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Gentamicin sulfate prepared through enhanced microbial fermentation, and application method of gentamicin sulfate

A technology of gentamicin and antibacterial drugs, which is applied in the field of directional high-yield gentamicin C2 engineering bacteria and antibiotic manufacturing, which can solve the problems of pollution, interference, low yield, etc., meet the requirements of industrialization and shorten the production cycle , the effect of reducing production costs

Active Publication Date: 2021-09-17
青岛安惠仕生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above two epimers have similar chemical structures and similar physical and chemical properties. It is very difficult to separate single-component gentamicin C2 from them, resulting in a shortage of gentamicin C2, high production costs and long chromatographic separation cycles. , low yield, high material consumption, serious pollution, complex extraction and refining process, and unstable product quality, and the by-product gentamicin C2a, which is similar in structure to gentamicin C2, interferes at any time, causing great problems for product quality control. Large uncertainties seriously affect the stability, safety and effectiveness of drugs, and are urgent scientific problems to be solved

Method used

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  • Gentamicin sulfate prepared through enhanced microbial fermentation, and application method of gentamicin sulfate
  • Gentamicin sulfate prepared through enhanced microbial fermentation, and application method of gentamicin sulfate
  • Gentamicin sulfate prepared through enhanced microbial fermentation, and application method of gentamicin sulfate

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Effect test

Embodiment 1

[0016] Example 1 Atmospheric room temperature plasma mutagenesis technology screening

[0017] 1) With the gentamicin production strain Micromonospora rubrum SD2018 in the applicant's strain bank as the starting strain, use a sterilized 1ml straw to draw 0.1ml and inoculate it on the sterilized slant medium for slanting Spore culture, culture temperature is 35~37 ℃, stand still, culture time is 4 days, obtain the slant spore of SD2020 strain;

[0018] 2) Take 1.0cm 2 Step 1) Add the slanted spores prepared into a sterilized Erlenmeyer flask containing 5ml of saline with a mass concentration of 0.9%, and shake for 20 minutes to obtain a spore suspension;

[0019] 3) Take 500 μl of the spore suspension prepared in step 2), add 250 μl of 20% lithium chloride aqueous solution and 250 μl of 0.9% normal saline, shake and mix for 5 minutes to obtain spores containing 5% lithium chloride Suspension; after shaking and mixing, draw 20 μl of spore suspension containing 5% lithium chlor...

Embodiment 2

[0021] The efficient fermentation of embodiment 2 gentamicin C2

[0022]The Plackett-Burman design method is a nearly saturated two-level experimental design method developed in the middle and late 20th century. Based on the principle of incomplete balance blocks, it can estimate the main effect of factors with the least number of trials, and quickly and effectively screen out the most important factors from many factors for further research. This method is simple in data processing. Compared with the currently commonly used partial factor test and random balance test, this method is the most efficient and accurate in screening important factors. Therefore, it has been widely used in the optimization of microbial culture conditions and achieved good results. In the embodiment, it is planned to investigate and evaluate many factors in the fermentation medium formula of gentamycin C2 through the Plackett-Burman experimental design of DesignExpert7.0, and screen out several impor...

Embodiment 3

[0036] The refining of embodiment 3 gentamicin C2

[0037] Utilize the above-mentioned fermentation method to carry out expanded cultivation, add 10% tap water to dilute the fermented liquid after expanded fermentation, add hydrochloric acid or sulfuric acid, acidify to pH1.0-3.0, fully stir 1-4 hour; pH5.0-7.0, put in 732NH 4+ Resin static adsorption for 6-8 hours. The amount of resin input is calculated at about 50,000 u / mL. Collect the adsorbed saturated resin and rinse it with tap water (until there is no floating mycelium). Pack the saturated resin into a column, pickle the saturated resin with 0.5M HCl solution twice the volume of the saturated resin, then wash it with ion-free water until neutral, and then use 0.01% ammonia water for alkaline washing. When the effluent reaches pH 9.0 or above, Connect in series to an equal volume of 711 resin column, use 4% ammonia water for elution, the amount of ammonia water is 8-10 times the volume of saturated resin, the elution...

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Abstract

The invention claims to protect gentamicin sulfate prepared through enhanced microbial fermentation, and an application method of the gentamicin sulfate. The enhanced microbial engineering bacterium is a Micromonospora purpurea C2 1591 strain which is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, and the Micromonospora purpurea C2 1591 strain is registered and collected in the China General Microbiological Culture Collection Center (CGMCC for short, the address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing) on June 13, 2021, and the collection number is CGMCC No.59450. The screened engineering bacterium for directionally producing gentamicin C2 in high yield can directionally produce the gentamicin C2 in the high yield, the component ratio of gentamicin C2 is increased to 85% or above from the original about 15%, and the highest fermentation titer of an optimized formula is 1184 U / mL through Plackett-Burman design and is increased by 42% than the titer of an original formula. Compared with an original strain, the yield of the gentamicin C2 is increased by three times, in addition, other isomers or impurities are hardly contained, the gentamicin C2 can be directly applied downstream, complex biotransformation and purification is not required, a large amount of cost is saved, and an industrialization requirement is met.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to an enhanced microbial fermentation to prepare gentamycin sulfate and its application method. Specifically, the invention relates to an engineering bacterium with a directional high-yield gentamicin C2 and its application. Antibiotic manufacturing. Background technique [0002] Gentamicin was first discovered by Weinstein in 1963. It is a multi-component aminoglycoside antibiotic produced by Micromonospora, including C1, C2, C1a, C2a and C2b components. The main components of gentamicin, C1, C2 and C1a, are widely used clinically, among which C1a has the highest antibacterial activity, which is the precursor of etimicin synthesis, followed by C2b, also known as sagamycin . These antibiotics can bind to 16SrRNA on the 30S subunit of bacterial ribosomes, causing misreading of the genetic code, thereby blocking the synthesis of bacterial proteins, so they are mainly used to treat b...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12P19/48C12R1/31
CPCC12N1/20C12N1/02C12P19/485
Inventor 王云松史京波张梵杨翠霞袁帅妮
Owner 青岛安惠仕生物制药有限公司
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