CN1940059A

CN1940059A - Fermentation of FW523 series compound from marine single-cell FIMO2-523

Info

Publication number: CN1940059A
Application number: CN 200510105771
Authority: CN
Inventors: 江红; 林如; 郑卫; 程元荣
Original assignee: Fujian Institute of Microbiology
Current assignee: Fujian Institute of Microbiology
Priority date: 2005-09-29
Filing date: 2005-09-29
Publication date: 2007-04-04

Abstract

Fermentation of FW523 series compound micromonosporin FIM 02-523 is carried out by slop culturing, seed culturing, fermentation culturing to obtain the fermentation liquid, and extraction purifying for metabolite and fermentation liquid to obtain the final product. It has excellent inhibition function for tumor cell.

Description

Sea micromonoad FIM 02-523 fermentative production FW523 series compound

Technical field

The invention belongs to biological technical field, particularly, the present invention relates to microbial fermentation and produce antitumor antibiotics FW523.

Background technology

The antitumor antibiotics FW523 that sea micromonoad FIM 02-523 produces has 5 components, FW523-1 (C ₃₂H ₅₄N ₄O ₇) with the antitumor antibiotics Rakicidin A homogeneity that the micromonospora of external report produces, FW523-2 is the isomers of Rakicidin A, FW523-3 (C ₃₃H ₅₆N ₄O ₇) and the RakicidinB homogeneity, FW523-4 (C ₃₄H ₅₈N ₄O ₇) and FW523-5 (C ₃₁H ₅₂N ₄O ₇) be the new homologue of Rakicidin class, abroad do not appear in the newspapers.

Summary of the invention

The objective of the invention is to use sea micromonoad FIM 02-523 fermentative production antitumor antibiotics FW523 series compound.For realizing above-mentioned target, the technical solution used in the present invention is as follows;

1. culture of strains fermentation; Sea micromonoad FIM 02-523 bacterial classification obtains fermented liquid through slant culture, seed culture, fermentation culture.

2. meta-bolites extracts purifying: fermented liquid obtains the FW523 series compound through extracting purifying.

Embodiment

Embodiment 1: the fermentation culture of sea micromonoad FIM 02-523

The Micromonospora sp.FIM02-523 Gao Shi asparagine slant culture of one platinum loop inserts liquid seed culture medium (Zulkovsky starch 1.5%, glucose 0.5%, peptone 0.5%, yeast powder 0.5%, (NH ₄) ₂SO ₄0.05%, K ₂HPO ₄3H ₂O 0.05%, and NaCl 0.05%, MgSO ₄7H ₂O 0.05%, CaCO ₃0.1%, tap water preparation, pH 7.5), 32 ℃ of shaking culture 48 hours change fermention medium (Zulkovsky starch 2%, sucrose 0.5%, casein food grade 1.0%, corn steep liquor 0.5%, MgSO over to 5% culture transferring amount ₄7H ₂O 0.04%, FeSO ₄7H ₂O 0.005%, CuSO ₄5H ₂O 0.005%, CoCl ₂6H ₂O 0.0005%, CaCO ₃0.3%, the tap water preparation, pH 7.5), 32 ℃, bottle is put on 96 ~ 120 hours biologically active tat peaks of 240rpm shaking culture.

The extraction purifying of embodiment 2:FW523 series compound

Above-mentioned fermented liquid is through the acidifying flocculation treatment, and the throw out that centrifugal back obtains amasss alcohol immersion twice, alcohol immersion liquid concentrating under reduced pressure with diploid.The ethanol concentrated solution is evaporated to dry doubling and dissolves with small amount of methanol with chloroform extraction twice.Silica gel column chromatography on the dissolve with methanol liquid (silica gel G 100-200 order, column length 50cm internal diameter 4cm), chloroform: the methyl alcohol gradient elution, HPLC detects, and merges the active principle concentrating under reduced pressure and gets the FW523 crude product.The FW523 crude product obtains FW523-1,2,3,4,5 five compounds through preparation property HPLC (Tianjin, island LC-8A preparation property HPLC, Tianjin, island PRO-ODS 10mm * 250mm post, 90% acetonitrile water elution, flow velocity 10ml/min, detection wavelength 260nm) separation.

The immunosuppressive activity of embodiment 3:FW523-3

10,50, the FW523-3 of 250ng/ml transforms inhibiting rate to ConA inductive mouse spleen lymphocyte and is respectively 16%, 40%, 100%, quite (CsA transforms the half-inhibition concentration IC that suppresses to its immunosuppressive activity to ConA inductive mouse spleen lymphocyte with immunosuppressor ciclosporin CsA commonly used clinically ₅₀Be about 35ng/ml).

The anti-tumor activity of embodiment 4:FW523-3

Adopt U.S. Cellomics company many indexs apoptosis kit measurement, control drug is Paclitaxol (taxol), uses the ArrayScan HCS system acquisition data of the said firm.The result shows that the above FW523-3 of 0.3 μ g/ml has the obvious suppression effect to the L929 growth of tumour cell, and the above FW523-3 of 1 μ g/ml obviously reduces nucleus size, F-Actin amount, plastosome quantity and the membrane potential of L929 tumour cell.Illustrate that cytotoxic compound FW523-3 (Rakicidin B) is suitable with control drug Paclitaxol to the restraining effect concentration of growth of tumour cell, but it is different fully with the mechanism of action of Paclitaxol.

Claims

1, the Micromonospora sp.FIM02-523 Gao Shi asparagine slant culture of a platinum loop inserts liquid seed culture medium, 32 ℃ of shaking culture 48 hours, change fermention medium over to 5% culture transferring amount, 32 ℃, bottle is put on 96 ~ 120 hours biologically active tat peaks of 240rpm shaking culture;

Above-mentioned fermented liquid is through the acidifying flocculation treatment, and the throw out that centrifugal back obtains amasss alcohol immersion twice, alcohol immersion liquid concentrating under reduced pressure with diploid; The ethanol concentrated solution is evaporated to dry doubling and dissolves with small amount of methanol with chloroform extraction twice; Silica gel column chromatography on the dissolve with methanol liquid, chloroform: the methyl alcohol gradient elution, HPLC detects, and merges the active principle concentrating under reduced pressure and gets the FW523 crude product; The FW523 crude product obtains FW523-1,2,3,4,5 five compounds through preparation property HPLC separation.