Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-08-22
2004-01-13
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S099000, C435S072000, C435S097000
Reexamination Certificate
active
06677142
ABSTRACT:
DESCRIPTION
The present invention relates to &agr;-1,4-glucan-chain-containing polysaccharides and to a process for their preparation.
Polysaccharides are polymers which are composed of numerous glycosidically bound monosaccharides. Polysaccharides occur both in higher organisms and in microorganisms such as bacteria and there fulfill, for example, the function of storage and framework substances. The polysaccharides are used commercially, inter alia, as aids and additives in the food industry, in light industry, in health care and in analysis.
Glucans are polysaccharides which solely consist of glucose monomers. In the &agr;-1,4-glucans, these glucose radicals are linked to one another by &agr;-1,4-glycosidic bonds. &agr;-1,4-Glucans, owing to their physicochemical properties, can be used to produce films which are color-free, odorless and tasteless, non-toxic and biodegradable. Already, there are numerous applications for such films, for example in the food industry, the textile industry and the glass fiber industry.
The most frequently occurring natural &agr;-1,4-glucan is amylose, a starch constituent. Amylose is already used to produce fibers whose properties resemble those of natural cellulose fibers and make possible their partial or complete replacement in paper making. In pharmacy, amylose is used as a filler for tablets, pastes and as additive to skin protection substances. In the food industry, it serves as thickener and binder for puddings, soups, sauces, mayonnaises, cream fillings and as a gelatin substitute. Amylose is also used as a binder in the production of sound-insulating wall panels.
Amylopectin, the main constituent of starch, and glycogen are further polysaccharides whose main chains consist of glucose radicals having &agr;-1,4-glycosidic linkage. These polysaccharides bear side chains which are linked to the main chain via &agr;-1,6-glycosidic bonds. These polysaccharides are also used to a great extent in industry.
The isolation of &agr;-1,4-glucans such as starch and glycogen from plant and animal organisms is complex and costly and does not always lead to products having reproducible properties. For this reason, bacteria which can produce such glucans have increasingly become the subject of attention.
In most bacteria, polysaccharides are synthesized in a similar manner to in higher organisms, via nucleotide-activated sugars. Thus, in most bacteria, the biosynthesis of glycogen involves three enzymes, that is to say ADP-glucose phosphorylase, which catalyzes the formation of ADP-glucose from glucose-1-phosphate and ATP, glycogen synthase which transfers the glucose from ADP-glucose to the growing glucan chain, and a branching enzyme which introduces &agr;-1,6-links into the linear &agr;-1,4-glucan chain. However, in some bacteria polysaccharide synthesis can also take place without the participation of activated sugars.
One of the bacterial systems which is able to synthesize polysaccharides without the participation of nucleotide sugars has been found in bacteria of the genus Neisseria. In these bacteria, polysaccharides having a similar structure to glycogen are synthesized by the enzyme amylosucrase directly from sucrose, the natural substrate of the enzyme [Okada, G., and E. J. Hehre, J. Biol. Chem. 249:126-135 (1974); MacKenzie, C. R. et al., Can. J. Microbiol. 23:1303-1307 (1977); MacKenzie, C. R. et al., Can. J. Microbiol. 24:357-362 (1978)].
Amylosucrase (sucrose: 1,4-&agr;-glucan 4-&agr;-glucosyltransferase, E. C. 2.4.1.4.) catalyzes the formation of &agr;-1,4-glycosidically linked glucans, by transferring the glucosyl radical of the sucrose molecule to the growing polymer chain, with the release of D-fructose, according to the following reaction equation
Sucrose+(&agr;-1,4-D-glucosyl)
n
→D-fructose+(&agr;-1,4-D-glucosyl)
n+1
.
Nucleotide-activated sugars or cofactors are not required in this reaction. However, the enzyme is stimulated by the presence of glucosyl group acceptors (or primers), for example oligo- and polysaccharides such as amylose or glycogen, to which the glucosyl radical of the sucrose is transferred according to the above reaction equation with &agr;-1,4-glucan chain extension [Okada, G., and E. J. Hehre, J. Biol. Chem. 249:126-135 (1974); Remaud-Simeon, M. et al., In S. B. Petersen, B. Svenson and S. Pedersen (editors), Carbohydrate bioengineering, pp. 313-320 (1995); Elsevier Science B. V., Amsterdam, Netherlands].
Amylosucrases have been found to date only in bacteria of the genus Neisseria. The enzyme which is expressed constitutively in the bacteria, is extremely stable and binds very firmly to its polymerization product. In most species investigated the enzyme is localized intracellularly, but in
Neisseria polysaccharea
, the amylosucrase is secreted. The gene for amylosucrase from
Neisseria polysaccharea
has in the interim been isolated and expressed using genetic engineering methods. It has been found that the enzyme highly probably only catalyzes the formation of linear &agr;-1,4-glucan chains (WO 95/31553).
The use of amylosucrase from
N. polysaccharea
for preparing linear &agr;-1,4-glucans has already been proposed in WO 95/31553. However, a problem in the use of amylosucrases for producing polysaccharides is that the polysaccharides usually formed in the presence of amylosucrase have highly variable molecular weights, i.e. a high polydispersity or broad molecular weight distribution. However, for an industrial application, because of their more homogeneous physicochemical properties, polysaccharide preparations having a molecular weight as uniform as possible, that is to say low polydispersity, are desired.
The object of the present invention was therefore to provide &agr;-1,4-glucan-chain-containing polysaccharides having a low polydispersity.
This object was achieved by the processes and polysaccharides described in the claims.
The present invention therefore relates to a process which comprises a glucosyl group acceptor being subjected to a chain extension reaction by reaction with sucrose in the presence of an amylosucrase, the amount of glucosyl group acceptor in the reaction mixture being chosen so that the molar ratio of glucosyl group acceptor ends available for chain extension to sucrose is at least 1:1 000 and/or the weight ratio of glucosyl group acceptor to sucrose is at least 1:50.
The invention also relates to a process which comprises subjecting a glucosyl group acceptor to a chain extension reaction by reaction with sucrose in the presence of an amylosucrase and with addition of fructose.
&agr;-1,4-Glucan-chain-containing polysaccharides which are available by these processes are also subject-matter of this invention.
The inventively used glucosyl group acceptors are compounds on which synthesis of &agr;-1,4-glucan chains, that is &agr;-1,4-glucan chain extension, can proceed under amylosucrase-catalyzed transfer of &agr;-D-glucosyl radicals originating from sucrose. Suitable glucosyl group acceptors are, in particular, short-chain and longer-chain oligo- and polysaccharides having terminal glucose radicals which are linked via &agr;-1,4-glycosidic bonds. Preferably, the inventively used glucosyl group acceptor is an unbranched, particularly preferably a branched, oligo- or polysaccharide. Examples of inventive glucosyl group acceptors are maltooligosaccharides such as maltopentaose, maltohexaose or maltoheptaose.
Preferred glucosyl group acceptors are dextrins, amylopectins, amyloses and amylose-like polysaccharides, for example from corn and potatoes, and glycogens and glycogen-like polysaccharides, for example from muscle tissue, mussels or bacteria.
Particularly preferred glucosyl group acceptors are branched polysaccharides such as glycogen. Such branched glucosyl group acceptors have more than one end at which chain extension can take place. Thus the glycogen chain bears approximately 7-12% of branches to which glucosyl radicals can be transferred.
Surprisingly, it has now been found that, in the amylosucrase-catalyzed synthesis of &agr;-1,4-g
Provart Nicholas
Quanz Martin
Weissmüller Max
Celanese Ventures GmbH
Marshall & Gerstein & Borun LLP
Prats Francisco
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