Abstract:
A substance GIF-2 having bacteriostatic activities and a process for producing the same are disclosed. 
     GIF-2 is produced by culturing bacteria capable of producing GIF-2 and belonging to genus Bacillus, and isolating GIF-2 from the culture medium. GIF-2 is presumed to have the following structure: ##STR1## wherein one of three X&#39;s is an amido bond and R is a β-amino acid residue represented by the formula ##STR2##

Description:
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° C. -Heat resistance test (10° 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 1polypepton         3%yeast extract    0.5%NaCl             0.5%deionized water   96%            (pH = 7.0)L-mediumbacto-trypton      1%yeast extract    0.5%NaCl             0.5%            (pH = 7.0)MY-mediumlactose            1%polypepton       0.5%yeast extract    0.3%malt extract     0.3%(The pH is adjusted to 7.0 with 0.05 M phosphoricacid 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° to 40° C., preferably at 25° to 30° 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 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 2  O. 
     Ethanol is added to the eluate solution to give 80% and the mixture in allowed to stand at 4° 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. 
     1. Molecular weight: 1057 by Mass spectrometer 
     2. Color and appearance: White prismatic microcrystals 
     3. Melting point: 210°-215° C. (light brown, slightly shrinked), 234°-239° C. (dark brown), 240°-245° C. (carbonized) 
     4. UV absorption spectrum: Shown in FIG. 1 measured as a 2.8% aqueous solution. 
     5. IR spectrum: Shown in FIG. 2 
     6. Solubilities in solvents: Soluble in water, methanol, ethanol, and t-butanol; insoluble in n-butanol, acetone, ethyl acetate, ether, chloroform, benzene, carbon tetrachloride, and petroleum ether 
     7. Color change test: 
     
         ______________________________________  CBB test   +  Xanthoprotein test             +  Adamkiewitz test             -  Earlich test             -  Biuret test             +______________________________________ 
    
     8. Other property: A solution of this substance in water (1.4 mg/ml) has a pH of 7.6 
     9. Amino acid composition: The amino acid composition by an amino acid analyzer is as follows: 
     
         ______________________________________   Amino Moler   acid  ratio______________________________________   AsX   2   Ser   1   GluX  1   Leu   1   Tyr   1   Pro   1______________________________________ 
    
      wherein one of three X&#39;s has an amido bond, and in addition to the above amino acids, one molecule of β-amino acid is found by a mass spectrometer. 
     10. Presumed Structure: ##STR3##  wherein one of three X&#39;s is an amido bond and R is a β-amino acid residue represented by the formula 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a chart showing U.V. absorption spectrum of GIF-2, and 
     FIG. 2 is a chart showing I.R. absorption spectrum of GIF-2. 
    
    
     The antimicrobial activities of GIF-2 are confirmed and shown hereunder. 
     The antimicrobial activities in relation to various microorganisms were tested by the use of the following culture media and the results are shown in Tables 1 to 4 below. 
     
         ______________________________________Culture medium for bacteria in generalMeat extract            10      gPeptone                 10      gNaCl                    5       gWater                   1       l                   (pH =   7.2)Culture medium for mycobacteriaGlycerol                10      gPolypepton              10      gCasamino acid           5       gYeast extract           5       gNa.sub.2 HPO.sub.4      0.5     gWater                   1       l                   (pH =   7.2)Culture medium for fungiLactose                 10      gPolypepton              5       gYeast extract           3       gMalt extract            3       g0.05 M phosphoric acid buffer                   1       l                   (pH =   7.0)______________________________________ 
    
     The following microorganisms were cultured on a microtiter plate at 27° C. for 3 to 7 days. The minimum inhibitory concentration (MIC) of GIF-2 with respect to the microorganisms is shown in Tables 1 to 4 below. 
     
                       TABLE 1______________________________________Strain                  M.I.C. (μg/ml)______________________________________Bacillus subtilis          IAM 1206     &gt;625          IAM 1069     &gt;625          IAM 1145     &gt;625          IAM 1168     &gt;625          IAM 1169     &gt;625          IAM 1207     &gt;625          IAM 1212     &gt;625          IAM 1213     &gt;625          IAM 1259     &gt;625          IAM 1260     &gt;625          IAM 11060    &gt;625          IAM 12021    &gt;625          IAM 12118    &gt;625Bacillus licheniformis          IAM 11054    &gt;625Bacillus polymixa          IAM 1210     &gt;625Bacillus amyloliquefaciens          IAM 1521     &gt;625Bacillus cereus          IAM 1029     &gt;625          IAM 1072     &gt;625          IAM 1110     &gt;625          IAM 1656     &gt;625          IAM 1729     &gt;625Bacillus coagulans          IAM 1194     &gt;625Bacillus megaterium          IAM 1166       19Bacillus cereus SW          &gt;625______________________________________ 
    
     
                       TABLE 2______________________________________Strain                 M.I.C. (μg/ml)______________________________________Escherichia coli IAM 1268  &gt;625Enterobacter aerogenes            IAM 12348 &gt;625Klebsiella pneumoniae            IAM 1063  &gt;625Serratia marcescens            IAM 12142 &gt;625Proteus vulgaris IAM 1025  &gt;625Pseudomonas aeruginosa            PAO 1     &gt;625Staphylococcus aureus            NIHJ 209P &gt;625Micrococcus luteus            IAM 1056  &gt;625Arthrobacter nicotianae            IAM 12342 &gt;625Nocardia opaca   IAM 12123 &gt;625Mycobacterium phlei            AU 3368   &gt;625&#34;                AU 1574   &gt;625______________________________________ 
    
     
                       TABLE 3______________________________________Strain                  M.I.C.(μg/ml)______________________________________Conidiobolus lamprauges            sp. No. 454                       19.5            ATCC 28996 39            ATCC 28997 19.5            CBS 153    19.5Conidiobolus thromboides            ATCC 12587 39Conidiobolus nanodes            CBS 154    19.5Conidiobolus nanodes            CBS 183    19.5Conidiobolus chlamydosporus            CBS 167    39Fusarium oxysporum            IAM 5009   39______________________________________ 
    
     
                       TABLE 4______________________________________Strain                 M.I.C. (μg/ml)______________________________________Aspergillus fumigatus           IAM 2004   78Aspergillus nidulans           IAM 2006   39Aspergillus oryzae           IAM 2640   156Chaetomium globosum           IAM 8059   78Eurotium chevalieri           IFO 4928   19.5           ATCC 16496 39Gliocladium virens           IAM 5061   39Mucor rouxianus IAM 6131   78Mucor javanicus IAM 6087   156Myrothecium verrucaria           IAM 5063   78Penicillium chrysogenum           IAM 7106   39Cryptococcus luteolus           IAM 12207  19.5Debaryomyces castellii           IAM 4977   78Hansenula anomala           IAM 4967   39Hansenula wingei           IAM 4983   19.5Kloeckera africana           IAM 4984   39Saccharomyces cerevisiae           IAM 4125   39Torulopsis colliculosa           IAM 4188   39______________________________________ 
    
     This invention is further illustrated by the following Example. 
     EXAMPLE 
     The strain Bacullus cereus SW, FERM BP-746 was inoculated on Medium 1 described above and cultured at 27° C. for 3 days by shaking culture. 
     The resulting culture medium was centrifuged at 7,500 rpm for 10 min., and to the supernatant was added calcium chloride to give 1%. The resulting precipitate was recovered by centifugation at 2,500 rpm for 10 min. The precipitate was dissolved in a 100 mM EDTA-0.05M tris-Hcl buffer (pH 8.0) and the solution was dialyzed against a 0.05M phosphoric acid buffer, and ethanol was added to the dialyzed solution to give a final concentration of 80%. 
     The resulting precipitate was removed, the supernatant was evaporated under reduced pressure to dryness and the residue was dissolved in deionized water. The pH of the solution was then adjusted to 3 by addition of hydrochloric acid. The resulting precipitate was dissolved in a 0.05M NaHCO 3  aqueous solution and the solution was dialyzed against deionized water. The dialyzate solution was then charged into and passed through a column with Sephadex G-100/H 2  O. Ethanol was added to the eluate to give a concentration of 80%, and the mixture was allowed to stand at 4° C. for 2 to 3 days to form a white crystalline precipitate. 
     The precipitate was evaporated under reduced pressure, to dryness and the presence of GIF-2 was confirmed as white prismatic microcrystals by microscopic observation.