Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2001-07-25
2002-11-12
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S101000
Reexamination Certificate
active
06479275
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a modified alternan having a lower apparent molecular weight than native alternan and to a method for producing the modified alternan. The modified alternan is produced by cultivation of native alternan in the presence of a Penicillium sp. isolate capable of essentially quantitative conversion of the native alternan to a polymeric modified form. The rheological properties of the modified alternan are similar to commercial gum arabic.
2. Description of the Prior Art
The polysaccharide alternan was first described by Jeanes et al. (1954,
J. Am. Chem. Soc.,
76:5041-5052) as one of two extracellular &agr;-D glucans, referred to as fraction S, produced by
Leuconostoc mesenteroides
NRRL B-1355. The structure of this fraction was later determined by Misaki et al. (1980, Carbohydr. Res., 84:273-285) to consist primarily of an alternating sequence of &agr;-1,3-linked and &agr;-1,6-linked D-glucose residues, with approximately 10% branching. Because the &agr;-1,3-linkages are part of the linear chain of the S fraction and there are not any consecutive &agr;-1,6-linkages, the fraction is not a true dextran, and Cote and Robyt (1982, Carbohydr. Res., 101:57-74) therefore named this fraction alternan. These authors also isolated the enzyme alternansucrase which synthesizes alternan from sucrose.
Native alternan has an apparent molecular weight average ({overscore (M
w
)}) of 10
6
−10
7
[Cote G. L.,(1992 ), Carbohydrate Polymers 19:249-252; Cote G. L. et al., (1997), Alternan and highly branched limit dextrans: Low-viscosity polysaccharides as potential new food ingredients. In: Spanier A. M. et al. (ed) Chemistry of Novel Foods, Carol Stream, Ill.: Allured Publishing Corp, pgs. 95-110]. Derivatives of alternan have been produced by ultrasonication, reducing the apparent molecular weight average to <10
6
and modifying the rheological properties of the polymer so that they more closely resemble gum arabic [Cote, 1992, Ibid]. However, ultrasonication is a relatively expensive process that would be difficult to carry out on an industrial scale. Although alternan is resistant to hydrolysis by most known endoglucanases, a “limit alternan” has also been produced by treatment of native alternan with isomaltodextranase from
Arthrobacter globiformis
[Cote, 1992, Ibid]. Limit alternan exhibited an apparent {overscore (M
w
)} of 3.5×10
3
and was rheologically similar to oligosaccharides of maltodextrin [Cote, 1992, Ibid]. Recently, Bacillus sp. isolates were described that produced an endoglucanase specific for alternan [Biely P. et al., (1994), Eur. J. Biochem., 226:633-639; Cote G. L. et al., (1998), U.S. Pat. No. 5,889,179; Wyckoff H. A. et al., (1996), Curr. Microbiol., 32:343-348]. This enzyme produced a novel cyclic tetrasaccharide from alternan [Cote G. L. et al., (1994), Eur. J. Biochem., 226:641-648].
The desire to further extend the range of properties of modified alternan and to expand the scope of new applications for these materials has led to a continued search for new organisms, particularly fungi, that could partially degrade or modify the native polysaccharide.
SUMMARY OF THE INVENTION
We have now discovered four new Penicillium spp. isolates capable of essentially quantitative conversion of the native alternan to a polymeric modified form (hereafter referred to as “modified alternan”) having a lower apparent molecular weight than native alternan. The rheological properties of the modified alternan are similar to commercial gum arabic. A fifth isolate having the same apparent ability to convert native alternan to a lower molecular weight form was found in a survey of isolates of Penicillium subgenus Biverticillium obtained from a culture collection.
In accordance with this discovery, it is an object of this invention to provide novel fungal organisms for reducing the apparent molecular weight of alternan.
Another object of this invention is to provide a method for reducing the apparent molecular weight of alternan by cultivating the native form of this polysaccharide with novel Penicillium spp. isolates.
It is also an object of the invention to provide a composition of modified alternan, composed of novel low molecular weight fractions and having functional properties similar to those of gum arabic.
A further object of the invention is to produce a polysaccharide product having properties similar to those of ultrasonicated alternan, but without the expense of ultrasonication.
Yet another object of the invention is to provide novel compositions for use as bulking agents and extenders in foods and cosmetics, especially as carbohydrate-based soluble food additives.
Other objects and advantages of this invention will become obvious from the ensuing description.
REFERENCES:
patent: 5889179 (1999-03-01), Cote et al.
Cote, Gregory L., “Low-viscosity &agr;-D-glucan fractions derived from sucrose which are resistant to enzymatic digestion”,Carbohydrate Polymers, 19, 1992 pp. 249-252.
Cote, Gregory L., et al., “Enzymatically produced cyclic &agr;-1,3-linked and &agr;-1,6-linked oligosaccharides of D-glucose”,Eur. J. Biochem., 226, 1994, pp. 641-648.
Biely, Peter, et al., “Purfication and properties of alternanase, a novel endo-&agr;-1,3-&agr;-1,6-D-glucanase”,Eur. J. Biochem., 226, 1994, pp. 633-639.
Wyckoff, H.A., et al., “Isolation and Characterization of Microoganisms with Alternan Hydrolytic Activity”,Current Microbiology, vol. 32, 1996, pp. 343,348.
Cote Gregory L.
Leathers Timothy D.
Nunnally Melinda S.
Fado John D.
Lilling Herbert J.
Ribando Curtis P.
Silverstein M. Howard
The United States of America as represented by the Secretary of
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