CN1008445B - Strain a2 with proper component ratio gentamycin and its selective culture - Google Patents
Strain a2 with proper component ratio gentamycin and its selective cultureInfo
- Publication number
- CN1008445B CN1008445B CN 86108119 CN86108119A CN1008445B CN 1008445 B CN1008445 B CN 1008445B CN 86108119 CN86108119 CN 86108119 CN 86108119 A CN86108119 A CN 86108119A CN 1008445 B CN1008445 B CN 1008445B
- Authority
- CN
- China
- Prior art keywords
- gentamicin
- strain
- component ratio
- strains
- gentamycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Abstract
The present invention belongs to the selection of microbe strains, which aims to obtain productive strains with a proper component proportion of gentamicin by starting from modification on strain properties and with a simple method of screening anti-methonine analogue mutant strains. Mutagens are used for mutagenizing gentamicin productive bacteria to screen out three mutants of micromonospora purpurea A2 from a culture medium containing methionine analogues of (such as ethionine). The proportion of gentamicin C1, C1a and C2 is measured by means of ferment liquid paper chromatography and biological development, obtained results perfectly meet the needs of pharmacopoeias.
Description
The invention belongs to microbial fermentation and produce the method for gentamicin.
Homemade gentamicin and external gentamycin (being gentamicin) all are the aminoglycosides antibiotics by the many components of gang of micromonospora generation.Its main component of the goods of Shi Yonging is C clinically
1, C
1a, C
2Because each component chemical structure difference, the toxicity that is showed are also different, the relative content of therefore controlling component in the gentamicin product is related to the result of treatment of medicine.
United States Patent (USP) (U.S.Patent 3,651,042(1972)) is introduced the main component of his mycin: C
1Content is about 25%-50%, is generally 40%; C
1aContent is about 15%-40%, is generally 25%; C
2Content is about 20%-50%, is generally 35%.Isolated in China in 1967 goes out gentamicin and produces the bacterium purple-red single-spore bacteria, since the operation in 1969, and gentamicinC that each relevant pharmaceutical factory of China produces
2Compare C
1The content height, so the toxicity of medicine is bigger.Kaifeng pharmaceutical factory willow generation scholar (＜microbiotic〉8(1) 28-32(1983)) according to C
1, C
1a, C
2Chemical structure in C
1Contain methyl number this fact at most, in shake flask fermentation, add the DL-methionine(Met), improved gentamicinC
1Content.But owing to methionine(Met) costs an arm and a leg, can't be in industrial popularization.
The objective of the invention is to propose a kind of suitable fermentation process of gentamicin component ratio that makes from changing strain properties.
The present invention is a kind of method of utilizing deep red micromonospora to produce the suitable gentamicin of component ratio, adopts common fermention medium and culture condition, and wherein used bacterial strain is deep red red little monospore saltant Micromonospora purpura A
2, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 21st, 1986, and preserving number is CGMCC0118.
The methionine(Met) analog is produced bacterial strain to gentamicin and is had the effect that suppresses growth, regulate theoretical according to metabolism, can expect that the saltant of anti-methionine(Met) analog can make synthetic more methionine(Met) in the thalline, become the source that more methyl are provided, thereby under the condition that does not change the substratum composition, make the gentamicinC that contains Quito
1Content increases.So strains A that the present invention is used
2There is following method to obtain: to adopt nitroso-group
Be mutagenic compound (other mutagenic compound also can) that the mutagenesis gentamicin is produced bacterial strain, again the bacterial strain of the anti-methionine(Met) analog of screening on the substratum that contains first sulphur analog (as ethionine).Concrete steps are:
Produce bacterial strain #72 with the nitrosoguanidine mutagenesis gentamicin, grow up to spore through the #72 of mutagenesis bacterial strain on minimum medium, sampling is placed on the substratum that contains ethionine and grows then, after the resistance bacterium colony that grows is purified, connects the long spore in inclined-plane.Above-mentioned culture temperature and incubation time needn't be very strict, can as long as can form the culture condition of bacterium colony; The concentration of ethionine is more than 50 mcg/ml in the substratum, generally in 50 mcg/ml to 5000 mcg/ml.With aforesaid method screen the saltant A of the anti-ethionine of three strains
2(Micromono-spora purpurea A
2).Visual inspection: no aerial mycelium, the bacterium colony projection, it is luxuriant to grow, surperficial dark red brown, reverse side russet; Microscopic examination: mycelium is long, and branch does not have and separates, and the footpath is about 0.5 micron.
Analyse with the fermented liquid ply of paper, the way of biological developing is measured the component C with the gentamicin of the inventive method fermentative production
1, C
2, C
1aRatio, the gained result reasonably well meets the pharmacopeia requirement.Data see Table 1.
A
2Bacterial strain shows through producing last four tons of fermentation test results: A
2The total unit of strain fermentation is not less than and produces bacterial strain #72, uses A
2The gentamicin that bacterial strain is produced is better than the gentamicin quality of producing bacterial strain production with #72.
Gentamicin is the active drug that control streptococcus aureus, Pseudomonas aeruginosa, intestinal bacteria and Bacillus proteus etc. infect, and also is one of microbiotic of China's outlet.The bacterial strain that the present invention uses the short-cut method of anti-methionine(Met) analog (as ethionine etc.) to screen can be produced the suitable gentamicin of component ratio, has reduced the toxicity of medicine, is of value to people's health; And can improve homemade gentamicin competitive capacity in the international market, increase national foreign exchange income.
Be embodiments of the invention below:
The strain inclined plane Micromonospora purpurea#72 that uses in the fresh production digs piece and is inoculated in the triangular flask that contains nutrient solution, with 230 rev/mins, 33 ℃ shaking culture 48 hours, and centrifugal collection mycelia; With the phosphoric acid buffer washed twice of 0.03M, mycelia is suspended from the phosphoric acid solution again; Break up mycelia with the glass strain, filter, obtain mycelia fragment suspension with the dual-layer sterilization toilet paper.Take by weighing 1mg NTG, be dissolved in the 0.1ml methane amide, add mycelia fragment suspension 4.9ml, put 37 ℃ and handled 90 minutes, with 3000 rev/mins centrifugal 10 minutes, supernatant liquor inclines; The mycelia fragment that mutagenic treatment is crossed is suspended from the phosphoric acid buffer, and sampling is coated on the minimum medium, cultivates 7 days for 37 ℃; Wash spore with sterile purified water, break up with the glass strain, with the mycelia of the netted growth of dual-layer sterilization toilet paper elimination, the preparation monospore suspension.Sampling is coated on the minimum medium that contains ethionine 3000 mcg/ml, cultivates 15 days for 37 ℃, connects the inclined-plane after the resistance bacterium colony of appearance is purified, and behind the long good spore, it is standby to put 4 ℃ of refrigerators.
Micromonospora purpurea A with fresh anti-ethionine
2The inclined-plane digs piece and is inoculated into 250ml and contains shaking in the bottle of seed culture medium 30ml, 33 ℃, 230 rev/mins shaking culture 2 days; Be transferred on the fermention medium shaking culture 5 days by 10% inoculum size; Fermented liquid is with the portable measuring method for measuring of Shanghai the 4th pharmaceutical factory's gentamicin component (＜medicine industry 〉) (9) 8-14(1979) carry out the quantitative assay each component.
Claims (1)
- A kind of method of utilizing deep red micromonospora to produce the suitable gentamicin of component ratio adopts common substratum and culture condition, it is characterized in that used bacterial strain is deep red red little monospore mutant bacterium Nicoao-nospora purpurea A 2(CGMCC 0118).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86108119 CN1008445B (en) | 1986-12-03 | 1986-12-03 | Strain a2 with proper component ratio gentamycin and its selective culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86108119 CN1008445B (en) | 1986-12-03 | 1986-12-03 | Strain a2 with proper component ratio gentamycin and its selective culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN86108119A CN86108119A (en) | 1988-06-15 |
| CN1008445B true CN1008445B (en) | 1990-06-20 |
Family
ID=4803807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 86108119 Expired CN1008445B (en) | 1986-12-03 | 1986-12-03 | Strain a2 with proper component ratio gentamycin and its selective culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1008445B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1040177C (en) * | 1993-04-23 | 1998-10-14 | 江苏省微生物研究所 | 1-N-ethyl gentamicin derivative and its preparing method |
| CN1063483C (en) * | 1995-08-23 | 2001-03-21 | 中国科学院上海植物生理研究所 | High-butanol ratio clostridium acetobutylicium and culturing method and use thereof |
| CN102586146B (en) * | 2011-12-19 | 2014-04-23 | 沈阳药科大学 | Engineering bacteria for generating gentamicin C1a and constructing method of engineering bacteria |
| CN103160552B (en) * | 2013-02-24 | 2015-01-21 | 烟台只楚药业有限公司 | Preparation method of gentamicin sulphate |
| CN103740627A (en) * | 2013-11-30 | 2014-04-23 | 福州市鼓楼区荣德生物科技有限公司 | Gentamycin JI-20B gene engineering bacterium, and construction and application thereof |
| CN113403237B (en) * | 2021-07-27 | 2021-12-28 | 青岛安惠仕生物制药有限公司 | Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof |
-
1986
- 1986-12-03 CN CN 86108119 patent/CN1008445B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| CN86108119A (en) | 1988-06-15 |
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| CF01 | Termination of patent right due to non-payment of annual fee |