CN103060235B - Preparing and regenerating method of tetracycline producing strain protoplast - Google Patents
Preparing and regenerating method of tetracycline producing strain protoplast Download PDFInfo
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- CN103060235B CN103060235B CN201210592699.6A CN201210592699A CN103060235B CN 103060235 B CN103060235 B CN 103060235B CN 201210592699 A CN201210592699 A CN 201210592699A CN 103060235 B CN103060235 B CN 103060235B
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Abstract
The invention aims to provide a preparing and regenerating method of a tetracycline producing strain protoplast. The method comprises the following steps: (a) preparing a tetracycline slant culture-medium; (b) preparing a spore suspension; (c) preparing a mycelium culture-medium; (d) preparing a regeneration medium; (e) culturing mycelium; (f) enzymatically hydrolyzing a cell wall; (g) separating a protoplast; and (h) regenerating the protoplast. A novel method for breeding tetracycline strains, provided by the invention, has the advantage as follows: problems that the tetracycline protoplast bacterial colony cannot be regenerated, the spore generating capacity of the tetracycline protoplast bacterial colony is low and the passage of the tetracycline protoplast bacterial colony cannot be made are solved.
Description
Technical field
The present invention relates to a kind of microorganism protoplastis preparation and renovation process, specifically a kind of tsiklomitsin produces the preparation of bacterium protoplastis and the method for regenerating.
Background technology
Tsiklomitsin (Tetracycline, TC) be a kind of Broad spectrum antibiotics of anti-bacteria protein synthesis, because its molecular structure is gained the name containing four acene basic frameworks, be mainly used in the treatment of infection of various gram-positive coccis and Gram-positive bacillus, medicine, herding and agriculture aspect all have been widely used.China, since the suitability for industrialized production tsiklomitsin sixties in 20th century, has the history of five more than ten years so far.It is the streptomyces (Streptomyces) in actinomycetes that tsiklomitsin produces bacterium, and at present, the seed selection of its excellent species mainly adopts traditional breeding method, and bacterial classification, through long-term induced mutations breeding and natural separation, has formed higher throughput.After but bacterial classification brings out sudden change through the multiple physics and chemistry factor, not only genetic background becomes quite complicated, and the tolerance of mutagenic compound has also been strengthened greatly, therefore, utilize the output of traditional breeding method continuation raising tsiklomitsin more and more difficult, induction mutation of bacterium rate is low, Breeding Effect is poor is the subject matter of puzzlement tsiklomitsin strain improvement work.
Protoplast Fusion Technique is a kind of new genetic breeding means that 20 century 70s grow up, the advantage of this breeding technique is the obstacle of having removed cell walls, adopt physical agent or chemokines to carry out mutagenic treatment to protoplastis, can improve the mutation frequency of protoplastis, in regeneration bacterium colony, screen mutant strain, can effectively improve the Breeding Efficiency of excellent species, utilize Protoplast Technique to cultivate the generally attention that the new bacterial strain of industry has been subject to domestic and international Microbial Breeding worker.In exploring the process of tsiklomitsin protoplastis breeding technique, the greatest problem existing is about the method for the preparation of tetracycline antibiotics protoplastis and regeneration, can not use for reference, after protoplastis is prepared successfully, the process of reconstruction of cell walls is very difficult, or cannot produce the regeneration bacterium colony of protoplastis, or only grow substrate mycelium and minute quantity aerial hyphae, can not produce fibrillae of spores, cause bacterium colony not go down to posterity normally.Greatly limited thus the application of protoplasma technology in the work of tsiklomitsin strain improvement.
Summary of the invention
Object of the present invention is just to provide preparation, the renovation process that a kind of tsiklomitsin produces bacterium protoplastis, to solve the problem that induction mutation of bacterium rate is low and Breeding Effect is poor existing in existing traditional breeding technology.
The present invention is achieved in that
Tsiklomitsin provided by the present invention produces the preparation of bacterium protoplastis and the method for regenerating, and it comprises the steps:
A, prepare tsiklomitsin slant medium: take Testa Tritici 3.0-3.5g, magnesium sulfate 0.004-0.006g, dipotassium hydrogen phosphate 0.01-0.12g, Secondary ammonium phosphate 0.013-0.015g, agar powder 1.3-1.5g, adding distil water is to 100ml; Under 120-124 ℃ of condition, moist heat sterilization is 30 minutes.Slant medium loading amount 60ml/ eggplant bottle.
B, prepare spore suspension: tsiklomitsin is produced to bacterium and be inoculated on slant medium, under 33-36 ℃ of condition, cultivate about 96 hours, grow the slant pore that visual appearance is qualified.Under aseptic condition, add sterile distilled water or Mycelium culture base to make spore suspension, make spore liquid concentration 1 * 10
8more than/ml.
C, prepare Mycelium culture base: take glucose 0.8-1.2g, peptone 0.3-0.5g, yeast extract paste 0.3-0.5g, potassium primary phosphate 0.1-0.2g, magnesium sulfate 0.03-0.05g, dipotassium hydrogen phosphate 0.3-0.4g, glycine 0.2-1g, adds water to 100ml; 120-124 ℃ of moist heat sterilization 30 minutes.
D, prepare regeneration culture medium: take flour 2-4g, peptone 0.3-0.5g, potassium primary phosphate 0.05-0.10g, calcium carbonate 0.1-0.4g, sucrose 0-11g, agar 1.0-2.5g, adds water to 100ml; 120-124 ℃ of ℃ moist heat sterilization made plate culture medium after 30 minutes.
E, cultured mycelia:
Tsiklomitsin spore suspension is inoculated in to c and walks in described Mycelium culture base, under 27 ℃ of-33 ℃ of conditions, 22-32 hour is cultivated in concussion, and centrifugal abandoning supernatant, obtains mycelium;
F, enzymolysis cell walls:
To in e step gained mycelium, add stable liquid, centrifuge washing 2-4 time, adds lysozyme soln, and the activity that makes enzyme is 2mg/ml-6mg/ml, and 30 ℃ of-37 ℃ of enzymolysis 1-3 hour, obtain enzymolysis solution;
G, separated protoplastis:
Enzymolysis solution is through the centrifugal precipitation (optimum condition when centrifugal is 500 revs/min, 2-4 minute) of first removing.And then by supernatant liquor centrifugal (the centrifugal optimum condition of supernatant liquor is rotating speed 3500-8000 rev/min, time 10-15 minute), obtain protoplastis;
H, protoplast regeneration:
By stable liquid centrifuge washing 2-4 time of the protoplastis of g step gained, add a certain amount of stable liquid to make protoplastis suspension, after blood counting chamber counting, by stable liquid, protoplastis suspension is carried out to gradient dilution, adopt sandwiching to be incubated on regeneration culture medium, under 33 ℃ of-36 ℃ of conditions, cultivate after 5-7 days and form regeneration bacterium colony.
Tsiklomitsin of the present invention produces the bacterial classification that bacterium can be selected bacterium numbering CPCC220020.
Stable liquid of the present invention is selected high concentration sucrose penetrating fluid, and wherein sucrose concentration is advisable with 10-11%.
The method of preparing tsiklomitsin spore suspension described in b step of the present invention is: tsiklomitsin is produced to bacterium and be inoculated on slant medium, under 33-36 ℃ of condition, cultivate about 96 hours, grow the slant pore that visual appearance is qualified.Under aseptic condition, add sterile distilled water or Mycelium culture base to make spore suspension, spore liquid concentration 1 * 10
8more than/ml.
In order to be more conducive to the formation of protoplastis, when the present invention is inoculated in tsiklomitsin spore suspension c and walks described Mycelium culture base, inoculum size is not less than 10
6individual/ml.
Because protoplastis is very sensitive to the osmotic pressure of solution and substratum, must under the environment of high osmotic pressure or isosmoticity, just can maintain its existence.Therefore, described in the inventive method, protoplast regeneration operation Chinese medicine is selected stablizer.The sucrose solution that the quality of wherein take is 10-11% than concentration is preferred stabilizer, and it can build a high environment oozing as protoplastis protective material, then adds lysozyme soln to carry out enzymolysis, plays the effect that protection protoplastis avoids swelling fracture.
In order to improve bacterial classification mutation rate, select better high-yield strains, the present invention carries out mutagenic treatment to prepared protoplastis.
Its preferred treatment process includes:
The bioblast of described acquisition, carries out mutagenic treatment (ultraviolet irradiation time 30 seconds to 120 seconds through physical mutagen ultraviolet ray; Irradiation distance 33-66cm);
The bioblast of described acquisition is processed 50-100 minute through chemical mutagen ethyleneimine (EI) under 1000ug/ml condition.
The invention provides a kind of novel method of tsiklomitsin strain improvement, solved the problem that tsiklomitsin protoplastis bacterium colony cannot be regenerated and sporiparous ability is low, can not go down to posterity.This breeding method has been removed the obstacle of somatic cells wall, has improved bacterial classification mutation rate, and regeneration bacterium colony can produce abundant fibrillae of spores, is suitable for connecing inclined-plane and goes down to posterity, and is worth carrying out promotion and application in the work of tetracycline antibiotics strain improvement.
Protoplast release prepared by the inventive method is up to 10
4individual/ml, its regeneration rate can reach more than 3%.
Embodiment
Below by specific embodiment, the present invention is described in further detail, but with this, the present invention is not carried out to any restriction.
N,O-Diacetylmuramidase in following embodiment derives from AMRESCO company, formulated by stable liquid, and enzyme adopts filter type degerming (the water system syringe filters of 0.22um) after dissolving, and N,O-Diacetylmuramidase compound concentration is 10%.
Stable liquid in following embodiment is 10.3% sucrose solution.
Embodiment 1
A, mycelial cultivation:
Tsiklomitsin is produced to bacterium (Streptomyces aureofaciens) slant pore and make spore suspension, be inoculated in Mycelium culture base, inoculum size is not less than 10
6individual/ml, under 30 ℃ of conditions, concussion is cultivated 26 hours, obtains Mycelium culture liquid, and sediments microscope inspection mycelia is netted, and centrifugal (3500 revs/min, 10 minutes) abandoning supernatant obtains mycelium.
Wherein the bacterial classification of tsiklomitsin slant pore derives from Chinese microorganism strain composite catalog, bacterial strain deposit number CPCC220020.
Mycelium culture base is formulated by distilled water, and in 100ml Mycelium culture base cumulative volume, it contains following component: glucose 1g, peptone 0.4g, yeast extract paste 0.4g, dipotassium hydrogen phosphate 0.4g, potassium primary phosphate 0.2g, magnesium sulfate 0.05g, glycine 1g.Sterilising conditions: 121 ℃, 30 minutes.
The preparation of b, protoplastis:
By a step gained mycelium by 10.3% sucrose stable liquid centrifugal (3500 revs/min, 10 minutes) wash 3 times, add lysozyme soln, making the activity of N,O-Diacetylmuramidase in reaction vessel (test tube) is 4mg/ml, be placed in 35 ℃ of constant water bath box and carry out enzymolysis, within every 10 minutes, shake once, enzymolysis 2 hours, microscopy protoplast release reaches 10
6individual/more than ml, reaction vessel (test tube) to be taken out from water bath, stop enzymolysis.Enzymolysis solution is removed precipitation through centrifugal (500 revs/min of rotating speeds, 3 minutes time), and supernatant liquor carries out again centrifugal (5000 revs/min of rotating speeds, 15 minutes time) obtains protoplastis.
C, by stable liquid centrifuge washing 3 times for protoplastis (stable liquid adds to centrifuge tube 10ml place, 3500 revs/min, centrifugal 10 minutes), add 5ml stable liquid to make protoplastis suspension.
D, protoplastis suspension dilute by stable liquid, via interlayer method is incubated on regeneration culture medium, cultivates 5-6 days for 34-35 ℃, forms regeneration bacterium colony, regeneration colony diameter 2-5mm, steamed bun shape or straw hat type, outward appearance Slate grey, spore is abundant, fibrillae of spores is just threaded to the spacious volution of pine, spore oval, smooth surface, is suitable for going down to posterity.
Distilled water and tap water that wherein regeneration culture medium is 1:1 by volume ratio are formulated, in 100ml regeneration culture medium cumulative volume, wherein contain following component: flour 2g, peptone 0.05g, potassium primary phosphate 0.08g, calcium carbonate 0.1g, sucrose 2g, agar 2.2g.Sterilising conditions: 121 ℃, 30 minutes.
Embodiment 2
A, mycelial cultivation:
Tsiklomitsin is produced to bacterium (Streptomyces aureofaciens) slant pore and make spore suspension, be inoculated in Mycelium culture base, inoculum size is not less than 10
6individual/ml, under 30 ℃ of conditions, concussion is cultivated 26 hours, obtains Mycelium culture liquid, and sediments microscope inspection mycelia is netted, and centrifugal (3500 revs/min, 10 minutes) abandoning supernatant obtains mycelium.
Wherein the bacterial classification of tsiklomitsin slant pore derives from Chinese microorganism strain composite catalog, bacterial strain deposit number CPCC220020.
Mycelium culture base is formulated by distilled water, and in 100ml Mycelium culture base cumulative volume, it contains following component: glucose 1g, peptone 0.4g, yeast extract paste 0.4g, dipotassium hydrogen phosphate 0.4g, potassium primary phosphate 0.2g, magnesium sulfate 0.05g, glycine 1g.Sterilising conditions: 121 ℃, 30 minutes.
The preparation of b, protoplastis:
By a step gained mycelium by 10.3% sucrose stable liquid centrifugal (3500 revs/min, 10 minutes) wash 3 times, add lysozyme soln, making the activity of N,O-Diacetylmuramidase in reaction vessel (test tube) is 4mg/ml, be placed in 35 ℃ of constant water bath box and carry out enzymolysis, within every 10 minutes, shake once, enzymolysis 2 hours, microscopy protoplast release reaches 10
6individual/more than ml, reaction vessel (test tube) to be taken out from water bath, stop enzymolysis.Enzymolysis solution is removed precipitation through centrifugal (500 revs/min of rotating speeds, 3 minutes time), and supernatant liquor carries out again centrifugal (5000 revs/min of rotating speeds, 15 minutes time) obtains protoplastis.
C, by protoplastis, with stable liquid centrifuge washing, 3 times (stable liquid adds to centrifuge tube 10ml place, 3500 revs/min, centrifugal 10 minutes), add 5ml stable liquid to make protoplastis suspension, adopt uviolizing (30 watts of ultraviolet lamps in 30 seconds, irradiation distance 42cm) carry out mutagenic treatment, promote it to undergo mutation.
Protoplastis suspension after d, processing dilutes by stable liquid, via interlayer method is incubated on regeneration culture medium, cultivates 5-6 days for 34-35 ℃, forms regeneration bacterium colony, regeneration colony diameter 2-5mm, steamed bun shape or straw hat type, outward appearance Slate grey, spore is abundant, fibrillae of spores is just threaded to the spacious volution of pine, spore oval, smooth surface, is suitable for going down to posterity.
Distilled water and tap water that wherein regeneration culture medium is 1:1 by volume ratio are formulated, in 100ml regeneration culture medium cumulative volume, wherein contain following component: flour 2g, peptone 0.05g, potassium primary phosphate 0.08g, calcium carbonate 0.1g, sucrose 2g, agar 2.2g.Sterilising conditions: 121 ℃, 30 minutes.
Embodiment 3
A, mycelial cultivation:
Tsiklomitsin is produced to bacterium (Streptomyces aureofaciens) slant pore and make spore suspension, be inoculated in Mycelium culture base, inoculum size is not less than 10
6individual/ml, under 30 ℃ of conditions, concussion is cultivated 26 hours, obtains Mycelium culture liquid, and sediments microscope inspection mycelia is netted, and centrifugal (3500 revs/min, 10 minutes) abandoning supernatant obtains mycelium.
Wherein the bacterial classification of tsiklomitsin slant pore derives from Chinese microorganism strain composite catalog, bacterial strain deposit number CPCC220020.
Mycelium culture base is formulated by distilled water, and in 100ml Mycelium culture base cumulative volume, it contains following component: glucose 1g, peptone 0.4g, yeast extract paste 0.4g, dipotassium hydrogen phosphate 0.4g, potassium primary phosphate 0.2g, magnesium sulfate 0.05g, glycine 1g.Sterilising conditions: 121 ℃, 30 minutes.
The preparation of b, protoplastis:
By a step gained mycelium by 10.3% sucrose stable liquid centrifugal (3500 revs/min, 10 minutes) wash 3 times, add lysozyme soln, making the activity of N,O-Diacetylmuramidase in reaction vessel (test tube) is 4mg/ml, be placed in 35 ℃ of constant water bath box and carry out enzymolysis, within every 10 minutes, shake once, enzymolysis 2 hours, microscopy protoplast release reaches 10
6individual/more than ml, reaction vessel (test tube) to be taken out from water bath, stop enzymolysis.Enzymolysis solution is removed precipitation through centrifugal (500 revs/min of rotating speeds, 3 minutes time), and supernatant liquor carries out again centrifugal (5000 revs/min of rotating speeds, 15 minutes time) obtains protoplastis.
C, by protoplastis, with stable liquid centrifuge washing, 3 times (stable liquid adds to centrifuge tube 10ml place, 3500 revs/min, centrifugal 10 minutes), add 5ml stable liquid to make protoplastis suspension, through chemical mutagen ethyleneimine (EI) under 1000ug/ml condition, carry out 50-100 minute mutagenic treatment, promote it to undergo mutation.
Protoplastis suspension after d, processing dilutes by stable liquid, via interlayer method is incubated on regeneration culture medium, cultivates 5-6 days for 34-35 ℃, forms regeneration bacterium colony, regeneration colony diameter 2-5mm, steamed bun shape or straw hat type, outward appearance Slate grey, spore is abundant, fibrillae of spores is just threaded to the spacious volution of pine, spore oval, smooth surface, is suitable for going down to posterity.
Distilled water and tap water that wherein regeneration culture medium is 1:1 by volume ratio are formulated, in 100ml regeneration culture medium cumulative volume, wherein contain following component: flour 2g, peptone 0.05g, potassium primary phosphate 0.08g, calcium carbonate 0.1g, sucrose 2g, agar 2.2g.Sterilising conditions: 121 ℃, 30 minutes.
Claims (4)
1. tsiklomitsin produces a Formation and regeneration method for bacterium protoplastis, it is characterized in that it comprises the steps:
A, prepare tsiklomitsin slant medium: take Testa Tritici 3.0-3.5 g, magnesium sulfate 0.004-0.006 g, dipotassium hydrogen phosphate 0.01-0.12 g, Secondary ammonium phosphate 0.013-0.015g, agar powder 1.3-1.5 g, adding distil water is to 100ml; Under 120-124 ℃ of condition, moist heat sterilization is 30 minutes; Slant medium loading amount 60ml/ eggplant bottle;
B, prepare spore suspension: tsiklomitsin is produced to bacterium and be inoculated on slant medium, under 33-36 ℃ of condition, cultivate about 96 hours, grow slant pore; Under aseptic condition, add sterile distilled water or Mycelium culture base to make spore suspension, make spore suspension concentration 1 * 10
8more than/ml;
C, prepare Mycelium culture base: take glucose 0.8-1.2 g, peptone 0.3-0.5 g, yeast extract paste 0.3-0.5 g, potassium primary phosphate 0.1-0.2 g, magnesium sulfate 0.03-0.05 g, dipotassium hydrogen phosphate 0.3-0.4 g, glycine 0.2-1 g, adds water to 100ml; Under 120-124 ℃ of condition, moist heat sterilization is 30 minutes;
D, prepare regeneration culture medium: the distilled water and the tap water that by volume ratio, are 1:1 are formulated, in 100ml regeneration culture medium cumulative volume, wherein contain following component: flour 2g, peptone 0.05g, potassium primary phosphate 0.08g, calcium carbonate 0.1g, sucrose 2g, agar 2.2g; Sterilising conditions: 121 ℃, 30 minutes;
E, cultured mycelia:
Tsiklomitsin spore suspension is inoculated in described in c step in Mycelium culture base, and under 27 ℃ of-33 ℃ of conditions, 22-32 hour is cultivated in concussion, and centrifugal abandoning supernatant, obtains mycelium;
F, enzymolysis cell walls:
E step gained mycelium is added to stable liquid, and centrifuge washing 2-4 time, adds lysozyme soln, and making N,O-Diacetylmuramidase activity is 2 mg/ml-6 mg/ml, and 30 ℃ of-37 ℃ of enzymolysis 1-3 hour, obtain enzymolysis solution;
G, separated protoplastis:
Enzymolysis solution precipitates through centrifugal removal, and then supernatant liquor is centrifugal, obtains protoplastis;
H, protoplast regeneration:
By after the protoplastis use stable liquid centrifuge washing of g step gained 2-4 time, add stable liquid to make protoplastis suspension, protoplastis suspension dilutes by stable liquid, adopts sandwiching to be incubated on regeneration culture medium, under 33 ℃ of-36 ℃ of conditions, cultivate 5-7 days, form regeneration bacterium colony.
2. tsiklomitsin according to claim 1 produces the Formation and regeneration method of bacterium protoplastis, it is characterized in that described stable liquid is the sucrose solution that quality is 10-11% than concentration.
3. tsiklomitsin according to claim 1 produces the preparation of bacterium protoplastis and the method for regenerating, and it is characterized in that the bioblast of described acquisition, through uviolizing 30 seconds to 2 minutes.
4. tsiklomitsin according to claim 1 produces the preparation of bacterium protoplastis and the method for regenerating, and it is characterized in that the bioblast of described acquisition, through ethyleneimine, under 1000ug/ml condition, processes 50-100 minute.
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| CN101343630A (en) * | 2007-07-12 | 2009-01-14 | 中国科学院上海生命科学研究院 | Method for improving doramectin preparing bacterium with genome reorganization technique |
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