Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-07
2001-07-31
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S114000, C536S119000, C536S124000
Reexamination Certificate
active
06268493
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method for preparing cellobiuronic acid, and more particularly to the hydrolysis of polysaccharide to prepare a hydrolysate that includes cellobiuronic acid.
BACKGROUND OF THE INVENTION
The gene encoding &bgr;-glucuronidase has gained widespread use as a versatile tool for use in a variety of recombinant DNA techniques. The most widely embraced utility of &bgr;-glucuronidase is as a reporter gene in &bgr;-glucuronidase-deficient cells for indicating patterns of gene expression mediated by sequence elements attached to the glucuronidase gene. In addition, it has been recognized that &bgr;-glucuronidase can play a role as a positive selection marker for cells carrying exogenous DNA containing an expressible glucuronidase gene. The utility of &bgr;-glucuronidase as selective marker relies on the fact that cells cannot grow on a &bgr;-glucuronide carbon source such as a glucuronide disaccharide unless &bgr;-glucuronidase is provided to cleave the &bgr;-glucuronide bond. The most useful example of such a disaccharide is cellobiuronic acid, which comprises &bgr;-glucuronic acid in [1-4] linkage to glucose. Only cells expressing &bgr;-glucuronidase can grow on a carbon source consisting only of cellobiuronic acid.
Unlike the toxic agents commonly used in conjunction with negative selection markers in recombinant DNA techniques, cellobiuronic acid is non-toxic. Although methods related to the use of &bgr;-glucuronidase are well known, including methods for introducing &bgr;-glucuronidase genes into cells, and for assaying &bgr;-glucuronidase activity, see, e.g., U.S. Pat. No. 5,599,670 to Jefferson, there are unfortunately few economical methods for preparing cellobiuronic acid.
One known method of preparing cellobiuronic acid is by exposing cellulose to nitrous oxide. The exposure to nitrous oxide results in random oxidation of a portion of the glucose residues to glucuronic acid residues. Subsequent acid hydrolysis can be used to produce cellobiuronic acid, which may be purified from the reaction mixture. This method of preparing cellobiuronic acid is deficient for several reasons. The oxidation step involves the expense and difficulties inherent in performing a controlled oxidation in the presence of a toxic gas. Also, the physical and chemical properties of cellulose usually necessitate a pre-treatment step such as grinding, milling or steam explosion in order to allow optimal accessibility of the acid to the fibrous material during acid hydrolysis. In addition, because the oxidation step is not readily controllable, the cellobiuronic acid produced after oxidation and then hydrolysis is part of a hydrolysate mixture that may contain several byproducts such as oligo-glucuronic acids of various lengths, glucuronic acid, and some gluconic acids. These negatively charged byproducts have chemical properties similar to cellobiuronic acid, which makes purification of cellobiuronic acid by simple procedures such as anion-exchange chromatography or crystallization more difficult and expensive than if cellobiuronic acid were the only negatively charged reaction product.
The preparation of cellobiuronic acid from glucose via a synthetic oxidation approach is thus deficient for several reasons. Therefore, there is a need in the art to provide a simple method for the rapid and economical preparation of cellobiuronic acid. The present invention satisfies this need and provides other related advantages as disclosed further herein.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a method of preparing cellobiuronic acid. The method includes the steps of exposing polysaccharide to partially hydrolyzing conditions to produce a hydrolysate that includes saccharides, where the saccharides include cellobiuronic acid; and isolating the cellobiuronic acid. The polysaccharide is formed from oxidized and nonoxidized monosaccharide residues where oxidized monosaccharide residues are not adjacent to one another, i.e., directly bonded together. In the polysaccharide, the oxidized monosaccharide residues provide at least 10% of the total number of residues.
In another embodiment, the invention provides a method of hydrolyzing polysaccharide. The method includes the step of contacting polysaccharide with a hydrolyzing agent selected from acid, base and hydrolytic enzyme, under conditions that provide a hydrolysate that includes disaccharide and monosaccharide. The disaccharide includes cellobiuronic acid, and the cellobiuronic acid is present in the hydrolysate at a concentration of at least 5 wt. % based on the total weight of polysaccharide. Preferably, the polysaccharide has not previously been subjected to oxidizing conditions such as nitric oxide oxidation.
In another embodiment, the invention provides a method of hydrolyzing g(ellan. The inventive method includes the steps of contacting gellan with an aqueous composition having a ply between 2 and 7. under conditions effective to partially hydrolyze the gellan to cellobiuronic acid, and separating the cellobiuronic acid from water.
These and other embodiments of the present invention will become evident upon reference to the following detailed description and examples. In addition, various references as identified herein which describe in more detail certain procedures or compositions (e.g., gellans, etc.), are incorporated by reference in their entireties.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of preparing cellobiuronic acid from a polysaccharide. The method of the invention will be described after a brief description of cellobiuronic acid and the polysaccharides used to prepare the cellobiuronic acid.
Cellobiuronic acid is the name by which the disaccharide having the following structure (1) is commonly known:
In the literature, the disaccharide of structure (I) is sometimes referred to by other names, including cellobiouronic acid, 4-O-(&bgr;-D-glucopyranuronosyl)-D-glucose, and &bgr;-glucuronosyl[1-4]glucose). See, e.g., Carbohydrates, P. M. Collins, ed. Chapman and hall, page 117, 1987. Regardless of the name, as shown in structure (I), cellobiuronic acid is a disaccharide formed between &bgr;-glucopyranuronic acid in &bgr;-linkage to a D-glucose, where the &bgr;-linkage is through carbon number 1 of D-glucopyranuronic acid and carbon number 4 of glucose (as identified in the structure (I)). A &bgr; linkage from a glucuronic acid to another sugar moiety (as seen in cellobiuronic acid) is referred to herein as a &bgr;-glucuronide linkage.
For clarity, it is noted that D-glucopyranuronic acid is a member of the uronic acid family of sugars, and is commonly known by several other names including “the pyranose form of D-glucuronic acid.” Because the pyranose form of D-glucuronic acid is far more common than other forms, D-glucopyranuronic acid is often referred to simply as “D-glucuronic acid”. D-glucopyranuronic acid, and saccharides containing D-glucopyranuronic acid such as cellobiuronic acid, are referred to herein as “oxidized” saccharides because they contain a carboxylic acid or carboxylic ester substituent.
The polysaccharide useful in the present invention comprises cellobiuronic residues, where a cellobiuronic acid residue is shown in structure (II). In structure (II), the wavy lines indicate attachment to adjacent saccharide residues. The term “R” in structure (II) designates either a hydrogen or an alkyl group, so that the residue of structure (II) is either a carboxylic acid or an ester, respectively. A suitable alkyl group has 1-10 carbon atoms, where the carbons may be connected in a cyclic, acyclic, linear or branched fashion. Methyl is a suitable R group. OHOH
The polysaecharide useful in the invention contains a high proportion of cellobiuronic residues, and can generally be described as a high cellobiuronic acid-containing polysaecharide. More specifically, cellobiuronic residues will constitute at least 5% of the entire weight of the polysaccharide. The cellobiuronic residues will preferably constitute
Center for the Application of Molecular Biology to International
Foley & Lardner
Geist Gary
White Everett
LandOfFree
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