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Microbial fermentation preparation method of bleomycin derivatives

A bleomycin family and microbial fermentation technology, applied in the field of biomedicine, can solve the problems of low fermentation yield and low efficiency

Active Publication Date: 2014-08-27
CHANGSHA CHARISM BIOSCIENCES CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fermentation yield of 6'-dehydroxyl-BLM S in Streptomyces chrysanthemum recombinant engineered strain SB9026 is extremely low, and there is also the accompanying by-product BLM S, the yield of both is equivalent, and the total yield is only about 10mg / L. Among them, the 6'-dehydroxyl-BLM S is about 5 mg / L, and the whole fermentation preparation cycle is more than 12 days, which is inefficient

Method used

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  • Microbial fermentation preparation method of bleomycin derivatives
  • Microbial fermentation preparation method of bleomycin derivatives
  • Microbial fermentation preparation method of bleomycin derivatives

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Experimental program
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Effect test

Embodiment 1

[0054] Both Streptomyces chrysogenum SB9001 and the genetically engineered mutant strain obtained in the experiment were cultured on ISP4 agar solid medium at 30°C to obtain spores. Streptomyces verticillium ATCC15003 was grown in TSBY liquid medium containing 0.5% glycine at 28°C and 250 rpm to obtain genomic DNA. Escherichia coli was grown in LB liquid medium at 37°C and 250rpm for daily plasmid cloning and BAC library establishment.

[0055] 1) Construction of Streptomyces chrysanthemum SB9025 mutant strain

[0056] A 10kb DNA fragment containing the complete zbmVIII gene was isolated from the cosmid containing a part of the zobermycin biosynthetic gene cluster zbm, and cloned into the Litmus 28 plasmid through BamHI and BglII restriction sites superior. The resulting plasmid was digested with Sbf1 to remove the insert fragment with a size of about 3.7 kb, and then the plasmid was self-ligated to complete the knockout of the zbmVIII gene. The modified DNA insert was dige...

Embodiment 2

[0063] Separation, purification and detection of novel bleomycin analogs BLM S (Tianci 102, compound 1) and 6’-dehydroxy-BLM S (Tianci 103, compound 2)

[0064] The supernatant solution obtained after the fermentation broth was centrifuged was detected and analyzed by high-performance liquid phase (HPLC). The mobile phase A was 99.9% deionized water and 0.1% acetic acid, and the mobile phase B was 99.9% methanol and 0.1% acetic acid. The flow rate was 0.8mL / min, the UV detector wavelength is 300nm. The linear gradient analysis program is: 0-30 minutes, 100%A / 0%B to 0%A / 100%B. The rest of the supernatant was adjusted to pH 7.0 with 1N hydrochloric acid and loaded into Amberlite IRC50 (NH 4 + type) resin column, washed with 10 times the column volume of deionized water, and then eluted with 2 liters of 20% ammonium acetate solution. The resulting eluate was mixed with Diaion HP-20 resin and gently shaken at room temperature for 45 minutes for adsorption, then the HP-20 resin...

Embodiment 3

[0077] BLM S (Tianci 102, compound 1) and 6’-dehydroxy-BLM S (Tianci 103, compound 2) DNA cleavage activity test

[0078] in Fe 2+ In the presence of conditions, respectively for BLM S, 6'-dehydroxy-BLM S and bleomycin A 2 (BLMA 2 ) to test and compare their biological activity. Based on the analysis of plasmid relaxation activity of supercoiled plasmid DNA pBluescript II SK(+), single-strand cleavage mediated by bleomycin family compounds first converts supercoiled plasmid DNA (form A) into open circular plasmid DNA (form B ), followed by double-strand cleavage to convert it into linear plasmid DNA (form C). The total reaction volume of DNA cleavage activity test experiment is 10μl, which contains 25mM Tris-HCl buffer (pH 7.5), about 0.65μg of pBluescript SK II (+) plasmid DNA, 5μM Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O (in 1mM H 2 SO 4 freshly prepared in solution) and a certain concentration of active compounds to be tested (BLM S, 6'-dehydroxy-BLM S and BLM A 2 ). After...

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Abstract

The invention relates to the technical field of biomedicines, and concretely relates to a microbial fermentation preparation method of bleomycin derivatives. The method is characterized in that SB9026 (with the preservation number of CCTCC M2011292) fermentation strains are cultured, an appropriate seed culturing medium, a fermentation culturing medium and culturing conditions are selected, pilot scale amplification of the above microbial fermentation preparation method is carried out in a fermentation tank, and the temperature, the rotating speed, the pH value and raw material supplementation are regulated to adjust in order to establish the large-scale microbial fermentation preparation method of novel active bleomycin derivatives 6'-dehydroxy-BLMs. The preparation method shortens the fermentation preparation period of the 6'-dehydroxy-BLMs, improves the fermentation yield of the 6'-dehydroxy-BLMs, increases the proportion of the 6'-dehydroxy-BLMs in total metabolites, and is convenient for subsequent separation and purification. The chemical structure of the bleomycin derivatives is represented by a general formula shown in the specification.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a microbial fermentation preparation method of novel bleomycin derivatives. Background technique [0002] Bleomycin (BLM) is a class of glycopeptide antitumor antibiotics isolated from the fermentation product of Streptomyces verticillus in the 1960s. At present, more than ten kinds of natural components of bleomycin have been discovered, and their structures differ only in the terminal amino groups, the main components of which are bleomycin A2 (55%-70%) and bleomycin B2 ( 25% to 32%). In addition, natural products including Tallysomycin, Zorbamycin and Phleomycin are very similar in structure and activity to bleomycin, so they are collectively called bleomycin Glycopeptide antitumor antibiotics. [0003] Bleomycin has a unique molecular structure and biological activity, can mediate the activation of oxygen molecules, and selectively cause single-stranded and...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12R1/465
Inventor 段燕文沈奔朱湘成杨虎
Owner CHANGSHA CHARISM BIOSCIENCES CO LTD
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