CN104531535A - Genetic recombination strain for producing pneumocandins B0, breeding method and application - Google Patents

Genetic recombination strain for producing pneumocandins B0, breeding method and application Download PDF

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CN104531535A
CN104531535A CN201410555356.1A CN201410555356A CN104531535A CN 104531535 A CN104531535 A CN 104531535A CN 201410555356 A CN201410555356 A CN 201410555356A CN 104531535 A CN104531535 A CN 104531535A
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kangding
knob
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黄和
宋萍
沈文和
秦婷婷
章人川
纪晓俊
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Nanjing Tech University
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Abstract

The invention discloses a genetic recombination strain for producing pneumocandins B0, a breeding method and applications. The bacterial strain classification name of the genetic recombination strain is Glarea lozoyensis Q45; the preservation registration number is CCTCC NO: M2014416; and the preservation date is September 14, 2014. The method for breeding the strain includes the steps that an original strain is manufactured into a protoplast; a mutation library composed of a plurality of mutant strains is obtained through ion implantation and lithium chloride processing; the mutation library is manufactured into a protoplast again; random fusion is carried out on the protoplasts after ion implantation inactivation and heat inactivation are carried out; fermentation screening is carried out on high-yield genetic recombinant bacteria; and protoplast preparation and fusion and fusant screening are carried out on the screened genetic recombinant bacteria, and therefore the genetic recombination strain is obtained. The genetic recombination strain is excellent in performance and stable in production capacity; the yield of the pneumocandins B0 obtained through fermentation is 6 g/L; in addition, by-products are few; the post-extraction difficulty of the pneumocandins B0 is reduced; the application to high-quality caspofungin preparation is facilitated; and the genetic recombination strain has the important industrial value.

Description

A kind of production knob not Kangding B 0gene recombination bacterial strain and selection and application
Technical field
The present invention relates to microorganism field, produce knob not Kangding B in particular to one 0gene recombination bacterial strain and selection and application.
Background technology
Echinocandin antifungal agent thing is a kind of novel lipopeptide compound, the synthesis of and then Antifungi cell walls active by distinctive β-1-3-glucan synthase in noncompetitive inhibition fungal cell wall, and does not impact human body.This mechanism of action being obviously different from this kind of conventional antibiotic medicine of amphotericin, echinocandin class is had broad spectrum is strong, resistance is little, drug drug interaction is little feature, becomes the developing direction of new antibiotic of following low toxicity, safety, broad spectrum.
Caspofungin is first echinocandin antifungal agent going through to go on the market, by the secondary metabolite knob not Kangding B of filamentous fungus Glarea lozoyensis 0derivative and obtain through chemically modified, Merck & Co., Inc. develops its antimycotic/anti-lung sac worm medicine as a kind of wide spectrum.Caspofungin goes on the market February calendar year 2001 in the U.S., is mainly used in treating the patient suffering from infectious aspergillosis and don't applicable amphotericin B or curative effective of Itraconazole in curing.In addition, in non-responsiveness patient, this medicine can be used for the initial therapy medication of aspergillosis, candidiasis; Also can be used for the neutropenia patient and some the paediatrics indication that microbiotic are produced to resistance.
Glarea lozoyensis fermentative production knob not Kangding B 0process in, various structures analogue can be produced, comprise knob not Kangding A 0, knob not Kangding C 0, knob not Kangding B 1, knob not Kangding B 2with knob not Kangding D 0deng.Wherein main analog A 0and C 0great difficulty is caused to the separation and Extraction in later stage.Merck & Co., Inc. has carried out mutagenic and breeding to original strain ATCC20868, obtains knob not Kangding B 0superior strain ATCC20957, but the knob of this bacterial strain not Kangding B 0output is 285mg/L only, and knob not Kangding A 0content is higher, the ratio (B of both concentration 0/ A 0) be 10.Subsequently NTG mutagenesis is carried out to ATCC20957, obtain knob not Kangding B 0superior strain ATCC74030, knob is Kangding B not 0the lifting that obtains of output, knob is not Kangding A 0content reduce further, knob is Kangding B not 0/ knob is Kangding A not 0be 80, knob is Kangding C not 0<5%.Current knob is Kangding B not 0the production peak of fermentation is 5.2g/L.
Summary of the invention
The object of the present invention is to provide one can stablize high yield knob not Kangding B 0and the gene recombination bacterium that by product is few.
Another object of the present invention is to provide the method for this gene recombination bacterium of seed selection.
Another object of the present invention utilizes this gene recombination bacterium fermentative production knob not Kangding B 0method.
The object of the invention is to be realized by following technical proposal:
One, a kind of production knob not Kangding B 0gene recombination bacterial strain, its Classification And Nomenclature is Glarea lozoyensis Q45, and this bacterial strain is by China typical culture collection center preservation, and preservation registration number is CCTCC NO:M 2014416, and preservation period is September 14 in 2014.
Two, production knob of the present invention not Kangding B 0the selection of gene recombination bacterial strain Glarea lozoyensis Q45, comprise the following steps:
(1) preparation of protoplastis
Glarea lozoyensis original strain access seed culture medium is cultivated, collects mycelia, use enzymolysis solution enzymolysis, make protoplastis;
2) original mutation word bank
By step 1) the protoplastis volume prepared is divided into two groups, atmospheric pressure at room plasma body (ARTP) and lithium chloride is adopted to carry out mutagenic treatment respectively, coat on regeneration culture medium by the mutant strain of acquisition, the well-grown bacterium colony of picking carries out fermentation and measures knob not Kangding B 0, select the high mutant strain of output and carry out inclined-plane and to go down to posterity investigation stability, obtain the high productive mutant of many strains stable in properties, form sudden change word bank;
3) fusion of protoplastis
By step 2) mutant strain that the proterties that obtains is stable, according to step 1) prepare protoplastis, the protoplastis of every plant mutant strain all equal-volume is divided into two groups, one group adopts ARTP to inject deactivation, another group adopts hot deactivation, the protoplastis mixing after two groups of deactivations is added fusion liquid and merges at random;
4) screening of fusant
By step 3) protoplastis after the fusion that obtains with high ooze buffering centrifuge washing after, coat on regeneration culture medium, the well-grown bacterium colony of picking carries out fermentation mensuration knob not Kangding B 0, obtain knob not Kangding B 0yield Genes recombinant bacterial strain;
5) with step 4) the knob not Kangding B of gained 0yield Genes recombinant bacterial strain is starting strain, repeatedly repeats step 3)-5) protoplastis preparation, merge, screening operation;
6) genetic stability is investigated
To step 5) obtain knob not Kangding B 0yield Genes recombinant bacterial strain carries out genetic stability experiment and detects, and obtains a strain and produces knob not Kangding B 0high and stable mutant strain, i.e. gene recombination bacterial strain Glarea lozoyensis Q45 of the present invention.
Three, the strain fermentation of Glarea lozoyensis Q45 of the present invention is utilized to produce knob not Kangding B 0method: be by the mycelium inoculation of Glarea lozoyensis Q45 in seed culture medium cultivate after, be inoculated in fermention medium and carry out fermentative production knob not Kangding B 0.
Concrete steps comprise:
1) mycelia of Glarea lozoyensis Q45 bacterial strain good for slant culture is linked in seed culture medium, under 24-28 DEG C, 180-300rpm condition, cultivates 2-3 days, obtain seed liquor;
2) seed liquor is inoculated in fermention medium by the inoculum size of fermention medium volume 5-15%, under 24-28 DEG C, 200-500rmp condition, cultivates 9-11 days.
In order to improve knob not Kangding B 0output, in step 2) fermenting process in 3-11 days, with 0.05-0.4g -1l -1h -1speed stream add the N.F,USP MANNITOL that concentration is 10-20% (v/v).
Described substratum is: seed culture based formulas is (g/L): carbon source 10-70, nitrogenous source 10-80, KH 2pO 40-1, FeSO 47H 2o0.005-0.015, MnSO 44H 2o 0.005-0.015, ZnSO 47H 2o 0.001-0.003, CaCl 22H 2o 0.005-0.015, CuCl 22H 2o 0.0001-0.0005, H 3bO 30.0002-0.0006, (NH 4) 6mo 7o 244H 2o 0.0002-0.0006;
Fermentative medium formula is (g/L): carbon source 40-150, nitrogenous source 1-50, amino acid 0.1-20, KH 2pO 40-3, FeSO 47H 2o0.005-0.015, MnSO 44H 2o 0.005-0.015, ZnSO 47H 2o 0.001-0.003, CaCl 22H 2o 0.005-0.015, CuCl 22H 2o 0.0001-0.0005, H 3bO 30.0002-0.0006, (NH 4) 6mo 7o 244H 2o 0.0002-0.0006;
Described carbon source comprise in glucose, fructose, N.F,USP MANNITOL, Zulkovsky starch, glycerine, maltose, sucrose, tetradecanoic acid one or more; Described nitrogenous source comprises the mixture of one or more the arbitrary ratios in analysis for soybean powder, soybean cake powder, cottonseed meal, Zein powder, peptone, soy peptone, yeast powder, yeast extract powder, corn steep liquor, ammonium sulfate, ammonium chloride, urea; Described amino acid comprises the mixture of one or more the arbitrary ratios in L-glutamic acid, Sodium Glutamate, proline(Pro), Methionin, arginine, ornithine, Threonine, Isoleucine.
Beneficial effect of the present invention:
Knob not Kangding B after Glarea lozoyensis Q45 ferments 11 days 0output can reach 6g/L, and by product knob not Kangding C 0content be less than 3%, knob is Kangding A not 0content do not detect, thus this gene recombination bacterium is except can high yield knob not Kangding B 0outward, also there is the advantage that by product is few, fermentation period is short; In addition owing to utilizing the knob not Kangding B of this explained hereafter 0be conducive to follow-up separation and Extraction and technique amplification, therefore there is huge industrial value.
Embodiment
General explanation
PDA substratum (g/L): potato 200, sucrose 20.
Regeneration culture medium (g/L): NaCl 46.7, potato 200, sucrose 20, agar 2.2.
Lithium chloride regeneration culture medium (g/L): LiCl 3.5, NaCl 46.7, potato 200, sucrose 20, agar 2.2.
The compound method of high osmotic buffer I is: get 2.7218g KH 2pO 4, 4.64g NaCl adds 80ml heartily water dissolution, adjusts PH to 6.0, with water constant volume to 100ml with NaOH;
The compound method of high osmotic buffer II is: the glucose solution of 0.6mol/L;
The preparation method of enzymolysis solution is: get Yatalse 80g, melts wall enzyme 80g, helicase 80g, dissolves with 1L high osmotic buffer I.
The pre-treatment of fermented liquid:
Get 1ml fermented liquid, add 9ml ethanol, thermal agitation 10min, then centrifugal 3000rpm, 10min, the supernatant after centrifugal is used for detecting further.
Knob is Kangding A not 0, knob not Kangding B 0detection:
Employing is worn peace high performance liquid chromatography U3000 and is measured, and its concrete grammar is: chromatographic column, C18 post; Mobile phase A, acetonitrile; Mobile phase B, the phosphoric acid of 0.1%; Elution program, 0min-20min, A:B=40:60,20min-40min, A:B=50:50; Flow velocity 1.5ml/min, determined wavelength, 210nm; Detected temperatures, 25 DEG C.
Knob is Kangding C not 0detection:
Employing is worn peace high performance liquid chromatography P680 and is measured.Its concrete grammar is: chromatographic column, Inertsil 5u Si post; Moving phase ethyl acetate: methyl alcohol: water=84:9:7; Flow velocity, 1.2ml/min; Determined wavelength, 278nm; Detected temperatures, 25 DEG C.
The selection of embodiment 1 gene recombination bacterial strain Glarea lozoyensis
Starting strain: buy the Glarea lozoyensis 74030 from American Type Culture collection warehousing (ATCC).
(1) preparation of protoplastis
The most vigorous, knob not Kangding B will be grown after purifying 0the Glarea lozoyensis bacterial strain access that output is the highest is equipped with in the 250ml shaking flask of 50ml PDA liquid nutrient medium, in 26 DEG C, cultivate 3 days under 220r/min condition, get 8ml nutrient solution in aseptic 10ml centrifuge tube, 1800r/min, 4 DEG C, centrifugal 10min removes supernatant, collect mycelium after cleaning 2 times with high osmotic buffer I, in the mycelium collected, add the enzymolysis solution that 2ml newly configures, 26 DEG C, shaking culture 3h under 100r/min, obtains the liquid that enzymolysis is good; Get 2ml centrifuge tube, the liquid that enzymolysis is good adds wherein by the sucrose solution adding the 1.2M of 0.2ml in centrifuge tube, 1800rpm, 4 DEG C, centrifugal 10min, gently abandoning supernatant, then add 0.2ml high ooze buffering II, 1800rpm, 4 DEG C, centrifugal 10min, gently abandoning supernatant, the height that precipitation protoplastis is resuspended in 0.5ml is oozed in buffered soln II, is the protoplastis of gained.
(2) original mutation word bank preparation
(a) ARTP mutagenesis: protoplastis adjustment concentration step (1) prepared is 10 7cfu/ml, get 10 μ L protoplastiss and be placed on aseptic small iron plate, small iron plate is positioned on ARTP mutagenesis machine Stage microscope, and target chamber is evacuated to 10 -3pa, selects 15KeV helium ion, 50-300*10 14/ cm 2dosage processes these protoplastiss.Then taken off rapidly by small iron plate and be positioned over that 990ul is high to be oozed in buffering II solution, get 100 μ L and coat on regeneration culture medium and regenerate, 26 DEG C, incubator lucifuge is cultivated, observations after 7 days.Be inoculated on PDA solid medium by the bacterium colony that regenerated plate grows point, 26 DEG C, incubator lucifuge cultivates 11 days, and the well-grown bacterium colony of picking carries out liquid fermenting, measures knob not Kangding B after 11 days by HPLC 0output, obtains the higher bacterial strain of 2 strain output-ARTP-Q1, ARTP-Q2, and comparatively original strain improves 40% and 28% respectively, as shown in table 1.
(b) lithium chloride mutagenesis: protoplastis adjustment concentration step (1) prepared is 10 7cfu/ml, gets 100 μ L protoplastiss and coats on lithium chloride regenerated plate, 26 DEG C, and incubator lucifuge is cultivated, observations after 9 days.Be inoculated on PDA solid medium by the bacterium colony that regenerated plate grows point, 26 DEG C, incubator lucifuge cultivates 11 days, and the well-grown bacterium colony of picking carries out liquid fermenting, measures knob not Kangding B after 11 days by HPLC 0output.Enhanced variant goes down to posterity through inclined-plane and investigates stability 5 times, finally obtains the higher bacterial strain of 2 strain output-LiCl-Q1, LiCl-Q2, and comparatively original strain improves 26% and 22% (as shown in table 1) respectively.
Table 1 original mutation word bank
Numbering Knob is Kangding B not 0Concentration (g/L)
ATCC74030 0.81
ARTP-Q1 1.13
ARTP-Q1 1.04
LiCl-Q1 1.02
LiCl-Q2 0.99
(3) fusion of protoplastis
Two groups are divided into after equal-volume mixing after 4 bacterial strains step (2) obtained are prepared into protoplastis according to the method for step (1), one group is placed in ARTP instrument and processes 30-120s, another group is placed in after 60 DEG C of water-baths process 5-30min complete inactivation, the centrifugal 10min of 1800rpm after mixing, abandon supernatant, the PEG adding 26 DEG C of preheatings merges liquid 1ml makes two groups of protoplastiss merge.Wherein PEG merges the preparation method of liquid and is: 70g PEG-4000 is dissolved in 100ml deionized water, 121 DEG C of sterilizing 20min, 4 DEG C of preservation, use be prepended to 26 DEG C be incubated 20min, 4000rpm centrifugal after to get supernatant for subsequent use.
(4) screening of fusant
Protoplastis after fusion high osmotic buffer I centrifuge washing 3 times, and do suitably dilution, gets 0.2ml and coats on regeneration culture medium, cultivates to observe for 7 days for 26 DEG C to grow bacterium colony.By colony inoculation on PDA solid medium, the well-grown bacterium colony of picking, is inoculated in 1.5ml fermention medium, is placed on constant-temperature table that rotating speed is 250rpm, cultivates 11 days, measures its knob not Kangding B for 26 DEG C 0output.This fermentative medium formula is (g/L): glucose 20, N.F,USP MANNITOL 80, W-Gum 20, K 2hPO 42.5, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.001, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH 6.8.Select knob not Kangding B 04 strains that output is the highest, are respectively RHF1-14, RHF1-42, RHF1-75, RHF1-77.
Table 2 first round recombinant bacterial strain shaking flask primary dcreening operation
(5) by above-mentioned 4 strain knobs not Kangding B 0the preparation of the protoplastis described in RHF1 bacterial strain repeating step (3)-(4) that output is higher, the screening of protoplast fusion and fusant, carry out five altogether and take turns gene recombination, obtain 7 strain output and to suddenly change than RHF4 the fusant bacterial strain-RHF5-03, RHF5-07, RHF5-19, RHF5-32, RHF5-57, RHF5-68, RHF5-88 of plant height more than 30%.
(6) the genetic stability test of mutant strain
Take turns recombinant bacterial strain RHF5-03, RHF5-07, RHF5-19, RHF5-32, RHF5-57, RHF5-68, RHF5-88 to carry out PDA solid medium respectively go down to posterity the 5th 5 times, liquid fermenting is carried out to every generation bacterial strain and produces knob not Kangding B 0experiment, measures knob not Kangding B 0output.As shown in table 3, the output of RHF5-03 and RHF5-07 is comparatively stable, considers in industrial production the bacterial classification requiring stable hereditary property, therefore selects the RHF5-07 that output is more stable, and this bacterial strain renames as Glarealozoyensis Q45.The mean yield that Glarea lozoyensis Q45 produces Pneumocandin B0 is 6.12g/L.
Table 3 genetic stability is investigated
Embodiment 2 utilizes Glarea lozoyensis Q45 on 5L fermentor tank, carry out knob not Kangding B 0the method of producing.
1. inclined-plane seed culture
Solid culture method: inoculation, on PDA solid slant culture base, is cultivated 11 days for 25 DEG C.
2. shake-flask seed is cultivated
Seed culture medium (g/L): glucose 40, analysis for soybean powder 20, corn steep liquor 10, KH 2pO 41, FeSO 47H 2o 0.01, MnSO 44H 2o0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.001, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH is adjusted to 5.0.
Culture temperature: 25 DEG C
Incubation time: 3 days
Shaking speed: 220rpm
Shake-flask seed cultural method: take out one piece of 1cm from solid slant culture base 2the bacterium access liquid amount of size is in the 250ml shaking flask of 50ml, cultivates 3 days.
3,5L ferment tank
5L fermentation tank culture medium
Fermention medium (g/L): glucose 20, N.F,USP MANNITOL 80, peptone 30, proline(Pro) 2, K 2hPO 43, FeSO 47H 2o0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH 6.8.
The canned substratum 3L of liquid amount: 5L, 121 DEG C of sterilizing 30min.
The cultural method of 5L tank: accessed by cultured shake-flask seed liquid 300mL in fermentor tank, at 400rpm, 1vvm, cultivates 11 days under 26 DEG C of conditions.Within 3rd day, start with 0.15g in fermentation -1l -1h -1speed stream add the N.F,USP MANNITOL that stream adds 20%.
Fermentation termination judges: mycelia color is dark, and cavity is more, and mycelia has fracture, autolysis.
According to above-mentioned fermentation condition and technique, 5L tank carries out 3 batch fermentation experiments, and fermentation unit is respectively: 6.22g/L, 6.53g/L, 6.75g/L.
Embodiment 3-6Glarea lozoyensis Q45 utilizes other carbon source, nitrogenous source and nutritive ingredient metabolism to produce knob not Kangding B 0situation
Embodiment 3-6 cultural method adopts method used in example 2 to cultivate.
In embodiment 3-6 seed culture medium, carbon source adopts Zulkovsky starch 40g/L; Nitrogenous source adopts analysis for soybean powder 20g/L and yeast powder 2g/L; Other inorganic salt are (g/L): KH 2pO 41, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002; PH is adjusted to 5.2;
In embodiment 3 fermention medium, carbon source adopts fructose 80g/L; Nitrogenous source adopts peptone 20g/L; Amino acid adopts Sodium Glutamate 20g/L; Other inorganic salt (g/L): K 2hPO 43, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH 6.8.
In embodiment 4 fermention medium, carbon source adopts N.F,USP MANNITOL 150g/L; Nitrogenous source adopts Zein powder 50g/L; Amino acid adopts ornithine 0.1g/L; Other inorganic salt (g/L): K 2hPO 43, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH6.8.
In embodiment 5 fermention medium, carbon source adopts fruit sucrose 10g/L; Nitrogenous source adopts cottonseed meal 40g/L; Amino acid adopts arginine 1g/L; Other inorganic salt (g/L): K 2hPO 43, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o 0.0002, pH 6.8.
In embodiment 6 fermention medium, carbon source adopts maltose 60g/L; Nitrogenous source adopts ammonium chloride 1g/L; Amino acid adopts Isoleucine 2.5g/L or Methionin 0.5g/L; Other inorganic salt (g/L): K 2hPO 43, FeSO 47H 2o 0.01, MnSO 44H 2o 0.01, ZnSO 47H 2o 0.002, CaCl 22H 2o 0.01, CuCl 22H 2o 0.00025, H 3bO 30.00056, (NH 4) 6mo 7o 244H 2o0.0002, pH 6.8.
Measurement result is as shown in table 4:
Embodiment is numbered Knob is Kangding B not 0Concentration (g/L)
Embodiment 3 5.87
Embodiment 4 6.6
Embodiment 5 5.52
Embodiment 6 4.23

Claims (7)

1. a production knob not Kangding B 0gene recombination bacterial strain, its Classification And Nomenclature is Glarea lozoyensis Q45, and this bacterial strain is by China typical culture collection center preservation, and preservation registration number is CCTCC NO:M 2014416, and preservation period is on September 14th, 2014.
2. the strain fermentation of Glarea lozoyensis Q45 according to claim 1 produces knob not Kangding B 0method, it is characterized in that: by the mycelium inoculation of Glarea lozoyensis Q45 in seed culture medium cultivate after, be inoculated in fermention medium and carry out fermentative production knob not Kangding B 0.
3. produce knob not Kangding B according to the strain fermentation of the Glarea lozoyensis Q45 utilized described in claim 1 0method, it is characterized in that, concrete steps comprise:
1) mycelia of the bacterial strain of Glarea lozoyensis Q45 good for slant culture is linked in seed culture medium, under 24-28 DEG C, 180-260rpm condition, cultivates 2-3 days, obtain seed liquor;
2) seed liquor is inoculated in fermention medium by the inoculum size of fermention medium volume 5-15%, under 24-28 DEG C, 200-500rmp condition, cultivates 9-11 days.
4. the strain fermentation of Glarea lozoyensis Q45 according to claim 1 and 2 produces knob not Kangding B 0method, it is characterized in that, in step 2) fermenting process in 3-11 days, with 0.05-0.4g -1l -1h -1speed stream add the N.F,USP MANNITOL that concentration is 10-20% (v/v).
5. the strain fermentation of Glarea lozoyensis Q45 according to claim 4 produces knob not Kangding B 0method, it is characterized in that, described substratum is:
Seed culture based formulas is (g/L): carbon source 10-70, nitrogenous source 10-80g/L, KH 2pO 40-1, FeSO 47H 2o0.005-0.015, MnSO 44H 2o 0.005-0.015, ZnSO 47H 2o 0.001-0.003, CaCl 22H 2o 0.005-0.015, CuCl 22H 2o 0.0001-0.0005, H 3bO 30.0002-0.0006, (NH 4) 6mo 7o 244H 2o 0.0002-0.0006;
Fermentative medium formula is (g/L): carbon source 40-150, nitrogenous source 1-50, amino acid 0.1-20, K 2hPO 40-3, FeSO 47H 2o0.005-0.015, MnSO 44H 2o 0.005-0.015, ZnSO 47H 2o 0.001-0.003, CaCl 22H 2o 0.005-0.015, CuCl 22H 2o 0.0001-0.0005, H 3bO 30.0002-0.0006, (NH 4) 6mo 7o 244H 2o 0.0002-0.0006.
6. the strain fermentation of Glarea lozoyensis Q45 as claimed in claim 5 produces knob not Kangding B 0method, it is characterized in that described carbon source comprises in glucose, fructose, N.F,USP MANNITOL, Zulkovsky starch, glycerine, maltose, sucrose, tetradecanoic acid one or more; Described nitrogenous source comprises the mixture of one or more the arbitrary ratios in analysis for soybean powder, soybean cake powder, cottonseed meal, Zein powder, peptone, soy peptone, yeast powder, yeast extract powder, corn steep liquor, ammonium sulfate, ammonium chloride, urea; Described amino acid comprises the mixture of one or more the arbitrary ratios in L-glutamic acid, Sodium Glutamate, proline(Pro), Methionin, arginine, ornithine, Threonine, Isoleucine.
7. production knob according to claim 1 not Kangding B 0the selection of gene recombination bacterial strain Glarea lozoyensis Q45, it is characterized in that, comprise the following steps:
1) preparation of protoplastis
Glarea lozoyensis original strain access seed culture medium is cultivated, collects mycelia, use enzymolysis solution enzymolysis, make protoplastis;
2) original mutation word bank
By step 1) the protoplastis volume prepared is divided into two groups, atmospheric pressure at room plasma body (ARTP) and lithium chloride is adopted to carry out mutagenic treatment respectively, coat on regeneration culture medium by the mutant strain of acquisition, the well-grown bacterium colony of picking carries out fermentation and measures knob not Kangding B 0, select the high mutant strain of output and carry out inclined-plane and to go down to posterity investigation stability, obtain the high productive mutant of many strains stable in properties, form sudden change word bank;
3) fusion of protoplastis
By step 2) mutant strain that the proterties that obtains is stable, according to step 1) prepare protoplastis, the protoplastis of every plant mutant strain all equal-volume is divided into two groups, one group adopts ARTP to inject deactivation, another group adopts hot deactivation, the protoplastis mixing after two groups of deactivations is added fusion liquid and merges at random;
4) screening of fusant
By step 3) protoplastis after the fusion that obtains with high ooze buffering centrifuge washing after, coat on regeneration culture medium, the well-grown bacterium colony of picking carries out fermentation mensuration knob not Kangding B 0, obtain knob not Kangding B 0yield Genes resets bacterial strain;
5) with step 4) the knob not Kangding B of gained 0yield Genes reset bacterial strain be starting strain, repeatedly repeat step 3)-5) protoplastis preparation, merge, screening operation;
6) genetic stability is investigated
To step 5) obtain knob not Kangding B 0yield Genes is reset bacterial strain and is carried out genetic stability experiment detection, obtains a strain and produces knob not Kangding B 0high and stable mutant strain, i.e. gene recombination bacterial strain Glarea lozoyensis Q45 of the present invention.
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CN107201316A (en) * 2017-07-14 2017-09-26 浙江海正药业股份有限公司 A kind of Aspergillus and its production lung read rhzomorph B0Method
CN108048512A (en) * 2018-01-24 2018-05-18 湖南唯创前沿科技有限公司 Prepare the fermentation process of Pneumocandin B0
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ES2433142A1 (en) * 2012-06-04 2013-12-09 Fundación Medina, Centro De Excelencia En Investigación De Medicamentos Innovadores En Andalucía Use of ortho-depsid compounds as neuroprotective agents (Machine-translation by Google Translate, not legally binding)
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Publication number Priority date Publication date Assignee Title
CN106755223A (en) * 2017-01-20 2017-05-31 信泰制药（苏州）有限公司 A kind of fermentation process of Pneumocandin B0
CN107201316A (en) * 2017-07-14 2017-09-26 浙江海正药业股份有限公司 A kind of Aspergillus and its production lung read rhzomorph B0Method
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