Patent Application: US-28751599-A

Abstract:
the invention relates to two lipopeptides a1 and a2 produced by bacillus subtilis and their use as an anitfungal agent against aspergillus flavus . both peptides are cyclic , acidic and have broad range of antifungal and antimicrobial activity . both peptides belong to the bacillomycin d family . a method and composition for controlling aflatoxin contamination in plants susceptible to alflatoxin - producing fungi , like aspergillus flavus or aspergillus parasiticus is also disclosed .

Description:
the family of peptides , which have antifungal and antibacterial activity against a spectrum of fungi and bacteria include two small peptides designated herein as a1 and a2 . these peptides were found to be associated and isolated from b . subtilis au195 . they were characterized as iturin and belong to the bacillomycin d family . although as has been reported above , bacillomycin d peptides have been found to have antifungal activity , no peptide has been reported to date to have antifungal activity against certain specific fungi , to which plants are susceptible , specifically alternaria solani , fusarium monoliforme , pythium aphanidermatum , and especially a . flavus . other pathogenic fungi to which plants are known to be susceptible include puccinia ssp ., phytophthora ssp ., peronospora ssp ., rhizoctonia ssp ., botrytis ssp ., sclerotinia ssp . and colletotrichum ssp , which can be controlled with the peptides of the invention . further , as has been reported , certain peptides which were isolated from bacteria , have been found to have antibacterial activity , which are human pathogens . in contrast , the peptides of the invention have been found to be effective against plant pathogenic bacteria such as , xanthomonas campestris ( x . campestris ) is known to be pathogenic , for instance on cabbage plants and is responsible for black rot disease . further , the peptides are effective against certain bacteria - like clavibacter michiganensis and b . cereus . the composition of the invention comprises one or more of the principal two peptides discovered namely a1 and a2 in a mixture with conventional biologically acceptable inert or active carriers . the peptides can be used together or administered to the plant , or to the animal sequentially , or only one of the fungal peptides need to be used . the amount in which the peptide is used in a minimum amount effective to control or inhibit the target fungi or bacteria to a maximum as may be needed for control . instead of the isolated peptide , the microorganism , the b . subtilis can be used by itself or in the composition , preferably with an appropriate ingredient that promotes the release of the peptide ( s ). it is not excluded that the spores of b . subtilis ( e . g . b . subtilis au 195 ) may be used . these would be allowed to grow on the plant and release the peptides . the peptides a1 and a2 of the invention have been characterized as described hereafter . they have , respectively , a 1045 da and 1059 . 5 ba molecular weight , determined by mass spectroscopy . it is believed that a1 has a β - amino fatty acid of 15 carbons and that a2 has a β - amino fatty acid of 16 carbons . the tandem mass spectrometry of a2 indicated a sequence very similar to a1 with an additional ch 2 at some point other than the glu residue . it is not excluded that the length of these amino acids vary each by 1 - 3 carbons , i . e ., be shorter or longer accordingly . the mass shift of a2 may be accountable by a homoserine for the serine residue or a fatty acid modifier one carbon longer than a1 or a single amino acid substitution of some type . the target plant or mammal , which can be treated is any plant susceptible to the fungal or microbial infection to be controlled , like the major crops , whether monocotyledonous or dicotyledonous , like cereals , legumes , tubers , solanaceous plants , cucurbits , fibrous plants , corn , peanut , rice , wheat , etc . of particular interest at this time in the treatment of peanut plants which are susceptible to aflatoxin - producing fungi . peanut plants including the seeds can be treated in accordance with the invention . any mammal especially animals susceptible to a . flavus or and / or a . parasitus can also be treated with the compositions including the peptides of the invention . the peptides of the inventions are generally obtained as a mixture of isomers in a mixture with other peptides that do not interfere with the activity of the peptides of the invention . the isomeric mixture can be separated into the respective isomers by know inventions . upon isolation and sequencing of the gene ( s ) which codes the peptides of the invention , the plants like corn , soybeans , peanut and other crops can be readily transformed ( either by nucleus or chloroplast transformation ) to produce plants resistant to the target infection , and likewise produce progeny ( and maternally inherited ) generations , that are likewise stably resistant to the target pathogen ( s ). any mammal , especially animals susceptible to a . flavus and / or a . parasiticus can be treated on accordance with the invention . the peptides can be used for prophylactic control and / or therapeutic control . other embodiments of the invention will become apparent from the description herein . the following examples are not intended to limit the invention in any manner whatsoever , they are purely illustrative . description of the producing strain ( see table iii ). table iii gave the identification of the au 195 as a bacillus subtilis strain with gas chromatography of fatty acid methyl ester . au195 produces a pigment , which gave an orange color to the culture filtrate . fig1 shows the in vitro inhibitory activity of the strain b . subtilis au195 . description of the method for the antifungal assays . microtiter plate assay : samples were tested for their antifungal activity against aspergillus flavus in a microtiter plate ( falcon 3918 , becton - dickinson and company ). each well contained 100 μl potato dextrose agar ( pda , difco laboratories ) and 100 μl of sample and was inoculated with 500 spores of aspergillus flavus . fungal growth was monitored by reading optical density ( o . d .) at 560 nm ( dynatech microplate reader ). disc plate diffusion assay : antifungal activity of the different purification steps were carried out under sterile conditions using the disc plate diffusion assay . mycelium plugs from actively growing fungal cultures were placed in the center of the petri plate . after incubation at 27 ° c . to allow vegetative growth , samples were applied on sterile filter paper discs laid on the agar surface . production of the antifungal peptides . au195 has been cultured in lb medium and culture filtrate was sampled daily . samples were filter sterilized and incubated in presence of aspergillus flavus spores in microtiter plates which enabled us to measure fungal growth spectrophotometrically as described in example 2 . the antifungal peptides secreted into the culture filtrate by au195 increased every day ( fig2 ). temperature stability and sensitivity to enzyme . 7 - day culture filtrate was boiled for 20 minutes and then tested for antifungal activity . 50 μl of 7 - day culture filtrate were incubated with 200 μg of lipase from wheat germ ( sigma ) in 50 mm tris hcl ph 7 . 5 . incubation with enzymes was carried out at 37 ° c . for 2 hours . the activity of the samples was then tested with a disc diffusion assay as described in example 2 and compared with that of a control . there was no decrease in the antifungal activity when the 7 - day culture filtrate is boiled , kept at room temperature for 7 days or one month or incubated with lipase . this indicated that the peptides are very stable . solubility . 7 - day culture filtrate was freeze - dried and resupended in methanol , ethanol , butanol , ether , chloroform and acetone . solvent was removed by evaporation , samples were dissolved in water and tested using the disc filter assay described in example 2 . the antifungal peptides were soluble in water , methanol , ethanol , butanol and insoluble in ether , chloroform and acetone . solubility in polar solvent indicated that they are hydrophilic . precipitation with ammonium sulfate . the 7 th day culture filtrate was sequentially precipitated with 20 , 40 , 60 and 80 % ammonium sulfate . using a disk filter assay as described in example 2 , only fraction precipitated with 20 % ammonium sulfate exhibited antifungal activity against aspergillus flavus ( fig3 ). pi of the antifungal peptides . to characterize the antifungal peptides , the fraction precipitated with 20 % ammonium sulfate was further separated with isoelectro focusing ( ief ) tube gel electrophoresis . for this purpose , 1 mg of protein sample was loaded on a tube gel containing ampholytes ( ph 2 - 8 ) and after electrophoresis gels were cut in 1 cm pieces . each piece ( total of 15 ) was placed on an agar media that was already inoculated with the fungus . fig4 represents the result of an antifungal assay against aspergillus flavus after separation of the bacillus proteins depending on their pi . purification of the antifungal peptides . bacterial cells were removed by centrifugation after 7 days of culture and proteins were precipitated with ammonium sulfate . after centrifugation , the pellet was dialyzed against water , acidified to ph 3 with 12n hcl and centrifuged . after resuspending in water and adjusting ph to 7 , pellet was loaded on a 5 ml anion exchange column ( hitrap q pharmacia ). the column was connected to an fplc system ( consep lc 100 , perseptive biosystems ) and a linear gradient of 0 to 1 . 6m nacl including 20 mm phosphate buffer ph 7 was applied . 5 ml fractions were collected , tested for antifungal activity using a filter disk assay as described in example 2 and dialyzed against water . all the fractions with antifungal activity were pooled together and loaded on a preparative superdex peptide hi load 16 / 60 column ( pharmacia ). the antifungal fractions were separated twice on this column until the antifungal peaks were fully separated ( fig5 ). molecular weight of both antifungal peptides . the molecular weights of both peptides were determined by fab mass - spectrophometry . the spectra for the antifungal peptide a1 indicated a mass [ m + h ] of 1045 . 5 and for the antifungal peptide a2 1059 . 5 ( fig6 ). amino acid sequence of a1 and a2 . initial edman sequencing was unsuccessful and tandem mass spectrometry of the molecular ion resulted in a highly complex spectrum which suggested a cyclic structure for both peptides . a1 was digested to give a linear molecule and the sequence after digestion at the glu — c amino acid was : nh2 - stnynpe - oh . a modification of the molecule at the thr residue suggested the attachment of hydroxy fatty acid . a1 has the same amino acid sequence and mass than one of the bacillomycin d ( 21 ). tandem mass spectrometry of the cyclic form of a2 indicated a sequence very similar to a1 with an additional ch2 . a2 has an amino acid sequence similar to bacillomycin d but its mass is higher m / z 1059 . 5 than the one published by peypoux et al . ( 20 ) ( m / z 1031 and 1045 ). antifungal activity of the crude fraction . the crude fraction included all the protein secreted in the culture filtrate of b . subtilis au195 culture and precipitated with 20 % ammonium sulfate . both antifungal peptide a1 and a2 were present in the crude fraction in a mixture of 50 / 50 . antifungal activity of fraction obtained after ammonium sulfate precipitation was carried out under sterile conditions using the disc plate diffusion assay described in example 2 . mycelium plugs from actively growing cultures were placed in the center of a petri plate containing pda for alternaria solani , fusarium monoliforme and pythium aphanidermatum . after incubation at 27 ° c . to allow vegetative growth , samples were applied on sterile filter paper disc laid on the agar surface . the crude fraction is able to inhibit hyphal growth of alternaria solani , fusarium monoliforme and pythium aphanidermatum ( fig7 ). antifungal activity of the purified peptides . after complete purification of the antifungal peptides as described in example 8 , removing salt by purification on a sepack cartridge ( waters ) and freeze drying , both peptides were quantified by weight . table iv shows the result of the antifungal assay . the antifungal assays are described in example 2 . in a similar manner the purified peptides each one individually or in a mixture ( 40 / 60 ) control the growth of a . parasiticus . when peanut plants are treated with the compositions of examples 11 and 12 , the plants are expected to show improved resistance to a . flavus . similarly improved resistance to a . flavus is expected by corn plants . antimicrobial activity of the crude fraction 3 or 5 ml of bacterial cell suspension ( from 10 4 to 10 8 cells / ml ) was widespread at the surface of lb plate and the excess was withdrawn . after 15 min dessication of plates under the hood , 4 discs were distributed containing different amount of the ammonium sulfate precipitate fraction . plates were incubated for 48 hours at 27 ° c . and the inhibition zone was observed after 24 and 48 hours . results are given in table v for all the strains tested . all publications referenced herein are hereby incorporated by reference in their entirety . the invention is not limited to the embodiments described herein , but encompasses modifications with the scope of the following claims and equivalent thereof . 1 . nishio c ., komura s . and kurahashi k ., 1991 , biochem . biophys . res . commun ., 116 : 751 - 758 . 2 . gueldner r . c ., reuilly c . c ., pusey p . l . costello e . c ., arrendale r . f ., cox r . h ., himmelsbach d . s ., crumley f . g . and cutler h . g ., 1987 , j . agric . food chem ., 36 : 366 - 370 . 3 . arrendale r . f ., gueldner r . c ., choryk o . t . and crumley f . g ., 1988 , j . microbiol . methods 8 , 249 - 257 . 4 . peypoux f ., besson f ., michel g . and delcambe l ., 1981 , eur . j . biochem ., 118 : 323 - 327 . 5 . kajimura y ., sugiyama m . and kaneda m ., 1995 , j . antibiot ., 48 ( 10 ): 1095 - 103 . 7 . kluge b ., vater , j ., salnikow j . and eckart k ., 1988 , febs letter , 231 : 107 . 9 . tsuge k ., ano t . and shoda m ., 1996 , arch . microbiol ., 165 : 243 - 251 . 10 . klich m . a ., arthur k . s ., lax a . r . and bland j . m ., 1994 , 127 : 123 - 127 . french patent no . 2 , 644 , 038 ; 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