CN1793356A - Process for selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete - Google Patents
Process for selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete Download PDFInfo
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- CN1793356A CN1793356A CN 200510015847 CN200510015847A CN1793356A CN 1793356 A CN1793356 A CN 1793356A CN 200510015847 CN200510015847 CN 200510015847 CN 200510015847 A CN200510015847 A CN 200510015847A CN 1793356 A CN1793356 A CN 1793356A
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Abstract
The invention discloses a method used laser mutagenic streptomyces roseosporus to breed daptomycin high yield strain breeding. It belongs to strain mutagenesis breeding technology. The method includes the following steps: forming spore suspension; loading in aseptic quartz groove; irradiating by He-Ne laser for certain time; coating on Gao-NO.1 culture medium flat after diluting by aseptic water to culture pure streptomyces roseosporus; selecting out pure strain; and transferring it to liquid fermentation medium to culture high yield strain. The invention has the advantages of simple device, practical method, and safe operation. Its mutagenesis effect is far better than traditional physical chemistry mutagenesis method. Compared with original strain, the fermentation unit of the formed streptomyces roseosporus can rise by 0.5-3.3 times.
Description
Technical field
The invention belongs to microorganism induced-mutation technique field, particularly utilize mutation induced by laser means seed selection daptomycin high productive mutant technology.
Background technology
It is one of China and even global severe challenge that faces that pathogenic bacteria produces resistance to microbiotic, and the microbiotic that searching can effectively suppress anti-medicine bacterium is the fundamental way that addresses this problem.Vancomycin, once being known as by people is the last line of defense of resisting gram-positive bacteria, yet, find increasing anti-this medicine resistant organism at present in China and even the world wide.Development of new microbiotic of new generation efficiently is extremely urgent.
" Streptomyces roseosporus " (the Latin name is called " Streptomyces roseosporus ") is a kind of streptomyces actinomycetes, its tunning can extract daptomycin (English name " daptomycin "), the acid lipopeptide antibiotic that a kind of tryptophane of the ring-type beta-amino acids peptide chain N-end of being made up of 13 amino-acid residues a decane side chain and is formed by connecting.
Daptomycin is compared with vancomycin clinically and is shown advantages such as wide spectrum, drug effect is fast, administration is little, side reaction is little because its particular structure and mechanism of action, is that at present universally acknowledged vancomycin etc. is now used the antibiotic best substitute of a line.Therefore, daptomycin has vast market prospect and huge economic benefit, should accelerate the study on the industrialization of this product.
Daptomycin 2003 has been realized suitability for industrialized production in the U.S., and annual sales amount was above 8,000 ten thousand dollars in 2004.Although the bacterial classification of domestic existing preparation daptomycin, this bacterium are the common bacterial strain without effectively positive mutagenesis, a little less than the preparation daptomycin ability, usefulness is low.
The application of mutation induced by laser technology in microorganism mutation breeding is comparatively extensive, it acts on microorganism cells and produces light, heat, pressure, point, electromagnetic field, low-intensity magnetic field effect etc., has changed the order of DNA and RNA genetic code in the microorganism cells, causes variation.Teng Lirong etc. handle hyaluronic acid with the He-Ne mutation induced by laser and produce bacterium---streptococcus zooepidemicus, and the positive mutating strain hyaluronic acid volume of production that obtains improves 4.5 times than original strain.Employing laser such as Han Jianrong have carried out mutagenic treatment to the protoplastis of mould PT95 bacterial strain, and seed selection improves 28.3% mutant strain to a strain carotenoid.And laser is applied in the seed selection aspect that produces the daptomycin bacterial classification, then do not see relevant patent and bibliographical information.
Summary of the invention
The objective of the invention is to propose a kind of method of selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete, Using such method can improve the output of daptomycin effectively.
The objective of the invention is to be achieved through the following technical solutions: a kind of method of selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete, it is characterized in that comprising following process: with this pattern bacterium be Streptomyces roseosporus (Streptomyces roseosporus) at room temperature, get slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/ml, 0.1～0.3ml spore suspension is sub-packed in the aseptic quartz cell, adopting wavelength is that the He-Ne laser of 632.8nm is under irradiation power 5～30mW, to this spore irradiation 5～150min, then through the spore suspension of radiation treatment after 10～10000 times of sterilized water dilutions, coat 28～30 ℃ of cultivation 7～10d in the Gause I culture medium flat plate, choosing well-grown single strain is inoculated in the 250ml that fills the 30ml liquid fermentation medium and shakes bottle, in 28～30 ℃, cultivate 90～120h in 180～220rpm shaking table, thereby obtain the pure bacterium Streptomyces roseosporus of the stabilization characteristics of genetics of mutagenesis.
Above-mentioned liquid fermentation medium is formed and mass content is: glucose 0.6～1.0g, dextrin 2～4g, casein food grade 0.5～1.0g, groundnut meal 0.4～0.6g, L-asparagine 0.08～0.12g, K
2SO
40.4～0.6g is dissolved in the 1L distilled water, initial pH 7.5.
The mode of the He-Ne laser radiation that the present invention adopts is carried out the mutafacient system that the bacterial classification to the preparation daptomycin carries out mutation induced by laser, mutation induced by laser equipment is simple, easy to implement the method, operational safety, its mutagenesis effect is good far beyond traditional physical chemistry mutafacient system, and bigger promotional value is arranged in the microbiological pharmacy strain improvement.The present invention carries out mutagenesis by utilizing He-Ne laser irradiation, can select high yield microbiotic daptomycin streptomycete mutant strain.Mutant strain positive variation rate 5～30%.Fermentation unit improves 0.5～3.5 times than original strain.
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
Embodiment 1
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 5min, irradiation power 10mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 8～9%.Fermentation unit improves 1.3～1.4 times than original strain.
Embodiment 2
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 25min, irradiation power 30mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 18～19%.Fermentation unit improves 2.3～2.4 times than original strain.
Embodiment 3
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 30min, irradiation power 25mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 25～26%.Fermentation unit improves 3.0～3.3 times than original strain.
Embodiment 4
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 37.5min, irradiation power 20mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 23～24%.Fermentation unit improves 2.7～2.8 times than original strain.
Embodiment 5
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 50min, irradiation power 15mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 18～19%.Fermentation unit improves 1.7～1.8 times than original strain.
Embodiment 6
At room temperature, get the Streptomyces roseosporus inclined-plane and make 10 with sterilized water
4～10
5The spore suspension of individual spore/ml is drawn 0.2ml and is placed cumulative volume 3.5ml (in 10 * 10 * 35mm) the aseptic quartz cell, to carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, lasing beam diameter 10mm) irradiation 150min, irradiation power 5mW.After diluting 100～1000 times, suspension 0.1ml after the absorption radiation treatment coats the Gause I culture medium flat plate, cultivate 10d for 30 ℃, the well-grown single strain of picking is inoculated in and fills the 30ml liquid fermentation medium and (consist of: glucose 0.75g/L, dextrin 3g/L, casein food grade 1g/L, groundnut meal 0.5g/L, L-asparagine 0.1g/L, K
2SO
40.5g/L pH 7.5) 250ml shake in the bottle, in 30 ℃, cultivate 110h in the 200rpm shaking table, the HPLC method detects daptomycin output in the fermented liquid, selects the inheritance stability mutant strain of high yield daptomycin.Mutant strain positive variation rate 8～9%.Fermentation unit improves 0.8～0.9 times than original strain.
Claims (2)
1. the method for a selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete, it is characterized in that comprising following process: with this pattern bacterium be Streptomyces roseosporus (Streptomyces roseosporus) at room temperature, get slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/ml, 0.1～0.3ml spore suspension is sub-packed in the aseptic quartz cell, adopting wavelength is that the He-Ne laser of 632.8nm is under irradiation power 5～30mW, to this spore irradiation 5～150min, then through the spore suspension of radiation treatment after 10～10000 times of sterilized water dilutions, coat 28～30 ℃ of cultivation 7～10d in the Gause I culture medium flat plate, choosing well-grown single strain is inoculated in the 250ml that fills the 30ml liquid fermentation medium and shakes bottle, in 28～30 ℃, cultivate 90～120h in 180～220rpm shaking table, thereby obtain the pure bacterium Streptomyces roseosporus of the stabilization characteristics of genetics of mutagenesis.
2. by the described method of claim 1, it is characterized in that liquid fermentation medium is formed and mass content is: glucose 0.6～1.0g, dextrin 2～4g, casein food grade 0.5～1.0g, groundnut meal 0.4～0.6g, L-asparagine 0.08～0.12g, K
2SO
40.4～0.6g is dissolved in the 1L distilled water, initial pH7.5.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899410A (en) * | 2010-07-13 | 2010-12-01 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces parvus and application thereof for preparing daptomycin |
| CN101777094B (en) * | 2010-02-01 | 2011-11-16 | 天津大学 | Metabolic network analysis method for streptomyces roseosporus as daptomycin producing thalli |
| CN102242073A (en) * | 2010-05-12 | 2011-11-16 | 山东鲁北药业有限公司 | Method for preparing daptomycin by fermentation |
| CN102286497A (en) * | 2010-09-29 | 2011-12-21 | 天津大学 | Method for improving yield of daptomycin through expressing zwf2 genes and application of daptomycin genetic engineering |
| CN102399706A (en) * | 2010-09-17 | 2012-04-04 | 上海来益生物药物研究开发中心有限责任公司 | Streptomyces parvus and application thereof |
| CN102477067A (en) * | 2010-11-19 | 2012-05-30 | 上海来益生物药物研究开发中心有限责任公司 | 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine and preparation method and application thereof |
| CN102485902A (en) * | 2010-12-06 | 2012-06-06 | 北大方正集团有限公司 | Method for producing daptomycin by fermentation |
| CN101824452B (en) * | 2010-01-14 | 2012-07-04 | 天津大学 | Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently |
| CN102965304A (en) * | 2011-10-27 | 2013-03-13 | 四川大学 | Daptomycin high-producing strain and preparation method thereof |
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2005
- 2005-11-03 CN CN 200510015847 patent/CN1793356A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824452B (en) * | 2010-01-14 | 2012-07-04 | 天津大学 | Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently |
| CN101777094B (en) * | 2010-02-01 | 2011-11-16 | 天津大学 | Metabolic network analysis method for streptomyces roseosporus as daptomycin producing thalli |
| CN102242073B (en) * | 2010-05-12 | 2013-03-20 | 山东鲁北药业有限公司 | Method for preparing daptomycin by fermentation |
| CN102242073A (en) * | 2010-05-12 | 2011-11-16 | 山东鲁北药业有限公司 | Method for preparing daptomycin by fermentation |
| CN101899410B (en) * | 2010-07-13 | 2012-05-16 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces parvus and application thereof for preparing daptomycin |
| CN101899410A (en) * | 2010-07-13 | 2010-12-01 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces parvus and application thereof for preparing daptomycin |
| CN102399706A (en) * | 2010-09-17 | 2012-04-04 | 上海来益生物药物研究开发中心有限责任公司 | Streptomyces parvus and application thereof |
| CN102399706B (en) * | 2010-09-17 | 2014-08-20 | 上海来益生物药物研究开发中心有限责任公司 | Streptomyces parvus and application thereof |
| CN102286497A (en) * | 2010-09-29 | 2011-12-21 | 天津大学 | Method for improving yield of daptomycin through expressing zwf2 genes and application of daptomycin genetic engineering |
| CN102477067A (en) * | 2010-11-19 | 2012-05-30 | 上海来益生物药物研究开发中心有限责任公司 | 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine and preparation method and application thereof |
| CN102477067B (en) * | 2010-11-19 | 2014-12-10 | 上海来益生物药物研究开发中心有限责任公司 | 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine and preparation method and application thereof |
| CN102485902B (en) * | 2010-12-06 | 2014-08-13 | 北大方正集团有限公司 | Method for producing daptomycin by fermentation |
| CN102485902A (en) * | 2010-12-06 | 2012-06-06 | 北大方正集团有限公司 | Method for producing daptomycin by fermentation |
| CN102965304A (en) * | 2011-10-27 | 2013-03-13 | 四川大学 | Daptomycin high-producing strain and preparation method thereof |
| CN102965304B (en) * | 2011-10-27 | 2014-03-26 | 四川大学 | Daptomycin high-producing strain and preparation method thereof |
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