Production of chitosan-and chitin-like exopolymers

Details Production of chitosan-and chitin-like exopolymers Production of chitosan-and chitin-like exopolymers

2000-11-28

2003-03-18

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C536S123000, C536S056000, C536S102000, C536S112000, C536S126000

Reexamination Certificate

active

06534294

ABSTRACT:

BACKGROUND OF THE INVENTION
Cellulose, (1-4)-linked-&bgr;-D-glucan, is a major structural component of the cell walls of higher plants (Delmer, D. P., and Amor, Y.,
Plant Cell
7:987-1000 (1995)). Some microorganisms also produce unbranched (1-4)-linked-&bgr;-D-glucan, named microbial cellulose (MC) (Schramm, M. and Hestrin, S.,
Biochem. J
., 56:163-166 (1954); Carr, J. G.,
Nature
(London), 182:265-266 (1958) and Canale-Parola, E. and Wolfe, R. S.,
Biochim. Biophys. Acta
. 82:403-405 (1964)). Cellulose is important industrially, for example, in the production of paper. Cellulose can be chemically detergents, varnishes, adhesives and for gelling or thickening of food stuffs or pharmaceuticals, depending on the extent of etherification.
Structurally related polysaccharides, such as chitin and chitosan, are also found in the biosphere. Chitin occurs as a major cuticular or skeletal component in all arthropods, in some invertebrates, and in the cell walls of some fungi. Chitin is a polysaccharide of high molecular weight and consists of unbranched chains of (1-4)-linked 2- acetamino-2-deoxy-&bgr;-D-glucose residues (Hackman, R. H. and Goldberg, M.,
Carbohydr. Res
. 38:35-45 (1974)). Because of its abundance as a waste material from the canning food industry from crab, shrimp and lobster, chitin is an attractive starting material for the production of chitosan. Chitosan is the fully or partially deacetylated form of chitin (Anthosen, M. W., et al.,
Carbohydr. Polym
. 22:193-201 (1993)). It contains &bgr;-(1-4)-linked 2-amino-2-deoxy-&bgr;-D-glucopyranose and 2-acetamido-2-deoxy-&bgr;-D-glucopyranose residues (Hirano, S., et al.,
Carbohydr. Res
. 47:315-320(1976)). Chitosan is found in the cell walls of some fungi such as
Mucor rouxii
(Bartnicki-Garcia, S. and Nickerson, W. J.,
J. Bacteriol
. 84:841-858 (1962)). However, like cellulose, chitin is generally insoluble in water and in most conventional solvent systems. Furthermore, the starting material, chitin, is easily degraded in the presence of acid.
Commercially, chitosan is derived by the chemical deacetylation of chitin from waste crustacean exoskeletons with strong alkali. This harsh conversion process, as well as variability in source material, leads to inconsistent physicochemical characteristics (Arcidiacono, S. and Kaplan, D. L.
Biotechnol. Bioeng
., 39:281-286 (1992).). The purification of chitosan derived from the cell wall of some fungi also requires strong alkaline treatment with heat, which leads to inconsistent material (White, S. A., et al.,
Environ. Microbiol
., 38:323-328 (1979); Arcidiacono, S. and Kaplan, D. L.
Biotechnol. Bioeng
., 39:281-286 (1992)).
Glucose-rich polysaccharides such as cellulose and curdlan have been post-biosynthetically modified by nonspecific chemical means to change physical properties (Yamamoto, I. et al.,
Carbohydr. Polym
., 14:53-63 (1991); Osawa, Z., et al.,
Carbohydr. Polym
., 21:283-288 (1993)). For example, chemically modified cellulose and curdlan exhibited strong antiviral activity in vitro (Yamamoto, I. et al.,
Carbohydr. Polym
., 14:53-63 (1991); Osawa, Z. et al.,
Carbohydr. Polym
., 21:283-288 (1993)). Selective chemical modification of polysaccharides under homogeneous conditions also has been reported (Roesser, D. S. et al.,
Macromol
., 29:1-9 (1996)). However, it is extremely difficult to regiospecifically modify cellulose in the secondary hydroxyl position, to chemically generate glucosamine or N-acetylglucosamine, for example. Disadvantages of these synthetic approaches as well as purification of chitin from crustacean exoskeleton and plant and fungi cell walls include low yields, side reactions, the use of toxic solvents, and purification requirements.
Biosynthesis of polysaccharides has traditionally been studied using unmodified simple sugars such as glucose and sucrose, or complex carbon sources such as wheat gluten and molasses (Kaplan, D. L. et al., “Biosynthetic Polysaccharides In
Biomedical polymers: designed
-
to
-
degrade systems
,” edited by S. W. Shalaby, Hanser Publishers, New York. pp. 189-212 (1994)). Alternatively, microbial mutants have been used to manipulate biopolymer molecular weight, yield, and main chain or branch composition (Thorne, L., et al.,
J. Bacteriol
. 169:3593-3600 (1987); Hassler, R. A. and Doherty, D. H.,
Biotechnol. Prog
. 6:182-187 (1990)). Yet polysaccharides have not been well studied with respect to the incorporation of modified or non-native building blocks, unlike the extensive work with proteins for the incorporation of unnatural amino acids (Chung, H., et al.,
Science
259:806-809 (1993)), and bacterial polyesters with incorporation of a wide range of novel monomers (Brandl, H., et al., “Plastics from Bacteria and for Bacteria: Poly(&bgr;-Hydroxyalkanoates) as Natural, Biocompatible and Biodegradable Polyesters In
Advances in Biochemical Engineering/Biotechnology
,” Vol. 41, edited by T. K. Ghose and A. Fiechter. Springer, Berlin. pp.77 (1990); Steinbüchel, A., “Polyhydroxyalkanoic Acids In
Biomaterials: Novel Materials from Biological Sources
, edited by D. Byrom, Stockton Press, New York. pp. 123 (1991); Gross, R. A., “Bacterial Polyesters: Structural Variability in Microbial Synthesis In
Biomedical Polymers: Designed
-
to
-
Degrade Systems
, edited by S. W. Shalaby. Hanser Publishers, New York, pp. 173-188 (1994)).
Direct incorporation of glucose-related sugar monomers, 3-O-methyl-D-glucose (3-O-methylglucose) and 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine), into the main chain of biosynthesized curdlan has been reported (Lee, J. W. et al.,
Can. J. Microbiol
. 43:149-156 (1997)). In related studies, direct incorporation of specific fatty acid pendent groups on a main chain polysaccharide such as emulsan has been demonstrated (Gorkovenko, A. et al.,
Proc. Am. Chem. Soc. Div. Poly. Sci. Eng
., 72:92-94 (1995); Gorkovenko, A. et al.,
Can. J. Microbiol
., 43:384-390 (1997); Zhang, J., et al.,
Int. J. Biol. Macromol
., 20:9-21 (1997)).
Bacterial cellulose containing a limited amount of N-acetylglucosamine has been described, however, the method to produce the copolymer required serial adapation of the bacteria in N-acetylglucosaime containing medium (Ogawa and Tokura,
Carbohydrate Polymers
, 19:171-178 (1992)). Furthermore, the copolymer produced only contained the glucose analog N-acetyglucosamine, at a mole percentage in liquid culture no greater than 4.5% (Ogawa and Tokura). Incorporation of up to 6.3% of N-acetylglucosamine has been achieved when bacteria were serially adapted to culture in N-acetylglucosamine and cultured in the presence of phosphorylated chitin (Shirai et al.,
Int. J. Biol. Macromol
., 16:297-300 (1994)).
Therefore, a method is needed to produce polysaccharides comprising useful glucose analogs such as glucosamine and N-acetylglucosamine that does not require harsh extraction protocols and such that variable levels of glucose analog incorporation can be achieved. Further, a method for the production of polysaccharides comprising glucose and glucose analogs other than N-acetylglucosamine is needed.
SUMMARY OF THE INVENTION
In the present invention, microbially produced polysaccharide copolymers are provided. Specifically, glucose analogs such as aminosugars are incorporated into polysaccharides produced as exopolymers (also referred to herein as copolymers or terpolymers) by
A. xylinum
. Examples of aminosugars are glucosamine and N-acetylglucosamine.
Polymer blends of cellulose-chitin and cellulose-chitosan have been reported; however, the availability of copolymers with the level of incorporation of these monomers as provided herein is novel and expected to result in new properties as well as enhanced control over structural features of the polysaccharide. Furthermore, the method of the present invention does not require adaptation of the polymer producing microbe to growth in glucose analog containing medium. Copolymers described herein possess new properties such as unique solubility behavior. In addition, direct formation of fibers by the bacteria are useful in t

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