Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1998-07-10
2001-07-31
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S911000, C424S093500
Reexamination Certificate
active
06268202
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to strains of the aerobic mycoparasitic fungus
Coniothyrium minitans
having &bgr;-glucanase activity.
2. Description of the Related Art
A major factor affecting animal production is a efficiency of feed utilization. Efficient feed utilization requires efficient digestion of complex &bgr;-glucan substrates that are present in forage. &bgr;-glucans are one of the most abundant groups of naturally-occurring polysaccharides. &bgr;-glucans with (1,3), (1,4), (1,6) and (1,2) linkages have been identified in both bacteria and plants (Stone and Clark 1992), while (1,3) and (1,6) linkages are abundant in fungal cell walls (Wessels and Sietsma 1981). Fibre and &bgr;-glucans in feed are often poorly digested by animals, especially monogastric animals, resulting in waste. Diets containing certain forms of glucan (such as arabinoxylan in wheat and rye, or &bgr;-glucan in barley and oats) may also have deleterious effects on nutrient absorption and may promote intestinal disturbances by enteric pathogens.
The digestion of fibre and &bgr;-glucans in feed by livestock may be improved by enzyme supplementation (Shuttle 1995). Glycanolytic enzyme feed additives are thought to enhance fibre degradation and/or reduce viscosity in the gastrointestinal tract, thereby increasing feed intake and passage rate (Sears 1993). Currently, feed enzyme supplementation has been limited by the high cost of enzyme production. Industry relies on mass fermentation to produce feed enzymes and is limited to a small group of fungal (mainly Aspergillus, Penicillium and Trichoderma) and bacterial (
Bacillus coagulans, B. lichenformis, B. circulans
and
B. subtilis var.
) taxa as a source for these enzymes (Sears 1993). There is, therefore, an ongoing need for new microbial sources of &bgr;-glucan degrading enzymes (&bgr;-glucanases). Ideally, such microorganisms could be easily and inexpensively grown by liquid or solid state fermentation, and would have high levels of &bgr;-glucanase activity. Endo- and exo-1,3-&bgr;-glucanase activity have been reported in the aerobic mycoparasitic fungus
Coniothyrium minitans
(Jones et al. 1973). However, the Jones et al. reference does not teach combined endo-1,3- and endo-1,4- &bgr;-glucanase activity of
C. minitans
. Combined endo-1,3- and endo-1,4-&bgr;-glucanase activity is the &bgr;-glucanase activity that is most useful for degrading &bgr;-glucans in animal feeds. Further, as the medium on which the
C. minitans
cultures of Jones et al. were grown consisted substantially of sclerotia of
Sclerotinia sclerotiorum
, a natural &bgr;-glucan source, the Jones et al. reference does not teach constitutive &bgr;-glucanase activity of
C. minitans
on a rich medium without a &bgr;-glucan source. Such a medium would be preferred for industrial fermentation applications. Teaching neither combined endo-1,3- and endo-1,4-&bgr;-glucanase activity nor constitutive &bgr;-glucanase activity of
C. minitans
, the Jones et al. reference does not suggest the utility of
C. minitans
as a useful source of &bgr;-glucanase enzymes.
SUMMARY OF THE INVENTION
The present invention provides biologically pure strains of the aerobic mycoparasitic fungus
Coniothyrium minitans
having high levels of &bgr;-glucanase activity when grown by liquid or solid state fermentation (
C. minitans
strains ATCC 74415, 74416 and 74417). Through mutagenesis of strain ATCC 74415, additional strains of
C. minitans
having high levels of &bgr;-glucanase activity have also been obtained (
C. minitans
strains ATCC 74418, 74419, 74435 and 74436).
A method for obtaining
C. minitans
strains having &bgr;-glucanase activity is also provided. Broadly stated, this method involves:
1. providing a plurality of cells of
Coniothyrium minitans
ATCC 74415;
2. exposing the cells to ultraviolet radiation to mutagenize the cells;
3. culturing the mutagenized cells on or in a culture medium to produce a plurality of cultures of
Coniothyrium minitans;
4. testing the cultures of mutagenized cells for &bgr;-glucanase activity; and,
5. recovering from step (d) those cultures that have &bgr;-glucanase activity.
Solid or liquid culture media may be used. The culture medium used may be a minimal medium wherein the only carbohydrate source is a source of &bgr;-glucan. Such a medium will select for mutants having a mutation increasing &bgr;-glucanase activity and which can therefore utilize the &bgr;-glucan in the medium as a carbohydrate source and will therefore out-compete
C. minitans
cells which have a mutation down-regulating &bgr;-glucanase activity or no mutation. Alternatively, a rich medium containing sources of starch and simple sugars but no source of &bgr;-glucan can be used. Mutagenized
C. minitans
cells which still exhibit &bgr;-glucanase activity on the rich medium have constitutive &bgr;-glucanase activity, as no source of &bgr;-glucan is required to induce &bgr;-glucanase activity, and do not suffer &bgr;-glucanase activity repression due to the presence of starches and simple sugars. Starches and simple sugars are readily metabolized nutrient sources that would be ordinarily utilized before &bgr;-glucan, resulting in repression of &bgr;-glucanase activity.
Through UV mutagenesis, mutant strains of
C. minitans
having levels of &bgr;-glucanase activity on average 10 times higher than the parental source strain
C. minitans
ATCC 74415 have been obtained. These mutants (ATCC 74435 and 74436) possess constitutive &bgr;-glucanase activity which does not require induction by a source of a &bgr;-glucan substrate, and which is not repressed by the presence of carbohydrate sources such as starches or simple sugars. These characteristics make the
C. minitans
strains of the present invention excellent candidates for commercial &bgr;-glucanase production in that the high &bgr;-glucanase activity strains can be grown on an inexpensive solid medium such as waste potato peelings. Such a medium is high in simple sugars and starch and does not contain a source of &bgr;-glucan to act as an inducer. The solid growth medium can be inoculated with spores or mycelium of
C. minitans
strains of the present invention having high constitutive &bgr;-glucanase activity, and over the course of culture growth, the growth medium will be digested and replaced by a mass of
C. minitans
mycelia possessing &bgr;- glucanase activity. The &bgr;-glucanase activity can then be recovered by freeze-drying the mycelia and grinding it to a powder. Such culture conditions are much less expensive and easier to work with than liquid fermentation systems, particularly if specialized minimal media containing only &bgr;-glucan as a carbohydrate source were required to grow
C. minitans
strains which possess &bgr;-glucanase activity that requires the presence of a source of &bgr;-glucan as an inducer, and which is repressed by the presence of simple sugars or starches.
REFERENCES:
patent: 4395490 (1983-07-01), Cooney et al.
patent: 4472504 (1984-09-01), Gallo
patent: 5409820 (1995-04-01), Gerson et al.
patent: 1950265 A1 (1996-01-01), None
patent: WO 90/09436 (1990-08-01), None
Huang et al., “Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria,” (1988) J. Phytopathology 123:133-39.
Tu et al., “Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum sclerotiorum and its effect on Sclerotinia Germination,” (1984) Phytopath.Z 109:261-268.
Jones, D., Gordon, A.H., and Bacon, J.S.D. 1974. Cooperative action by endo- and exo-beta- (1,3) -glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum. (Lib.) de Bary, Biochem. J. 140:47-55.
Jones, D. and Watson, D. 1969. Parasitism and lysis by soil fungi ofSclerotinia sclerotiorum(Lib.) de Bary, a Phytopathogenic Fungus. Nature 24:287-288.
Domsch, K.H., Gams, W., and Anderson, T.H. (eds.) 1980,Coniothyrium corda1840 emend. Sacc. 1880 (nomen conservadum). In: Compendium of Soil Fungi. Academic Press, New York. vol. 1:228-231.
S
Cheng Kuo Joan
Huang Hung Chang
Laroche Andre J.
Zantinge Jennifer L.
Greenlee Winner and Sullivan P.C.
Her Majesty the Queen in right of Canada as represented by the D
Marx Irene
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