Patent Publication Number: US-4929548-A

Title: Substance GIF-2 and process for production of the same

Description:
This is a Rule 60 Divisional Application of Ser. No. 807,066 filed Nov. 27, 1985, now Patent No. 4,771,121. 
    
    
     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 on 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 bacteria 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: 
     
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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       +                                                 
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     Bacteria such as Bacillus cereus SW are cultured in a suitable media. Some examples of the media are as follows: 
     
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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 0.05M phosphoric                          
acid buffer.)                                                             
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     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 obtained as white prismatic microcrystals by microscopic observation. 
     GIF-2 has the following physicochemical properties. 
     1. Molecular weight: 1057 by Mass spectrometer 
     2. Color and appearance: White prismatic microcrystals 
     3. Melting point: 210°-215° C. (light brown, slightly shrunk), 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                                                        
                  +                                                       
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     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: 
     
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        Amino Moler                                                       
        acid  ratio                                                       
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        AsX   2                                                           
        Ser   1                                                           
        GlX   1                                                           
        Leu   1                                                           
        Tyr   1                                                           
        Pro   1                                                           
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      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: ##STR1##  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. 
     
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Culture medium for bacteria in general                                    
Meat extract             10     g                                         
Peptone                  10     g                                         
NaCl                     5      g                                         
Water                    1      l                                         
                       (pH = 7.2)                                         
Culture medium for mycobacteria                                           
Glycerol                 10     g                                         
Polypepton               10     g                                         
Casamino acid            5      g                                         
Yeast extract            5      g                                         
Na.sub.2 HPO.sub.4       0.5    g                                         
Water                    1      l                                         
                       (pH = 7.2)                                         
Culture medium for fungi                                                  
Lactose                  10     g                                         
Polypepton               5      g                                         
Yeast extract            3      g                                         
Malt extract             3      g                                         
0.05M phosphoric acid buffer                                              
                          1     l                                         
                       (pH = 7.0)                                         
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     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                                                     
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Strain                 M.I.C.(μg/ml)                                   
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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; 625                                          
Bacillus licheniformis                                                    
                 IAM 11054 &gt; 625                                          
Bacillus polymixa                                                         
                 IAM 1210  &gt; 625                                          
Bacillus amyloliquefaciens                                                
                 IAM 1521  &gt; 625                                          
Bacillus cereus  IAM 1029  &gt; 625                                          
                 IAM 1072  &gt; 625                                          
                 IAM 1110  &gt; 625                                          
                 IAM 1656  &gt; 625                                          
                 IAM 1729  &gt; 625                                          
Bacillus coagulans                                                        
                 IAM 1194  &gt; 625                                          
Bacillus megaterium                                                       
                 IAM 1166    19                                           
Bacillus cereus SW         &gt; 625                                          
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                       TABLE 2                                                     
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Strain                 M.I.C.(μg/ml)                                   
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Escherichia coli IAM 1268  &gt; 625                                          
Enterobacter aerogenes                                                    
                 IAM 12348 &gt; 625                                          
Klebsiella pneumoniae                                                     
                 IAM 1063  &gt; 625                                          
Serratia marcescens                                                       
                 IAM 12142 &gt; 625                                          
Proteus vulgaris IAM 1025  &gt; 625                                          
Pseudomonas aeruginosa                                                    
                 PAO 1     &gt; 625                                          
Staphylococcus aureus                                                     
                 NIHJ 209P &gt; 625                                          
Micrococcus luteus                                                        
                 IAM 1056  &gt; 625                                          
Arthrobacter nicotianae                                                   
                 IAM 12342 &gt; 625                                          
Nocardia opaca   IAM 12123 &gt; 625                                          
Mycobacterium phlei                                                       
                 AU 3368   &gt; 625                                          
Mycobacterium phlei                                                       
                 AU 1574   &gt; 625                                          
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                       TABLE 3                                                     
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Strain                   M.I.C.(μg/ml)                                 
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Conidiobolus lamprauges                                                   
                  sp. No. 454                                             
                             19.5                                         
                  ATCC 28996 39                                           
                  ATCC 28997 19.5                                         
                  CBS 153    19.5                                         
Conidiobolus thromboides                                                  
                  ATCC 12587 39                                           
Conidiobolus nanodes                                                      
                  CBS 154    19.5                                         
Conidiobolus nanodes                                                      
                  CBS 183    19.5                                         
Conidiobolus chlamydosporus                                               
                  CBS 167    39                                           
Fusarium oxysporum                                                        
                  IAM 5009   39                                           
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                       TABLE 4                                                     
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Strain                 M.I.C.(μg/ml)                                   
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Aspergillus fumigatus                                                     
                IAM 2004   78                                             
Aspergillus nidulans                                                      
                IAM 2006   39                                             
Aspergillus oryzae                                                        
                IAM 2640   156                                            
Chaetomium globosum                                                       
                IAM 8059   78                                             
Eurotium chevalieri                                                       
                IFO 4928      19.5                                        
                ATCC 16496 39                                             
Gliocladium virens                                                        
                IAM 5061   39                                             
Mucor rouxianus IAM 6131   78                                             
Mucor javanicus IAM 6087   156                                            
Myrothecium verrucaria                                                    
                IAM 5063   78                                             
Penicillium chrysogenum                                                   
                IAM 7106   39                                             
Cryptococcus luteolus                                                     
                IAM 12207     19.5                                        
Debaryomyces castellii                                                    
                IAM 4977   78                                             
Hansenula anomala                                                         
                IAM 4967   39                                             
Hansenula wingei                                                          
                IAM 4983      19.5                                        
Kloeckera africana                                                        
                IAM 4984   39                                             
Saccharomyces cerevisiae                                                  
                IAM 4125   39                                             
Torulopsis colliculosa                                                    
                IAM 4188   39                                             
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     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 centrifugation 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.