CN103740614A - Actinoplanes of high-yielding sirolimus - Google Patents
Actinoplanes of high-yielding sirolimus Download PDFInfo
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- CN103740614A CN103740614A CN201310728024.4A CN201310728024A CN103740614A CN 103740614 A CN103740614 A CN 103740614A CN 201310728024 A CN201310728024 A CN 201310728024A CN 103740614 A CN103740614 A CN 103740614A
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Abstract
The invention discloses actinoplanes CGMCC NO.8430 of high-yielding sirolimus, belonging to the technical field of fermentation engineering. The bacterium is generated by mutating actinoplanes hn-016 of high-yielding sirolimus screened in a soil sample of Hainan Island as an original strain by plasmas at constant pressure and room temperature. Compared with conventional mutation methods, the mutant strain BCStr-096 disclosed by the invention is higher in hereditary stability and higher in production capacity of sirolimus, and the output is improved by 175% compared with the output 200mg/L of the original strain hn-016. The output is improved by about 37% in the highest fermentation level of existing sirolimus, so that the production cost is greatly lowered.
Description
Technical field
The invention belongs to fermentation engineering field, be specifically related to the actinoplanes mutagenic strain BCStr-096 that a plant height produces rapamycin.
Background technology
Rapamycin (rapamycin, RPM) having another name called sirolimus, is the nitrogenous triolefin macrolide antibiotics of tool unique effect mechanism, has antimycotic, antiproliferative effect, it is reported, the people's such as Luan FL experimentation on animals finds that rapamycin also has antineoplastic action.Rapamycin is the most promising novel potent immunosuppressor in the world at present, its molecular structure is similar to FK506, it is another close immune protein bonding agent, can be used for the anti-repulsive interaction of organ transplantation, its immunosuppressive action, than the strong decades of times of S-Neoral, is the immunosuppressor that renal toxicity is minimum, also can be used for treating the autoimmune disorders such as rheumatoid arthritis, lupus erythematosus, develop at present gene therapy and antitumor drug, have broad application prospects.
At first, RPM, as hypotoxicity antifungal antibiotic, finds that it has immunosuppressive action to autoimmune disorder for 1978.Inspired by this, Mortis in 1989 formally starts RPM to try out as the neotype immunosuppressant of the anti-repulsion of organ transplantation.In September, 1999 U.S. FDA official approval rapamycin is put on market as the anti-rejection drugs of renal transplantation, by Cordis company, researched and developed simultaneously using rapamycin as the intravascular stent Cypher of coating medicine in 2002 in succession in Europe, the U.S. and Japan's listing.The domestic rapamycin of in June, 2005 China Fujian Microorganism Inst. research and development is put on market through SFDA approval, and the rapamycin of Er You U.S. Wyeth company exploitation has now entered clinical experimental stage as anticarcinogen.Before 2006, rapamycin is 1 gram, 6,000 U.S. dollar in the price of American market, at present, the sales volume of rapamycin increases rapidly, its market price also slightly reduces, estimate that it will compete effectively with S-Neoral, little by little divide the market of anti-transplant organ rejection medicine equally, Pharmaprojects(is to grind new drug dynamic) predict that its sales volume reaches hundred million dollars of 5-20.Rapamycin belongs to six kind new medicines at home, and rapamycin in 2009 the at home sales volume in market surpasses 500,000,000 yuans, has wide market outlook.
At present, the suitability for industrialized production of rapamycin is mainly to produce by microbial fermentation processes, but this antibiotic fermentation level is also quite low at present, has the highest fermentation level of bibliographical information also only to reach 400mg/L left and right.Therefore, rapamycin biological fermentation is produced the deficiency existing and is mainly that the biological fermentation unit of rapamycin is not high, is unfavorable for extracting purifying, can not fully meet the requirement of suitability for industrialized production.
The present invention utilizes atmospheric pressure at room plasma body mutagenesis technology, filters out a strain rapamycin superior strain, has improved the output of rapamycin, effectively reduces production cost.
Summary of the invention
Problem to be solved by this invention is to utilize atmospheric pressure at room plasma body mutagenesis technology, filters out the novel rapamycin superior strain of a strain.
Bacterial strain provided by the present invention is actinoplanes (Actinoplanes sp.) mutagenic strain BCStr-096, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO.8430, Classification And Nomenclature: actinoplanes Actinoplanes sp.; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101; Preservation date on November 05th, 2013.This bacterial strain is to take the actinoplanes hn-016(Actinoplanes sp. of the product rapamycin that filters out from the pedotheque in Hainan Island) be starting strain, through many group Mutation screenings, obtain.
Actinoplanes of the present invention, specifically carries out seed selection by the following technical solutions:
1. adopt atmospheric pressure at room plasma body mutagenesis technology seed selection rapamycin superior strain
(1) select the actinoplanes hn-016 inclined-plane bacterial strain of well-grown product rapamycin, with physiological saline, wash lower spore, through filtered through gauze, make 10
6the spore suspension of individual/mL;
(2) by the spore suspension in (1), with liquid-transfering gun, draw on the round iron plate that 10 μ L are 1cm in diameter, be placed in and take helium as working gas, power 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, processing distance is 2mm, processes respectively 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s, the spore suspension of processing is carried out to gradient dilution and be coated with flat board, make lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of high, medium and low three different lethal doses, the spore suspension of bacterial strain hn-016 is carried out to plasma body mutagenesis;
(4) spore suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5) by the mutagenesis spore suspension in (4), carry out after gradient dilution, coat respectively and contain 2mg/L paraxin, 0.8mg/L Streptomycin sulphate, 4mg/L erythromycin, in the resistant panel of 1mg/L gentamicin;
(6) by behind the single bacterium colony switching inclined-plane growing in each resistant panel in (5), the inoculum size with 1%, is inoculated in seed culture medium, 28 ℃, 220r/min, cultivates after 45-50h, inoculum size with 10% is inoculated in fermention medium by seed liquor, and 28 ℃, 220r/min cultivates 7-8d;
(7) get fermented liquid in (6) in centrifuge tube, the centrifugal 10min of 5000r/min, get thalline methanol extraction, pour in the triangular flask that 10-15 granulated glass sphere is housed, on the shaking table of 180-200r/min, shake lixiviate 1h, get extract, the centrifugal 10min of 10000r/min, cross after organic filter membrane, utilize the output of high effective liquid chromatography for measuring rapamycin, by this method, filter out rapamycin superior strain BCStr-096.
2. the cultural method of actinoplanes BCStr-096 is:
(1) actication of culture: actinoplanes BCStr-096 is forwarded to the separated slant medium of preservation, 28 ℃ of constant temperature culture 7-15 days;
(2) seed culture: choose well-grown inclined-plane, wash lower spore with the physiological saline of sterilizing, preparation concentration approximately 10
6the spore suspension of individual/mL, the inoculum size with 1% is inoculated in the 500mL triangular flask that 60mL seed culture medium is housed, and 28 ℃, 220r/min, 45-50h is cultivated in concussion;
(3) fermentation culture: by the seed culture fluid in (2), the inoculum size with 10% is inoculated in the 500mL triangular flask that 60mL fermention medium is housed, 28 ℃, 220r/min, 7-8d is cultivated in concussion;
3. actinoplanes BCStr-096 mutagenesis and culturing process used medium consist of:
(1) preservation isolation medium: rolled oats 3%, glucose 2%, yeast powder 1%, peptone 1%, calcium carbonate 0.25%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min;
(2) seed culture medium: Zulkovsky starch 2.8%, peptone 0.8%, glucose 2%, soybean cake powder 1%, calcium carbonate 0.3%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min;
(3) fermention medium: W-Gum 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.5%, glycerine 1%, calcium carbonate 0.3%, bubble enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min.
Beneficial effect:
Mutagenic strain BCStr-096 of the present invention obtains by plasma body mutagenesis method, the bacterial strain genetic stability obtaining than conventional mutafacient system is higher, the throughput of rapamycin is higher, its output is than the output 200mg/L of starting strain hn-016, improved 175%, and on the highest fermentation level of existing rapamycin, improved 37% left and right, greatly reduced production cost.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, and following examples are descriptive, is not restrictive, can not limit protection scope of the present invention with this.
Embodiment 1
The bacterial strain hn-016 of the product rapamycin filtering out from soil
1. thalli morphology: thalline is gram-positive microorganism, without aerial hyphae, breeds in the mode of spore, and spore is perfectly round, the extremely raw feathering of most of tool, can move about.
2. colonial morphology: on flat board, the base silk surface of bacterium colony is particulate state, orange-yellow, and along with the prolongation of incubation time, bacterium colony can be by the orange-yellow tawny that becomes.
3. cultural characteristic: this bacterium is high oxygen consumption bacterium, and optimum growth temperature is 28 ℃, and the most suitable growth pH is 7.0-7.2; In shaking speed, be 210-240r/min, while cultivating 7-8d, rapamycin production is the highest, and in fermenting process, dissolved oxygen has remarkable effect to the generation of rapamycin.
Embodiment 2
Take actinoplanes hn-016 as starting strain, adopt atmospheric pressure at room plasma body mutagenesis technology screening bacterial strain BCStr-096
1. the acquisition of mutagenic strain
(1) select the actinoplanes hn-016 inclined-plane bacterial strain of well-grown product rapamycin, with physiological saline, wash lower spore, through filtered through gauze, make 10
6the spore suspension of individual/mL;
(2) by the spore suspension in (1), with liquid-transfering gun, draw on the round iron plate that 10 μ L are 1cm in diameter, be placed in and using helium as working gas, power is 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, processing distance is 2mm, processes respectively 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s, the spore suspension of processing is carried out to gradient dilution and be coated with flat board, make lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of 40s, 80s, tri-different lethal doses of 100s, the spore suspension of bacterial strain hn-016 is carried out to plasma body mutagenesis;
(4) spore suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5), by the mutagenesis spore suspension in (4), carry out gradient dilution 10
-1-10
-6, get 10
-4, 10
-5, 10
-6three dilution spore suspensions, coat respectively and contain 2mg/L paraxin, 0.8mg/L Streptomycin sulphate, and 4mg/L erythromycin, in the resistant panel of 1mg/L gentamicin;
(6) by behind the single bacterium colony switching inclined-plane growing in each resistant panel in (5), the inoculum size with 1%, is inoculated in seed culture medium, 28 ℃, 220r/min, cultivates after 45-50h, inoculum size with 10% is inoculated in fermention medium by seed liquor, and 28 ℃, 220r/min cultivates 7-8d;
Described seed culture medium consists of: Zulkovsky starch 2.8%, and peptone 0.8%, glucose 2%, soybean cake powder 1%, calcium carbonate 0.3%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min;
Described fermention medium consists of: W-Gum 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.5%, glycerine 1%, calcium carbonate 0.3%, bubble enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min.
(7) get fermented liquid in (6) in centrifuge tube, the centrifugal 10min of 5000r/min, get thalline methanol extraction, pour in the triangular flask that 13 granulated glass spherees are housed, on the shaking table of 190r/min, shake lixiviate 1h, get extract, the centrifugal 10min of 10000r/min, cross after organic filter membrane, utilize the output of high effective liquid chromatography for measuring rapamycin, by this method, filter out the mutagenic strain BCStr-096 of high yield rapamycin.
2. the characteristic of mutagenic strain BCStr-096
(1) thalli morphology: thalline is gram-positive microorganism, without aerial hyphae, breeds in the mode of spore, and spore is perfectly round, and the extremely raw feathering of most of tool, can move about.
(2) colonial morphology: on flat board, the base silk surface of bacterium colony is particulate state, orange-yellow, along with the prolongation of incubation time, bacterium colony can be by the orange-yellow tawny that becomes.
(3) cultural characteristic: this bacterium is high oxygen consumption bacterium, and optimum growth temperature is 28 ℃, and the most suitable growth pH is 7.1; In shaking speed, be 230r/min, while cultivating 7-8d, rapamycin production is the highest.
(4) can in the substratum that contains 0.8mg/L Streptomycin sulphate, grow, and well-grown.
Embodiment 3
The cultivation of actinoplanes BCStr-096
1. the preparation of substratum
(1) preservation isolation medium: rolled oats 3%, glucose 2%, yeast powder 1%, peptone 1%, calcium carbonate 0.25%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min;
(2) seed culture medium: Zulkovsky starch 2.8%, peptone 0.8%, glucose 2%, soybean cake powder 1%, calcium carbonate 0.3%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min;
(3) fermention medium: W-Gum 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.5%, glycerine 1%, calcium carbonate 0.3%, bubble enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min.
2. actication of culture
The bacterial classification BCStr-096 that glycerine is preserved is forwarded to preservation isolation medium inclined-plane, in constant incubator, cultivates 7-15d for 28 ℃;
3. seed culture
Select well-grown activated inclined plane, be inoculated in (the in-built 60mL substratum of 500mL triangular flask) in the seed culture medium making in above-mentioned 1 is housed, in 28 ℃, 220r/min, cultivates 45-50h.
4. fermentation culture
By the seed liquor making in 3 with 10%(v/v) inoculum size is inoculated in (the in-built 60mL substratum of 500mL triangular flask) in fermention medium, in 28 ℃, 220r/min condition bottom fermentation is cultivated 8d.
Detected result: the output of actinoplanes (Actinoplanes) BCStr-096 rapamycin on fermention medium is 535-565mg/L.
Claims (6)
1. a plant height produces the actinoplanes mutagenic strain of rapamycin, described actinoplanes BCStr-096(Actinoplanes), deposit number is CGMCC NO.8430.
2. the actinoplanes mutagenic strain of high yield rapamycin according to claim 1, is characterized in that, this bacterium is to take actinoplanes hn-016(Actinoplanes) be starting strain, raw through the sell of one's property of atmospheric pressure at room plasma inducing.
3. the actinoplanes mutagenic strain of high yield rapamycin according to claim 1, is characterized in that, the output of this bacterium rapamycin on fermention medium is 535-565mg/L.
4. the actinoplanes mutagenic strain of high yield rapamycin according to claim 3, is characterized in that fermention medium consists of: W-Gum 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.5%, glycerine 1%, calcium carbonate 0.3%, bubble enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 ℃ of high pressure steam sterilization 20min.
5. the actinoplanes mutagenic strain of high yield rapamycin according to claim 1, is characterized in that, can in the substratum that contains 0.8mg/L Streptomycin sulphate, well grow.
6. the application of the actinoplanes mutagenic strain BCStr-096 of high yield rapamycin according to claim 1 in fermentative production rapamycin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105420222A (en) * | 2015-12-04 | 2016-03-23 | 帝斯曼江山制药（江苏）有限公司 | Mutation screening method of ketogulonigenium vulgare |
| CN107164260A (en) * | 2017-05-18 | 2017-09-15 | 福建省微生物研究所 | A kind of Actinolpanes teichornyceticus mutagenic strain of high yield teicoplanin and its application |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
| CN109321560A (en) * | 2018-10-31 | 2019-02-12 | 成都雅途生物技术有限公司 | A kind of selection of high yield rapamycin streptomycete |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420222A (en) * | 2015-12-04 | 2016-03-23 | 帝斯曼江山制药（江苏）有限公司 | Mutation screening method of ketogulonigenium vulgare |
| CN107164260A (en) * | 2017-05-18 | 2017-09-15 | 福建省微生物研究所 | A kind of Actinolpanes teichornyceticus mutagenic strain of high yield teicoplanin and its application |
| CN107164260B (en) * | 2017-05-18 | 2019-12-31 | 福建省微生物研究所 | Tencel actinoplanes mutant strain for high-yield teicoplanin and application thereof |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
| CN109321560A (en) * | 2018-10-31 | 2019-02-12 | 成都雅途生物技术有限公司 | A kind of selection of high yield rapamycin streptomycete |
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