CN1181189C - Pantemperature streptomycete C-3662 capable of secreting proteinase with fibrinolytic activity - Google Patents
Pantemperature streptomycete C-3662 capable of secreting proteinase with fibrinolytic activity Download PDFInfo
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- CN1181189C CN1181189C CNB011360062A CN01136006A CN1181189C CN 1181189 C CN1181189 C CN 1181189C CN B011360062 A CNB011360062 A CN B011360062A CN 01136006 A CN01136006 A CN 01136006A CN 1181189 C CN1181189 C CN 1181189C
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Abstract
The present invention relates to a streptomycete strain C-3662 separated from soil, which belongs to a streptomycete strain of eurythermic streptomycete by classification and identification. A slant culture only produces a transparent ring with fibrinolysis activity on a flat fibrin plate, and protease CGW-3 with fibrinolysis activity can be produced and secreted in an optimized fermentation culture medium.
Description
Invention field
The present invention relates to a kind of general temperature streptomycete bacterial strain C-3662 that screening obtains from soil with generation fibrinolytic protein enzyme CGW-3.
Background technology
Streptomycete is the microorganism that a class especially extensively exists in soil at nature.Streptomyces G
+Bacterium is with respect to G
-Its cell wall structure of bacterium (having double-layer outer membrane) is fairly simple, wall matter is thinner.Though most of bacterium all secretes multiple proteins (comprising various enzymes, as proteolytic enzyme, amylase, cellulase or the like),, generally, G
+Bacterium is than G
-The easier excretory protein of will treating of bacterium is sent outside the thalline, thereby G
+Bacterium is in general than G
-Bacterium secretes more protein in the environment of its life.It is pointed out that most G
-Bacterium is pathogenic bacterium for the mankind, and under normal circumstances most G
+Bacterium does not belong to pathogenic bacterium.
Though the various existence of streptomycete kind is extensive, but most streptomycetes are harmless, and, because streptomycete is the most antibiotic generation bacterium of finding up to now and is widely used in the microbiotic pharmaceutical industry, streptomycete has been made huge contribution for improving human life health level.
The cardiovascular and cerebrovascular thrombotic disease is the disease of a class serious threat human health, and its sickness rate still has the trend that progressively rises in the crowd.Current, an effective therapy for the treatment of this class disease is a thrombolytic therapy, promptly this patient is used the medicine with thrombolysis activity, for example urokinase, tissue-type plasminogen activator or the like.This class medicine produces certain even curative effect preferably in clinical use, but also has the problem of some simultaneously, is still waiting to improve such as the thrombolysis specificity of medicine; This class medicine especially the price of tissue-type plasminogen activator's or derivatives thereof also than higher, sometimes or even be difficult to accept.In addition, use this class medicine thrombolysis, the interval when taking place embolism to administration is short more, and expected effect is also just good more, but the route of administration of this class medicine is mainly intravenous injection, so bring certain inconvenience.Also has a class thrombolytic drug, they derive from animal or microorganism, has the active or direct fibrin degradation activity of Profibrinolysin in the human activin, as streptokinase, snake venom antithrombotic enzyme, Lumbrukinase etc., the curative effect that has in this class medicine is still imprecise, the side effect that has is bigger, and what have is subjected to certain restriction etc. on the raw-material source of production.
A direction of current research exploitation thrombolytic drug is that plasmin and plasminogen activator are extracted in screening, in the hope of finding novel thrombolytic drug from the biology in difference source especially microorganism.
Streptomycete had been a non-pathogenic bacteria both, produced, secretes multiple compound and/or protein again, also streptomycete have the favorable industrial production basis, so screening and to produce the effort of the required material of people constant always from streptomycete.As if yet the direction of this effort mainly concentrates in the screening such as the proteolytic enzyme of microbiotic, cellulase, amylase and some kind, report is seldom seen in the screening of proteolytic enzyme with fibrinolytic.
Obviously, screening has the bacterial strain that produces fibrinolytic protein enzyme ability from streptomycete, is a research and development that is worth carrying out.French scientist was in report in 1996: find the fermented liquid of the streptomycete bacterial strain of not naming from a strain and separation and purification have the proteolytic enzyme of fibrinolytic.Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences utilizes the fibrinolytic screening substances model of design voluntarily the same year, screening has obtained producing the streptomycete bacterial strain of fibrinolytic material from the soil of Yunnan, and separates from the fermented liquid of wherein Y405 bacterial strain and obtained SW-1.After preliminary study finds though SW-1 has good thrombolysis activity, to have apparent in view bleeding tendency side effect simultaneously.
Summary of the invention
The objective of the invention is, from soil, screen streptomycete bacterial strain, and the streptomycete bacterial strain by fermenting and being screened, generation has fibrinolytic and the low proteolytic enzyme of toxic side effect, in the hope of obtaining to have the thrombolytic drug of excellent development application prospect.
Technical scheme of the present invention mainly may further comprise the steps: streptomycete bacterial strain that will be to be screened digs piece and places fibrin plate and casein flat board respectively after cultivating well on the agar plate, after 37 ℃ of incubated overnight, filters out the piece that digs that only has fibrinolytic; Correspondence being dug the streptomycete bacterial strain of piece cultivates in fermention medium; After obtaining fermented supernatant fluid, after ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography etc. separate the purification process processing, obtain the pure product of fibrinolytic protein enzyme CGW-3.
Content of the present invention and main points:
One, the screening of streptomycete C-3662 bacterial strain
To separate the multiple streptomycete bacterial strain that obtains from soil is inoculated into respectively on the agar slants (glucose 1.0%, yeast extract 0.1%, extractum carnis, lactalbumin hydrolysate 0.2%, agar 1.2%) such as Bennett, cultivated 7-10 days at 28 ℃, digging piece then places the casein flat board (to contain agarose 0.6%, casein 1.5%, in the pH7.5 of 50mM sodium phosphate buffer) and fibrin plate (contain agarose 0.6%, Fibrinogen 0.6mg/ml and zymoplasm 0.28u/ml) on, spend the night in 37 ℃ of placements.If the bacterial strain secretion produces the tool fibrinolytic protein enzyme higher to fibrin-specific, transparent circle is then arranged on fibrin plate, and on the casein flat board, do not have transparent circle.Observe the dull and stereotyped production of going up transparent circle in second day, therefrom screen the bacterial strain C-3662 that on the casein flat board, does not have or almost do not have transparent circle producing the obvious transparent circle on the fibrin plate.
Two. the classification of streptomycete bacterial strain C-3662:
1, morphological specificity: on No. 1 agar of Gao Shi and the glucose asparagine agar 28 ℃ cultivate 7 days after, the substrate mycelium of bacterial strain C-3662 does not have tabula, does not rupture, spore in silk ripple song to volution, spore ellipse, smooth surface (Fig. 1,2).
2, cultural characteristic: select eight kinds of Different Nutrition substratum commonly used of streptomycete, cultivated 7-15 days at 28 ℃.The appearance features of the mycelium of bacterial strain C-3662 in various different substratum sees Table 1.
3, physiological and biochemical property: bacterial strain C-3662 of the present invention is a kind of aerophil, at 25-30 ℃, preferably demonstrates the optimum growh ability at 28 ℃.Reference " uncle Jie Shi bacterial system identification handbook Volume Four " (Bergey ' s Mannual of Systematic Bacteriology, Vol.IV), bacterial strain C-3662 has been carried out the physiological and biochemical property evaluation, the results are shown in Table 2.
Table 1: the cultural characteristic of bacterial strain C-3662
But substratum aerial hyphae substrate mycelium lysochrome
Gao Shi synthesizes the light brown nothing of agar dark grey look cassia bark
The colourless nothing of grey in the glucose asparagine agar
The brown nothing of the taupe gray mustard of glycerine nitrate agar
The light grey little brown nothing of starch ammonium agar
The pale yellow brown nothing of calcium malate agar canescence
The taupe gray dirt of nutrient agar medium is brown shallow brown
The pale yellow brown nothing of oatmeal agar cinder grey
It is taupe gray shallow brown shallow brown that potato soaks juice agar
Table 2: the physiological and biochemical property of bacterial strain C-3662
| Feature result | Feature result |
| Sugar utilize D-Glucose+Arabinose-D-wood sugar+D-Fructose+L-rhamnose-D-MANNOSE+sucrose+raffinose+L-inositol- | Gelatin liquefaction+milk solidifies-milk peptonizes+Starch Hydrolysis+nitrate reduction-cellulose on growth-H2The generation of S generation+class melanochrome generation+tyrosine oxidase+ |
1, chemical classification: (1) cell walls chemical composition is analyzed according to Hasegawa, and the quick thin plate chromatography (TLC) of T. etc. is carried out the amino acid and the sugared type analysis of full cell hydrolyzed solution to bacterial strain C-3662.The result shows: bacterial strain C-3662 contains LL-diaminopimelic acid and glycine, atypism sugar (sugared type C).The cell walls chemical composition belongs to the I type.(2) experimental technique according to Lechevalier etc. carries out, and finds that bacterial strain C-3662 contains the phosphatidyl thanomin, and phosphate lipid belongs to the II type.
2,16S rDNA analyzes: the total DNA that extracts bacterial strain C-3662 according to a conventional method, adopt universal primer to carry out the pcr amplification of 16S rDNA, the purified back of PCR product directly with TaqDyeDeoxy Terminator Cycle Sequencing Kit order-checking, carried out automatically by Applied Biosystems DNA Sequencer (model 377) by electrophoresis and data analysis.Relevant kind, the sequence that belongs in measured 16S rDNA sequence (Fig. 3) and the GeneBank database being compared, show that the 16S rDNA sequence homology of bacterial strain C-3662 and general temperature streptomycete is very high, is 98.19% (Fig. 4).
The principle of planting surely according to polyphase sort, take all factors into consideration cultural characteristic, Physiology and biochemistry and the 16S rDNA sequential analysis comparative result of bacterial strain C-3662, think that bacterial strain C-3662 and general temperature streptomycete (Streptomyces eurythermus) are very approaching, be general temperature streptomycete (Streptomyces eurythermus) C-3662 so streptomycete C-3662 bacterial strain named.This bacterial classification is delivered the common micro-organisms center preservation of Beijing China Committee for Culture Collection of Microorganisms September 11 calendar year 2001, is numbered CGMCC No.0629.
Three. general temperature streptomycete C-3662 fermentation plasmin CGW-3-3 Optimum of culture medium
The inclined-plane seed culture medium of general temperature streptomycete C-3662 adopts synthetic No. 5 substratum well known in the art, and (it consists of: Zulkovsky starch 2.0%, KNO
30.1%, NaCl 0.05%, K
2HPO
43H
2O0.05%, MgSO
47H
2O 0.05%, FeSO
47H
2O 0.001%).General temperature streptomycete C-3662 preferably 28 ℃, cultivated 7-10 days the grey spore that generation is abundant in 25-30 ℃ in synthetic No. 5 medium slant.
The inclined-plane seed is inoculated in the fermention medium, 28 ℃ and pH near the neutral condition under shaking culture 3-5 days.
Fibrinolytic in the fermented supernatant fluid, adopt the fibrin plate method to measure: flat board contains agarose 0.6%, Fibrinogen 0.6mg/ml and zymoplasm 0.28u/ml; Be incubated 16h in 37 ℃ behind the application of sample.
At first select following 6 kinds of substratum, carried out the desk study that fermention medium is formed.
6 kinds of cultivations
The composition of base is respectively:
No. 1: glucose 1.0%, starch 3.0%, soybean cake powder 2.0%, NaCl 0.4%, CaCO
30.5%.
No. 2: glucose 2.5%, starch 1.0%, soybean cake powder 2.0%, corn steep liquor 0.5%, KH
2PO
40.05%, MgSO
47H
2O 0.05%, CaCO
30.3%.
No. 3: glucose 2.0%, starch 2.0%, soybean cake powder 2.0%, (NH)
4SO
40.05%, KH
2PO
40.05%, MgSO
40.05%, CaCO
30.15%.
No. 4 (Bennett): glucose 1.0%, yeast extract 0.1%, extractum carnis, lactalbumin hydrolysate 0.2%.
No. 5 (ISP2): glucose 1.0%, malt extract 0.3%, yeast extract 0.3%, peptone 0.5%.
No. 6 (GGCY): glucose 10%, glycine 0.1%, Difco casein hydrolysis aminoacids solution 0.4%, KH
2PO
40.2%, Na
2HPO
412H
2O 0.8%.Trace element solution wherein is: ZnCl
22H
2O 0.004%, FeCl
36H
2O 0.02%, CuCl
22H
2O 0.001%, MnCl
24H
2O0.001%, Na
2B
4O
710H
2O 0.001%, (NH
4)
6Mo
7O
24H
2O 0.001%.
By fermentation, finding that the fermented supernatant fluid fibrinolytic of No. 2 substratum is the highest, secondly is No. 3 substratum.Common component soybean cake powder-starch-glucose-potassium primary phosphate-sal epsom-lime carbonate based in No. 2 and No. 3 substratum carries out the optimization of fermention medium.Based on soybean cake powder 2.0% (nitrogenous source), starch 1.0% and glucose 2.5% (carbon source), potassium primary phosphate 0.05% and sal epsom 0.05% and lime carbonate 0.3% (inorganic salt), fibrinolytic in its fermented supernatant fluid is decided to be 100%, fixedly nitrogenous source conversion carbon source reaches fixedly carbon source conversion nitrogenous source respectively, and the research inorganic salt are to the influence that fermentation produces fibrinolytic, the results are shown in Table 3 and table 4.
Consolidated statement 3 and table 4 as can be seen, Optimal compositions of fermentation medium consist of soybean cake powder 2.0%, starch 1.0%, glucose 2.5%, potassium primary phosphate 0.05%, sal epsom 0.05%, lime carbonate 0.3%.
The resulting general temperature streptomycete C-3662 of the present invention, culture condition is simple, preservation is convenient, can secrete to produce a large amount of fibrinolytic protein enzyme CGW-3 in nutrient solution in the fermention medium of optimizing, and the separation and purification of CGW-3 is also fairly simple.In addition, because general temperature streptomycete C-3662 is the natural separation bacterial strain, so through after repeatedly going down to posterity, it is secreted the characteristic that produces fibrinolytic protein enzyme CGW-3 in large quantities and does not degenerate.Because CGW-3 behind animal experiment, finds that its thrombolysis is respond well, side effect is little, is expected to be developed as the new thrombolytic drug of a class, so general temperature streptomycete C-3662 has the potential commercial application value.
Table 3: different nitrogen, carbon source substratum
Influence to general temperature streptomycete C-3662 fermentation generation fibrinolytic
| Medium component changes | Relative fibrinolytic |
| Nitrogenous source soybean cake powder 2% corn steep liquor 2% yeast extract 2% fish meal 2% carbon source glucose 2.5% starch 2.5% corn flour 2.5% lactose 2.5% sucrose 2.5% dextrin 2.5% glycerine 2.5% glucose 2.5%+ starch 1.0%+dextrin 1.0%+corn flour 1.0%+sucrose 1.0%+lactose 1.0%+maltose 1.0%+glycerine 1.0% | 100％ 65％ 38％ 30％ 43％ 64％ 30％ 61％ 30％ 50％ 30％ 100％ 69％ 69％ 61％ 71％ 61％ 70％ |
Table 4: different minimal mediums
Influence to general temperature streptomycete C-3662 fermentation generation fibrinolytic
| Medium component changes | Relative fibrinolytic |
| Inorganic salt KH 2PO 40.05％+MgSO 40.05% does not add any inorganic salt KH 2PO40.05％ MgSO 40.05％ KH 2PO 40.05％+MgSO 40.05％+FeSO 4 0.001％ KH 2PO 40.05％+MgSO 40.05％+FeSO 4 0.2％ KH 2PO 40.05％+MgSO 40.05％+CuSO 45 mcg/ml KH 2PO 40.05％+MgSO 40.05％+CuSO 450 mcg/ml KH 2PO 40.05％+MgSO 40.05％+ZnCl 25 mcg/ml KH 2PO 40.05％+MgSO 40.05％+ZnCl 250 mcg/ml KH 2PO 40.05％+MgSO 40.05％+MnCl 25 mcg/ml KH 2PO 40.05％+MgSO 40.05％+MnCl 250 mcg/ml KH 2PO 40.05％+MgSO 40.05％+LiCl 25 mcg/ml KH 2PO 40.05％+MgSO 40.05％+LiCl 250 mcg/ml | 100％ 50％ 105％ 46％ 100％ 30％ 90％ 50％ 100％ 70％ 100％ 100％ 100％ 90％ |
Description of drawings
Fig. 1: the mycelium of general temperature streptomycete C-3662 and fibrillae of spores form (* 750).
Fig. 2: the spore surface feature (* 6000) of general temperature streptomycete C-3662
Fig. 3: the 16S rDNA complete sequence of general temperature streptomycete C-3662
Fig. 4: the comparison of the general temperature streptomycete 16S rDNA complete sequence in the 16S rDNA complete sequence of general temperature streptomycete C-3662 and the GeneBank database, the homology of the two is 98.19%; Query among the figure is the 16S rDNA sequence of streptomycete C-3662, and Sbjct is the 16S rDNA sequence of general temperature streptomycete.
Embodiment
Following examples are illustrative, are not intended to restriction.
Streptomycete bacterial strain to be screened is inoculated on the agar slants such as Bennett (glucose 1.0%, yeast extract 0.1%, extractum carnis, lactalbumin hydrolysate 0.2%, agar 1.2%), cultivates 10 days for 28 ℃.Then, dig piece from the inclined-plane, place the casein flat board (to contain agarose 0.6% respectively, casein 15%, in the pH7.5 of 50mM sodium phosphate buffer) and fibrin plate (contain agarose 0.6%, Fibrinogen 0.6mg/ml and zymoplasm 0.28u/ml) on, at every internal diameter be on the flat board of 8.5cm evenly 25 agar placed apart dig piece.37 ℃ are incubated 16 hours.Select and on fibrin plate, produce transparent circle, and on the casein flat board, do not produce the bacterial strain C-3662 of transparent circle.
Embodiment 2
Will be on synthetic No. 5 slant mediums 28 ℃ of general temperature streptomycete C-3662 that cultivate 7 days, dig piece and be inoculated in 100 milliliters of substratum (glucose 2.5%, starch 1.0%, soybean cake powder 2.0%, KH are housed
2PO
40.05%, MgSO
40.05%, CaCO
30.3%, in 500 milliliters of triangular flasks pH7.0), 28 ℃ of shaking culture 48 hours, 200 rev/mins; Then with 5% inoculum size transferred species to 5000 milliliters of triangular flasks that 1000 milliliters of same medium are housed, 28 ℃ of round shaking culture are about 96 hours; Fermented liquid is centrifugal or remove by filter mycelium, obtain supernatant liquor, use for further separation and purification CGW-3.
Embodiment 3
1 liter of fermented supernatant fluid is added ammonium sulfate to 80% saturation ratio, and 4 ℃ of placements are spent the night, then 8000 rev/mins centrifugal 10 minutes, supernatant discarded.To precipitate in water-soluble 50 milliliters, 50mM sodium phosphate (pH8.0) damping fluid will be dialysed.To dialyse the back solution centrifugal to remove insolubles, and cross DEAE Sepharose post (25 millimeter * 200 millimeter) decolouring with dialyzate this moment, then goes up CMSepharose post (25 millimeters * 200 millimeters), with 0.0-1.0M NaCl salt concn linear gradient elution.The mixed active elutriant adds 3M NH
4(SO
4)
2To final concentration be 1M, go up PhenylSepharose post (15 millimeters * 200 millimeters) then, use 1.0-0.0M NH
4(SO
4)
2The salt concn linear gradient elution.The mixed active elutriant, 4 ℃ of dialysed overnight of water desalt, and lyophilize then gets the pure product of CGW-3.Every liter of fermented supernatant fluid gets 8 milligrams of pure product of left and right sides CGW-3, and 15 aminoacid sequences of its N-end are VVGGTRAAQGEFPFM after testing.
A kind of secretion has the sequence table .txt of the general temperature streptomycete C-3662 of fibrinolytic protein enzyme
Sequence table
＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
＜120〉a kind of secretion has the general temperature streptomycete C-3662 of fibrinolytic protein enzyme
<140>01136006.2
<141>2001-09-28
<160>1
<210>1
<211>1450
<212>DNA
＜213〉general temperature streptomycete (Streptomyces eurythermus C-3662)
<220>
<221>gene
<222>(1)…(1450)
＜223〉homology of this sequence and general temperature streptomycete 16S rDNA sequence is 98.19%
<400>1
ttgacgaagc tgcggcgtgc ttaacacatg caagtcgaac gatgaaccac ttcggtgggg 60
attagtggcg aacgggtgag taacacgtgg gcaatctgcc ctgcactctg ggacaagccc 120
tggaaacggg gtctaatacc ggatacgagc ctcctccgca tggtgggggt tggaaagctc 180
cggcggtgca ggatgagccc gcggcctatc agcttgttgg tgaggtaacg gctcaccaag 240
gcgacgacgg gtagccggcc tgagagggcg accggccaca ctgggactga gacacggccc 300
agactcctac gggaggcagc agtggggaat attgcacaat gggcgaaagc ctgatgcagc 360
gacgccgcgt gagggatgaa ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg 420
aaagtgacgg tacctgcaga agaagcgccg gctaactacg tgccagcacc gcggtaatac 480
gtagggcgca agcgttgtcc ggaattattg ggcgtaaaga gctcgtaggc ggcttgtcgc 540
gtcggttgtg aaagcccggg gcttaacccc gggtctgcag tcgatacggg caggctagag 600
ttcggtaggg gagatcggaa ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa 660
caccggtggc gaaggcggat ctctgggccg atactgacgc tgaggagcga aagcgtgggg 720
agcgaacagg attagatacc ctggtagtcc acgccgtaaa cggtgggcac taggtgtggg 780
cgacattcca cgtcgtccgt gccgcagcta acgcattaag tgccccgcct ggggagtacg 840
gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg gagcatgtgg 900
cttaattcga cgcaacgcga agaaccttac caaggcttga catacaccgg aaagctctgg 960
agacagagcc ccccttgtgg tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcccgtgtt gccagcaggc 1080
ccttgtggtg ctggggactc acgggagacc gccggggtca actcggagga aggtggggac 1140
gacgtcaagt catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggccggtac 1200
aatgagctgc gataccgtga ggtggagcga atctcaaaaa gccggtctca gttcggattg 1260
gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcattgctg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca 1380
cccgaagccg gtggcccaac ccttgtggag ggagctgtcg aaggtgggac tggcgattgg 1440
gacgaaaggg 1450
Claims (1)
1, a kind of general temperature streptomycete (Streptomyces eurythermus) bacterial strain C-3662, its preserving number is CGMCC No.0629, it is characterized in that said bacterial strain separates, screens and can produce through fermentation the proteolytic enzyme CGW-3 of tool fibrinolytic from soil.
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