Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof

A technology of endophytic bacteria and camptothecin, applied in the field of medical biology, can solve the problems of difficult industrial production of production costs, large environmental pollution, cumbersome steps, etc., and achieve simple and easy-to-control separation methods, simple and easy-to-control methods, and rapid growth and reproduction Effect

Inactive Publication Date: 2013-06-12
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the use of a large amount of organic solvents in the extraction process leads to large environmental pollution and cumbersome steps
The chemical total synthesis of camptothecin is synthesized by different ring-building methods, such as split method, asymmetric oxidation method, etc. Most methods have shortcomings such as too long routes and low total yields, making it difficult to realize industrialization
The use of plant cell culture to produce high value-added plant secondary metabolites (such as camptothecin and its derivatives) was once considered a promising alternative, but due to the slow growth of plant cells and the heterogeneity of the culture process Factors such as severe globalization, unstable output, and production costs make it difficult to realize industrialized production
In short, the sources of camptothecin are limited and cannot meet the market demand at all (the annual demand for camptothecin in the international market is 3000kg, but the global annual output of camptothecin is only 600kg), and it is necessary to discover and develop alternative resources and technologies

Method used

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  • Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof
  • Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof
  • Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Isolation and screening of camptothecin-producing endophytes from camptothecin

[0032] The endophytic bacterial strain Paenibacillus polymyxa LY214 was isolated from camptophyllum.

[0033] 1. Test material

[0034] 1.1 Plant material:

[0035] Select the organs or tissues of philodendron that grow normally and healthy as plant materials, such as tree roots, bark, fruits, leaves, branches and other parts.

[0036] The collected plant materials were cut into sections, cleaned and disinfected according to routine operations. In this embodiment, tap water is used to thoroughly rinse to remove dust and other sundries, and the plant material is cut into small pieces of 10mm×10mm with a sterile scalpel, and soaked in 75% ethanol for 5 minutes, 10% sodium hypochlorite solution for 5 minutes, and 1% Triton. 5min, rinse with sterile water 5 times, 1min each time, and finally use sterile filter paper to absorb the surface moisture of each tissue part of Campylocarp...

Embodiment 2

[0045] Camptothecin was obtained from the camptothecin endophytic bacteria strain Paenibacillus polymyxa LY214.

[0046] 1. Culture medium and extraction solvent

[0047] Culture medium: Sabouraud liquid medium (SBA liquid medium), the components are the same as the SBA solid medium in Example 1, but no agar is added.

[0048] Extraction solvent: chloroform-methanol mixed solvent (V / V=4:1)

[0049] 2. Extraction method of camptothecin

[0050] 2.1 Fermentation culture

[0051] The strain Paenibacillus polymyxa LY214 preserved in Example 1 was prepared according to conventional methods to obtain seed liquid. Inoculate 50ml of seed liquid into 500ml of SBA liquid medium, culture at 28±2°C, shaker 160±20rpm, for 8-15 days, and obtain a fermentation mixture.

[0052] 2.2 Preparation of fermented extract

[0053] The fermentation mixture was centrifuged at 4000rpm for 15min, filtered, and the filtrate and mycelia were collected respectively.

[0054] The obtained filtrate was...

Embodiment 3

[0057] Camptothecin was obtained by using the camptothecin endophytic bacterial strain Paenibacillus polymyxa LY214, which is the same as Example 2 and will not be repeated. The difference is that a seed liquid preparation step is added before the fermentation culture, including strain activation and seed culture.

[0058] Activation of strains: the strain Paenibacillus polymyxa LY214 preserved in Example 1 was inoculated on SBA solid medium for activation, and cultured for 5-7 days to obtain activated strains;

[0059] Seed culture: Inoculate the activated strains into 50ml SBA liquid medium, culture at 28±2°C, shaker 160±20rpm, for 2-3 days to obtain seed liquid.

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Abstract

The invention relates to a camptotheca endophytic bacterium which is obtained from camptotheca acuminate through separation and purification, and an application of the camptotheca endophytic bacterium. The camptotheca endophytic bacterium LY214 involved by the invention is identified to be paenibacillus polymyxa by morphology and molecular biology; and the bacterial strain is preserved in the China General Microbiological Culture Collection Center, the preservation date is November 19th, 2012, and the preservation number is CGMCC 6841. The camptotheca endophytic bacterium is characterized in that the high performance liquid chromatography, the ultraviolet spectrum and the mass spectrometry show that the camptotheca endophytic bacterium can generate an antineoplastic active component which is camptothecin through liquid fermentation. The invention discloses for the first time that the endophytic bacterium for producing camptothecin can be separated and cultured from the camptotheca acuminate, and provides a new way for the preparation of camptothecin.

Description

technical field [0001] The invention relates to the isolation, cultivation and application of a camphor tree endophytic bacterium, belonging to the field of medical biotechnology. Background technique [0002] Camptothecin (CPT) is a terpenoid indole alkaloid formed by the condensation of tryptamine and split loganin. It was first isolated from the bark of the unique plant Camptotheca acuminata in my country, and was later found in many plants. Such as Ervatamia heyneana of Apocynaceae, Nothapodytes foetida of Tea Cornaceae, and Ophiorrhiza pumila of Rubiaceae. Camptothecin has a unique anti-tumor mechanism, and exerts anti-tumor activity by inhibiting topoisomerase I. Camptothecin series derivatives are effective against pancreatic cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, colon cancer, cervical cancer, gastric cancer and blood tumors, and are the main varieties in the global anti-tumor drug market. [0003] The main sources of camptothecin are 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12P17/18C12R1/01
Inventor 罗应刚蒲祥陈菲屈细兴张国林
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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