Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1999-04-07
2001-02-06
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S405000, C424S406000, C435S071100, C435S170000, C435S252500, C435S267000, C435S839000
Reexamination Certificate
active
06183736
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the control of pathogenic microorganisms, particularly pathogenic fungi and certain bacteria. The invention also relates to lipopeptides having such antipathogenic activity i.e. high antifungal particularly against the Aspergillus genus and antibacterial activity. The invention also relates to the plants which have been treated with the lipopeptides or the microorganisms that synthesize and produce the lipopeptides. The invention also relates to other aspects further described herein.
An important objective of the invention is to address and to contribute to solve the aflatoxin problem that is caused by the aflatoxin-producing fungi,
Aspergillus flavus
and
Aspergillus parasiticus.
2. Description of the Related Art
Bacillus ssp is known to produce a variety of peptide antibiotics that are antibacterial and/or antifungal. Although the peptides antibiotics are composed of amino acids, they often differed from gene-encoded polypeptides in their structure and mechanism of biosynthesis. Some are gene-encoded and synthesized ribosomally, but these often undergo posttranslational processing and modifications. Antibiotics produced non ribosomally are composed of 2 to 20 amino acids organized in a linear, cyclic or branched cyclic structure.
Bacillus subtilis
produced gene-encoded antibiotics and a variety of small antibiotic peptides with a molecular weight less than 2000 daltons, synthesized non-ribosomally. Subtilin is one gene-encoded lantibiotic peptide synthesized by
B. subtilis
as a prepropeptide that undergoes posttranslational processing (1). Among the antibiotics synthesized non ribosomally are two family: the lipopeptides including iturin, surfactin, fengycin, plistatin and the small hydrophilic di- and tripeptides. Iturin is a group of cyclic lipopeptides produced by
Bacillus subtilis
including iturin A, C, D and E (2,3), bacillomycin D, F and L (4), Bacillopeptin (5) and mycosubtlin (6). All contains a &bgr;-amino fatty acid linked by amide bonds to the constituent amino acid residues of the iturin group. Iturin lipopeptide share a common sequence [&bgr;-hydroxy fatty acid-Asx-Tyr-Asx] and show variation at the other four positions. Surfactin is also a cyclic lipopeptide containing seven residues of D- and L-amino acids and one residue of a &bgr;-hydroxy fatty acid (7) with an amino acid sequence completely different from the iturin group. It is a powerful surfactant and has been described as an antifungal agent. Fengycin (8) and plipastatin (9) are lipopeptide with ten amino acid and a lipid attached to the N-terminal end of the molecule. They differed from iturin and surfactin by the presence of unusual amino acid such as ornithine and allo-threonine.
Table 1 shows the amino acid residues of the iturins and the chemical structure of Bacillomycin D.
TABLE I
Amino acid residues
Antibiotic
L
D
D
L
L
D
L
Major &bgr; amino acid
Iturin A
Asn
Tyr
Asn
Gln
Pro
Asn
Ser
n-C
14
, i-C
15
, a-C
15
Iturin C
Asp
Tyr
Asn
Gln
Pro
Asn
Ser
n-C
14
, i-C
15
, a-C
15
Bacillopeptin
Asn
Tyr
Asn
Ser
Glu
Ser
Thr
n-C
14
, i-C
15
, i-C
16
Bacillomycin D
Asn
Tyr
Asn
Pro
Glu
Ser
Tbr
n-C
14
, i-C
15
, a-C
15
Bacillomycin F
Asn
Tyr
Asn
Gln
Pro
Asn
Thr
i-C
16
, i-C
17
, a-C
17
Bacillomycin L
Asp
Tyr
Asn
Ser
Gln
Ser
Thr
n-C
14
, i-C
15
, a-C
15
Mycosubtilin
Asn
Tyr
Asn
Gln
Pro
Ser
Asn
i-C
16
, a-C
17
Primary structure of iturins.
Bacillomycin D
Iturin exhibit a restricted antibacterial activity and a broad range of antifungal activity against fungi and yeast but none of them has been shown to have antifungal activity against
A. flavus
although iturin A has already been patented for control of aflatoxin (10). Culture filtrate from
B. subtilis
were described in 1948 as antifungal against important dermatophytes and systemic fungi, and the undescribed antibiotic was named “Bacillomycin” (11). Since then 3 class of bacillomycin D (12), F (13) and L (14), have been characterized according to their amino acid sequence. Among each class, different antibiotics have been reported such as bacillomycin Fa, Fb, Fc, and Lc. Bacillomycin Fb and Fc differ from bacillomycin Fa by the presence of one or two carboxyl group respectively instead of carboxamide groups and bacillomycin Lc from bacillomycin L only by the sequence positions of a side chain amide and a carboxylic acid (15). An exhaustive study of the literature show that bacillomycin F has been tested against a wide range of fungi and bacteria (Table II).
TABLE II
MIC (ug/ml)
Bacillomycin
Bacillomycin
Bacillomycin
Test Organisms
Fb
Fc
Fa
Aspergillus niger
25
30
40
Botrytis cinerea
20
Cladosporium cladosporioides
25
40
Fusarium oxysporum
>200
>200
>320
Mycosphaerella pinodes
100
30
10
Neurospora crassa
80
Penicillium chysogenum
20
Pleospora herbarum
10
Rhodotorula pillimanae
80
Sclerotina fructigena
40
Sclerotina sclerotiorum
50
Stemphylium radicinum
320
Trichophyton mentagrophytes
20
Candida albicans
>200
>200
40
Candida tropicalis
>200
>200
40
Saccharomyces cerevisiae
25
30
10
Azotobacter vinelandii
>400
Brucella brochiseptica
>400
Escherichia coli
K12
>400
Streptomyces albus
G
>400
Bacillus cereus
>400
Micrococcus luteus
200
Sarcinia lutea
>400
Staphylococcus aureus
>400
Kluyveromyces bulgaris
75
40
Bacillomycin F showed a strong antifungal activity against various yeasts, fungi and phytopathogenic fungi but a weak antibacterial activity (16). Bacillomycin Lc has been described as a new antibiotic of the bacillomycin family with antifungal activity against phytopathogenic fungi such as
Ophiostoma ulmi, Verticillium dahliae, Ceratocystis fagacearum
and
Cryphonectria parasitica
the causal agents of Dutch elm disease, Verticillium wilt of maples, oak wilt and chestnut blight respectively. Bacillomycin D has been reported to be antifungal against
Absydia corymbifera, Aspergillus Niger, Candida Albicans, Fusarium oxysporum, Kluyveromyces bulgaris
and
Saccharomyces cerevisae
(17).
Mycosubtlin, iturin A and bacillomycin L inhibited the growth of
Micrococcus luteus,
their activity are different upon
Micrococcus luteus
protoplast. Mycosubtlin and Iturin A are able to lyse
Micrococcus luteus
protoplast and bacillomycin L has no effect (18). Even a slight modification of the molecule as the methylation of the aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity (19). The phenolic group of the tyrosine residue has been shown to be essential for the antifungal activity, when the tyrosine residue was substituted no activity was observed (20).
Aflatoxin Problem
The invention relates and contributes to solve an important scientific problem of serious economic and business consequences in the United States and in the world. To date, there is no acceptable and effective way and means to control aflatoxin on plants susceptible to aflatoxin-producing fungi. There are no known aflatoxin-resistant plants, genetically transformed or otherwise.
Drought stress and high temperatures at critical times during kernel or seed development and/or insect injury of crops can contribute heavily to aflatoxin contamination of corn by the aflatoxin-producing fungi,
A. flavus
and
A. parasiticus.
The aflatoxin-producing fungi,
A. flavus
and
A. parasiticus,
present health hazards to humans and animals through the toxic and carcinogenic properties of their secondary products. Even a very low level of aflatoxin contamination can lead to severe economic losses in the peanut industry. To control this problem, breeding peanut varieties resistant to Aspergillus sp. or which support less aflatoxin production has been attempted with limited success.
Cleveland Thomas E.
Moyne Anne-Laure
Tuzun Sadik
Lankford , Jr. Leon B.
Schnader Harrison Segal & Lewis LLP
USDA/ARS Southern Regional Research Center
Ware Deborah K.
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