en) * | 2015-09-30 | 2017-11-24 | 菏泽学院 | The extraction process of environment protecting blue pigment |
| CN110484576B (en) * | 2019-10-18 | 2020-01-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for increasing yield of duricin and duricin B |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088983A (en) * | 1992-11-30 | 1994-07-06 | 伊莱利利公司 | Produce streptomyces bacterial strain of antipyretic compound and preparation method thereof |
| CN1318642A (en) * | 2000-04-18 | 2001-10-24 | 财团法人生物技术开发中心 | New streptomycete strain and its use |
| WO2005038009A2 (en) * | 2003-10-17 | 2005-04-28 | Ranbaxy Laboratories Limited | Production of tacrolimus (fk-506) using new streptomyces species |
| CN1219877C (en) * | 2002-01-21 | 2005-09-21 | 云南省微生物研究所 | Streptomyces ces virdocyaneus sp nov |
-
2006
- 2006-03-07 CN CNB2006100341497A patent/CN100448984C/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088983A (en) * | 1992-11-30 | 1994-07-06 | 伊莱利利公司 | Produce streptomyces bacterial strain of antipyretic compound and preparation method thereof |
| CN1318642A (en) * | 2000-04-18 | 2001-10-24 | 财团法人生物技术开发中心 | New streptomycete strain and its use |
| CN1219877C (en) * | 2002-01-21 | 2005-09-21 | 云南省微生物研究所 | Streptomyces ces virdocyaneus sp nov |
| WO2005038009A2 (en) * | 2003-10-17 | 2005-04-28 | Ranbaxy Laboratories Limited | Production of tacrolimus (fk-506) using new streptomyces species |
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