Substance GIF-2 and process for production of the same

Details Substance GIF-2 and process for production of the same Substance GIF-2 and process for production of the same

1985-11-27

1988-09-13

Phillips, Delbert R.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Cyclic peptides

C07K 512

Patent

active

047711213

DESCRIPTION:

BRIEF SUMMARY
THE FIELD OF THE INVENTION

This invention relates to a substance GIF-2 having an antimicrobial activity and to a process for production of the substance.


DESCRIPTION OF THE INVENTION

The inventors of this invention have been undertaking studies on microorganisms which are parasitic to insects and have occasionally found certain bacteria having an antimicrobial activities to organisms. After screening such bacteria, they identified them as bacteria belonging to the species Bacillus cereus . They have continued their studies on the antimicrobial substances produced by the bacteria and isolated a novel substance which they have named GIF-2.
The bacteria which are useful in this invention involve any species of bacteria capable of producing GIF-2. One example of such bacteris is Bacillus cereus SW which was first screened by the inventors and deposited with the Fermentation Research Institute, Japan as International Deposition Acceptance No. FERM BP-746 under the Budapest Treaty. The microbiological characteristics of Bacillus cereus SW are as follows:


______________________________________ Gram stain + Spore stain + V-P reaction + Catalase test + Oxidase test + Growth at 50.degree. C. - Heat resistance test (10.degree. C., 30 min.) + (growing) Egg-Yolk reaction + ______________________________________
Bacteria such as Bacillus cereus SW are cultured in a suitable media. Some examples of the media are as follows:


______________________________________ Medium 1 polypepton 3% yeast extract 0.5% NaCl 0.5% deionized water 96% (pH = 7.0) L-medium bacto-trypton 1% yeast extract 0.5% NaCl 0.5% (pH = 7.0) MY-medium lactose 1% polypepton 0.5% yeast extract 0.3% malt extract 0.3% (The pH is adjusted to 7.0 with 0.05 M phosphoric acid buffer.) ______________________________________
In addition to these culture media exemplified above, suitable media such as media for bacteria which contain appropriate amounts of carbon source, nitrogen source, and micronutrients may be used in this invention.
The bacteria may be cultured at 20.degree. to 40.degree. C., preferably at 25.degree. to 30.degree. C. for 2 to 3 days under aerobic conditions such as by shaking culture, aeration culture, stationary culture and the like.
After culturing, the culture medium is centrifuged, for example, at 7,500 rpm for 10 min., to remove bacterial cells. The medium is preferably concentrated if necessary and, after adding calcium chloride to the medium in such an amount as to give 1%, the resulting precipitate is recovered by centrifugation, for example at 2,500 rpm for 10 min. The collected precipitate is dissolved in 100 mM EDTA-0.05M tris-HCl buffer (pH 8.0) and the solution is dialyzed against 0.05M phosphoric acid buffer (pH 7.0). After dialysis, ethanol is added to the dialyzate to give 80%, and the resulting precipitate is then removed. The supernatant is evaporated under reduced pressure to dryness and the residue is dissolved in a deionized water. The pH is adjusted to 3 by addition of hydrochloric acid to form a precipitate. The precipitate is recovered and dissolved in a 0.05M NaHCO.sub.3 aqueous solution, and the solution is dialyzed against deionized water, the dialyzate then being charged into and passed through a column with Sephadex G-100/H.sub.2 O.
Ethanol is added to the eluate solution to give 80% and the mixture in allowed to stand at 4.degree. C. for 2-3 days to give a white crystalline precipitate.
After drying the precipitate under reduced pressure, the product GIF-2 is given as white prismatic microcrystals by microscopic observation.
GIF-2 has the following physiochemical properties. shrinked), 234.degree.-239.degree. C. (dark brown), 240.degree.-245.degree. C. (carbonized) solution. t-butanol; insoluble in n-butanol, acetone, ethyl acetate, ether, chloroform, benzene, carbon tetrachloride, and petroleum ether


______________________________________ CBB test + Xanthoprotein test + Adamkiewitz test - Earlich test

REFERENCES:
Chem. Abstr. vol. 104 (1986), 67506.

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