CN103740614B - One plant height produces the actinoplanes mutagenic strain of rapamycin - Google Patents
One plant height produces the actinoplanes mutagenic strain of rapamycin Download PDFInfo
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Abstract
The invention discloses a plant height and produce actinoplanes mutagenic strain (Actinoplanes) the CGMCC NO.8430 of rapamycin, belong to fermentation engineering field.This bacterium is the actinoplanes hn 016(Actinoplanes producing rapamycin to filter out from the pedotheque in Hainan Island) as starting strain, sell of one's property raw through atmospheric pressure at room plasma inducing.The mutagenic strain BCStr 096 of the present invention, higher than the bacterial strain hereditary stability that routine mutagenesis method obtains, the production capacity of rapamycin is higher, its yield yield 200mg/L than starting strain hn 016, improve 175%, and improve about 37% on the fermentation level that existing rapamycin is the highest, greatly reduce production cost.
Description
Technical field
The invention belongs to fermentation engineering field, be specifically related to a plant height and produce the actinoplanes mutagenic bacteria of rapamycin
Strain BCStr-096.
Background technology
Rapamycin (rapamycin, RPM) has another name called sirolimus, in being the nitrogenous triolefin macro ring of tool unique mechanism of action
Esters antibiotic, has antifungal, antiproliferative effect, it was reported that the zoopery of Luan FL et al. finds thunder
Handkerchief mycin also has antineoplastic action.Rapamycin is the most promising novel potent immunosuppressant at present
Agent, its molecular structure is similar to FK506, is another parent's immune protein bonding agent, can be used for the anti-repulsion of organ transplantation
Effect, its immunosuppressive action decades of times stronger than cyclosporin, it is the immunosuppressant that nephrotoxicity is minimum, can also be used with
In the treatment autoimmune disease such as rheumatoid arthritis, lupus erythematosus, develop into gene therapy and antitumor at present
Medicine, has broad application prospects.
Initially, RPM, as hypotoxicity antifungal antibiotic, finds that autoimmune disease is had immunity to press down by it for 1978
Make and use.Being inspired by this, Mortis in 1989 formally starts using RPM as the Novel immune of the anti-repulsion of organ transplantation
Inhibitor is tried out.In JIUYUE, 1999 U.S. FDA official approval rapamycin is as the anti-rejection drugs of renal transplantation
Put on market, the intravascular stent Cypher using rapamycin as coating medicine simultaneously researched and developed by Cordis company
In 2002 in succession in European, the U.S. and Japan's listing.In June, 2005, Fujian Microorganism Inst. of China ground
The domestic rapamycin sent out is put on market through SFDA approval, and by the rapamycin conduct of Wyeth company of U.S. exploitation
Anticarcinogen has entered clinical experimental stage.Before 2006, rapamycin is 6,000 U.S. dollars 1 in the price of American market
Gram, at present, the sales volume of rapamycin increases rapidly, and its market price the most slightly reduces, and estimates that it will be with ring spore
Rhzomorph is competed effectively, little by little divides the market of anti-transplant organ rejection medicine equally, and Pharmaprojects(is grinding new drug
Dynamically) predict that its sales volume reaches 5-20 hundred million dollars.Rapamycin belongs to six kind new medicines at home, and within 2009, thunder handkerchief is mould
The sales volume in element market at home, more than 500,000,000 yuans, has wide market prospect.
At present, the industrialized production of rapamycin is mainly produced by microbial fermentation processes, but this antibiotic at present
Fermentation level the most at a fairly low, the highest fermentation level having document to report also only reaches about 400mg/L.Therefore, thunder
The biofermentation unit that the deficiency that the production of handkerchief mycin biofermentation exists essentially consists in rapamycin is the highest, is unfavorable for extracting
Purification, it is impossible to fully meet the requirement of industrialized production.
The present invention utilizes atmospheric pressure at room plasma mutation technology, filters out a strain rapamycin superior strain, improves
The yield 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 mutation technology, filters out a strain novel thunder handkerchief
Mycin superior 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: the Chaoyang District, Beijing City North Star
West Road 1 institute 3, Institute of Microorganism, Academia Sinica, postcode 100101；Preservation date 2013 11
The moon 05.This bacterial strain is the actinoplanes producing rapamycin to filter out from the pedotheque in Hainan Island
Hn-016(Actinoplanes sp.) it is starting strain, obtain through many group Mutation screenings.
Actinoplanes of the present invention, carries out selection-breeding the most by the following technical solutions:
1. use atmospheric pressure at room plasma mutation technology selection-breeding rapamycin superior strain
(1) the actinoplanes hn-016 inclined-plane bacterial strain producing rapamycin that growth selection is good, washes with normal saline
Lower spore, through filtered through gauze, makes 106The spore suspension of individual/mL；
(2) by the spore suspension in (1), 10 μ L are drawn on the round iron plate of a diameter of 1cm with liquid-transfering gun,
It is placed in helium as working gas, the atmospheric pressure at room plasma mutation of power 110W, working gas flow 10L/min
In system, process distance is 2mm, process respectively 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s,
160s, 180s, carry out the spore suspension of process gradient dilution and be coated with flat board, make fatality rate curve；
(3) according to the fatality rate curve of (2), the irradiation time of high, medium and low three different fatal dose is selected,
The spore suspension of bacterial strain hn-016 is carried out plasma mutation；
(4) spore suspension of three different disposal times in (3) is mixed in equipped with in the test tube of normal saline, mixed
Close uniformly；
(5), by the mutation spore suspension in (4), after carrying out gradient dilution, it is respectively coated in mould containing 2mg/L chlorine
Element, 0.8mg/L streptomycin, 4mg/L erythromycin, in the resistant panel of 1mg/L gentamycin；
(6), behind the single bacterium colony switching inclined-plane that will grow in each resistant panel in (5), with the inoculum concentration of 1%, connect
Plant in seed culture medium, 28 DEG C, 220r/min, after cultivating 45-50h, with the inoculum concentration of 10%, seed liquor is connect
Planting in fermentation medium, 28 DEG C, 220r/min cultivates 7-8d；
(7) taking the fermentation liquid in (6) in centrifuge tube, 5000r/min is centrifuged 10min, takes thalline methanol and extracts
Taking, pour into equipped with in the triangular flask of 10-15 bead, on the shaking table of 180-200r/min, concussion extracts 1h,
Taking extract, 10000r/min is centrifuged 10min, after crossing organic filter membrane, utilizes high effective liquid chromatography for measuring thunder handkerchief mould
The yield of element, filters out rapamycin superior strain BCStr-096 by the method.
2. the cultural method of actinoplanes BCStr-096 is:
(1) actication of culture: actinoplanes BCStr-096 is forwarded to preservation separation slant medium, 28 DEG C of perseverances
Temperature is cultivated 7-15 days；
(2) seed culture: choose well-grown inclined-plane, washes lower spore with the normal saline of sterilizing, prepares concentration
About 106The spore suspension of individual/mL, is inoculated in the 500mL triangle equipped with 60mL seed culture medium with the inoculum concentration of 1%
In Ping, 28 DEG C, 220r/min, 45-50h is cultivated in concussion；
(3) fermentation culture: by the seed culture fluid in (2), is inoculated in the inoculum concentration of 10% and sends out equipped with 60mL
In the 500mL triangular flask of ferment culture medium, 28 DEG C, 220r/min, 7-8d is cultivated in concussion；
3. actinoplanes BCStr-096 mutation and incubation used medium consist of:
(1) preservation isolation medium: oatmeal 3%, glucose 2%, yeast powder 1%, peptone 1%, calcium carbonate
0.25%, insufficient section distilled water is supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min；
(2) seed culture medium: soluble starch 2.8%, peptone 0.8%, glucose 2%, soybean cake powder 1%,
Calcium carbonate 0.3%, insufficient section distilled water supplies, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min；
(3) fermentation medium: corn starch 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, phosphorus
Acid hydrogen dipotassium 0.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.2%, sodium chloride 0.5%, glycerol 1%, calcium carbonate 0.3%,
Steeping enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
Beneficial effect:
The mutagenic strain BCStr-096 of the present invention is obtained by plasma mutation method, ratio routine mutagenesis method
The bacterial strain hereditary stability obtained is higher, and the production capacity of rapamycin is higher, and its yield is than starting strain hn-016
Yield 200mg/L, improve 175%, and improve about 37% on the fermentation level that existing rapamycin is the highest,
Greatly reduce production cost.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, and following example are illustrative, is not limit
Property processed, it is impossible to limit protection scope of the present invention with this.
Embodiment 1
The bacterial strain hn-016 producing rapamycin filtered out from soil
1. thalli morphology: thalline is gram positive bacteria, without aerial hyphae, breeds in the way of spore, spore
Perfectly round, major part tool extremely gives birth to feathering, can move about.
2. colonial morphology: on flat board, the base silk surface of bacterium colony is in granular form, orange-yellow, along with prolonging of incubation time
Long, bacterium colony can be become yellowish-brown by orange-yellow.
3. cultural characteristic: this bacterium is high oxygen consumption bacterium, optimum growth temperature is 28 DEG C, and the most suitable growth pH is 7.0-7.2；
Being 210-240r/min in shaking speed, when cultivating 7-8d, rapamycin production is the highest, dissolved oxygen pair in sweat
The generation of rapamycin has remarkable effect.
Embodiment 2
With actinoplanes hn-016 as starting strain, use atmospheric pressure at room plasma mutation technology screening bacterial strain
BCStr-096
1. the acquisition of mutagenic strain
(1) the actinoplanes hn-016 inclined-plane bacterial strain producing rapamycin that growth selection is good, washes with normal saline
Lower spore, through filtered through gauze, makes 106The spore suspension of individual/mL；
(2) by the spore suspension in (1), 10 μ L are drawn on the round iron plate of a diameter of 1cm with liquid-transfering gun,
Being placed in using helium as working gas, power is 110W, atmospheric pressure at room of working gas flow 10L/min etc. from
In daughter mutagenesis system, process distance is 2mm, process respectively 0s, 20s, 40s, 60s, 80s, 100s, 120s,
140s, 160s, 180s, carry out the spore suspension of process gradient dilution and be coated with flat board, make fatality rate curve；
(3) according to the fatality rate curve of (2), the irradiation of tri-different fatal dose of 40s, 80s, 100s is selected
Time, the spore suspension of bacterial strain hn-016 is carried out plasma mutation；
(4) spore suspension of three different disposal times in (3) is mixed in equipped with in the test tube of normal saline, mixed
Close uniformly；
(5) by the mutation spore suspension in (4), gradient dilution 10 is carried out-1-10-6, take 10-4、10-5、10-6Three
Individual dilution spore suspension, is respectively coated in containing 2mg/L chloromycetin, 0.8mg/L streptomycin, and 4mg/L is red mould
Element, in the resistant panel of 1mg/L gentamycin；
(6), behind the single bacterium colony switching inclined-plane that will grow in each resistant panel in (5), with the inoculum concentration of 1%, connect
Plant in seed culture medium, 28 DEG C, 220r/min, after cultivating 45-50h, with the inoculum concentration of 10%, seed liquor is connect
Planting in fermentation medium, 28 DEG C, 220r/min cultivates 7-8d；
Described seed culture medium consists of: soluble starch 2.8%, peptone 0.8%, glucose 2%, soybean cake powder
1%, calcium carbonate 0.3%, insufficient section distilled water supplies, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min；
Described fermentation medium consists of: corn starch 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%,
Dipotassium hydrogen phosphate 0.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.2%, sodium chloride 0.5%, glycerol 1%, calcium carbonate
0.3%, steep enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
(7) taking the fermentation liquid in (6) in centrifuge tube, 5000r/min is centrifuged 10min, takes thalline methanol and extracts
Taking, pour in the triangular flask equipped with 13 beades, on the shaking table of 190r/min, concussion extraction 1h, takes extracting
Liquid, 10000r/min is centrifuged 10min, after crossing organic filter membrane, utilizes the product of high effective liquid chromatography for measuring rapamycin
Amount, i.e. filters out the mutagenic strain BCStr-096 of high yield rapamycin by the method.
2. the characteristic of mutagenic strain BCStr-096
(1) thalli morphology: thalline is gram positive bacteria, without aerial hyphae, breeds in the way of spore,
Spore is perfectly round, and major part tool extremely gives birth to feathering, can move about.
(2) colonial morphology: on flat board, the base silk surface of bacterium colony is in granular form, orange-yellow, along with incubation time
Prolongation, bacterium colony can be become yellowish-brown by orange-yellow.
(3) cultural characteristic: this bacterium is high oxygen consumption bacterium, optimum growth temperature is 28 DEG C, and the most suitable growth pH is 7.1；
Being 230r/min in shaking speed, when cultivating 7-8d, rapamycin production is the highest.
(4) can grow in the culture medium containing 0.8mg/L streptomycin, and well-grown.
Embodiment 3
The cultivation of actinoplanes BCStr-096
1. the preparation of culture medium
(1) preservation isolation medium: oatmeal 3%, glucose 2%, yeast powder 1%, peptone 1%, calcium carbonate
0.25%, insufficient section distilled water is supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min；
(2) seed culture medium: soluble starch 2.8%, peptone 0.8%, glucose 2%, soybean cake powder 1%,
Calcium carbonate 0.3%, insufficient section distilled water supplies, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min；
(3) fermentation medium: corn starch 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, phosphorus
Acid hydrogen dipotassium 0.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.2%, sodium chloride 0.5%, glycerol 1%, calcium carbonate 0.3%,
Steeping enemy 0.2%, insufficient section distilled water is supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
2. actication of culture
The strain BCStr-096 that glycerol preserves is forwarded to preservation isolation medium inclined-plane, in constant incubator, 28
DEG C cultivate 7-15d；
3. seed culture
The activated inclined plane that growth selection is good, is inoculated in equipped with (500mL tri-in the seed culture medium prepared in above-mentioned 1
Angle bottle built-in 60mL culture medium), in 28 DEG C, 220r/min, cultivates 45-50h.
4. fermentation culture
By the seed liquor for preparing in 3 with 10%(v/v) inoculum concentration is inoculated in fermentation medium (in 500mL triangular flask
Dress 60mL culture medium), in 28 DEG C, fermentation culture 8d under the conditions of 220r/min.
Testing result: actinoplanes (Actinoplanes sp.) BCStr-096 thunder handkerchief on fermentation medium is mould
The yield of element is 535 565mg/L.
Claims (2)
1. the actinoplanes mutagenic strain of rapamycin is produced in a strain,
Described actinoplanes mutagenic strain (Actinoplanes) BCStr-096, deposit number is CGMCC NO.8430.
The actinoplanes mutagenic strain BCStr-096 of product rapamycin the most according to claim 1 application in fermenting and producing rapamycin.
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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 |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101886085A (en) * | 2009-05-15 | 2010-11-17 | 上海医药工业研究院 | Rapamycin biosynthesis gene from actinoplanes, and separation method and application thereof |
| CN102433364A (en) * | 2011-11-10 | 2012-05-02 | 中科医药行业生产力促进中心有限公司 | Process for preparing rapamycin by using microbial fermentation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101886085A (en) * | 2009-05-15 | 2010-11-17 | 上海医药工业研究院 | Rapamycin biosynthesis gene from actinoplanes, and separation method and application thereof |
| CN102433364A (en) * | 2011-11-10 | 2012-05-02 | 中科医药行业生产力促进中心有限公司 | Process for preparing rapamycin by using microbial fermentation method |
Non-Patent Citations (2)
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| 培养条件优化和菌种选育提高雷帕霉素发酵水平;陈希杨等;《化学工程》;20110131;第39卷(第1期);81-84 * |
| 高能电子诱变选育雷帕霉素产生菌;贾啸静等;《河北省科学院学报》;20051231;第22卷(第4期);27-29 * |
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