Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-06-25
2003-05-13
Wilson, James O. (Department: 1623)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S099000, C435S209000, C435S210000, C536S102000, C536S123120, C536S124000, C536S125000
Reexamination Certificate
active
06562600
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
This invention relates to a novel process for the production of cyclic alternan tetrasaccharide from alternanase using oligosaccharides as substrates.
2. Description of the Prior Art
Alternan, an extracellular D-glucan produced by
Leuconostoc mesenteroides
(Jeanes et al., 1954, J. Am. Chem. Soc., 76:5041-5052), and its lower molecular weight hydrolysis products have been previously described. Early studies of the alternan found that the compound was considerably resistant to microbial degradation and was also not attacked by enzymes that degrade starch, nigeran or pullulan (Cote, 1992, Carbohydrate Polymers, 19:249-252). The only enzymes that were reported to hydrolyze alternan to any significant extent were isomaltodextranases and alternanase. Of these, isomaltodextranases were not endo-hydrolases but rather exo-hydrolases or exo-dextranases. Two isomaltodextranases were examined for hydrolysis of alternan (referred to as B-1335 fraction S), the isomaltodextranases produced by
Arthrobacter globiformis
(Sawai et al., 1978, Carbohydrate Res., 66:195-205) and by an actinomycete Actinomadura (Sawai et al., 1981, Carbohydrate Res., 89:289-299). The authors concluded that the isomaltodextranases release mainly isomaltose units from the non-reducing ends of alternan chains that are terminated with an &agr;-1,6-linked D-glucopyranosyl residues. Later, studies with
A. globiformis
isomaltodextranase purified in this laboratory according to Okada et al. (1988, J. Biol. Chem., 5:495-501) confirmed that this enzyme also was not capable of endo-hydrolytic cleavage of alternan, but functioned in an exo-fashion.
Alternanase was described more recently by Cote et al. as an endo-&agr;-D-glucanase specific for alternan, having substantially greater activity toward alternan than dextran (U.S. Pat. Nos. 5,786,196, 5,888,776, and 5,889,179). This enzyme is produced and secreted extracellularly by a plurality of soil bacteria. Among the fractions present in the thinned alternan resulting from hydrolysis with alternanase are a previously unknown cyclic tetrasaccharide, cyclo{-6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1-} and derivatives thereof. This cyclic tetrasaccharide may be used as a metal salt complexing agent, a soluble, low- or non-caloric substitute for sucrose, and as bulking agents or extenders in foods and cosmetics.
SUMMARY OF THE INVENTION
I have now discovered that the cyclic tetrasaccharide, cyclo{-6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1-}, may be produced by alternanase hydrolysis of complex carbohydrates other than alternan. Surprisingly, panose, pullulan, &agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-D-Glc, and D-glucans having alternating &agr;-(1,6) and &agr;-(1,4) linkages, are not only hydrolyzed by alternanase, but the hydrolysis of these complex carbohydrates also produces the cyclic tetrasaccharide cyclo{-6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1-}. In this process, the cyclic tetrasaccharide is produced by contacting a solution of one or more of the above-mentioned complex carbohydrates with an amount of alternanase under conditions effective for activity of the enzyme. Furthermore, the substrate panose used in the reaction may be produced from a variety of polysaccharides or oligosaccharides, including starch, maltose, pullulan, and mixtures thereof.
In accordance with this discovery, it is an object of this invention to provide an improved, novel process for producing the cyclic tetrasaccharide cyclo{-6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1-}.
Another object of this invention is to provide a process for the production of the cyclic tetrasaccharide from common, readily available polysaccharides or oligosaccharides, particularly starch.
Other objects and advantages of this invention will become obvious from the ensuing description.
DETAILED DESCRIPTION OF THE INVENTION
The process of the invention produces the cyclic tetrasaccharide, cyclo{-6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-&agr;-D-Glcp-(1-}, which has the following structure (I):
This structure is intended to show linkage only, and no particular conformation is implied. As noted above, this cyclic tetrasaccharide, as well as the enzyme alternanase, were described in the previous patents of Cote et al. (U.S. Pat. Nos. 5,786,196, 5,888,776, and 5,889,179, the contents of each of which are incorporated by reference herein). In the processes disclosed in those prior patents, the cyclic tetrasaccharide was formed by hydrolysis of alternan, a polysaccharide of alternating glucose units linked in an alternating &agr;-1,3- and &agr;-1,6-fashion, with alternanase.
In contrast with the previously described process, I have surprisingly found that not only will alternanase hydrolyze polysaccharides other than alternan, but it will hydrolyze selected complex carbohydrates to produce the same cyclic tetrasaccharide. Starting complex carbohydrates suitable for use herein as substrates for production of the cyclic tetrasaccharide include panose, pullulan, &agr;-D-Glcp-(1,6)-&agr;-D-Glcp-(1,3)-D-Glc, D-glucans having alternating &agr;-(1,6) and &agr;-(1,4) linkages, and mixtures thereof, with use of panose being preferred. However, although pullulan may be used, reaction rates and yields of the cyclic tetrasaccharide are significantly lower than those obtained using panose. The activity of alternanase toward these substrates is particularly unexpected as these polysaccharides do not contain the alternating &agr;(1,3),&agr;(1,6)-D-glucosidic linkage sequences previously thought to be necessary for alternanase activity.
In accordance with the process of this invention, a catalytically effective amount of alternanase may be contacted with one or more of the above-mentioned selected polysaccharide or oligosaccharide substrates in an aqueous solution under conditions effective to hydrolyze the polysaccharide. Alternanase generally retains hydrolytic activity over pH and temperature ranges between about 4.5 to 9 and about 0° to at least 50° C., respectively, with optima at about pH 7 and about 40° C. for the enzyme from strain NRRL B-21195. At a pH of 7.0, enzyme activity decreases rapidly as the temperature is increased to 60° C. The presence of calcium ions in the reaction mixture is required for optimal activity. Addition of the calcium binding agent EDTA has been found to inhibit activity.
Yields of cyclic tetrasaccharide will vary considerably with the particular substrate utilized, with optimal yields being obtained from use of panose. The extent of reaction may be controlled by terminating the reaction at any time. A variety of techniques which are conventional in the art may be used to stop the reaction, including heating to denature the enzyme, addition of inhibitors (e.g. EDTA), or adjusting the pH.
Following completion of the reaction, the cyclic tetrasaccharide produced may used in crude form, although it is preferably recovered from the reaction mixture in pure or substantially pure form. The particular technique for separation is not critical, and a variety of techniques are suitable for use herein. With the exception of the cyclic tetrasaccharide, the reaction products are all reducing sugars and thus may be removed using conventional ion-exchange resins. In the preferred embodiment, the cyclic tetrasaccharide may be selectively isolated from the other components of the reaction mixture by a single pass through a basic ion-exchange resin, which will bind the reducing sugars such as panose and maltose, but will allow the non-reducing cyclic tetrasaccharide to pass therethrough. Strong anionic exchange resins are particularly preferred for use herein, including, for example, AMBERLITE IRA-400 (Rohm & Haas, Philadelphia, Pa.) and DOWEX AG 1X (Dow Chemical, Midland, Mich.). Alternatively, the cyclic tetrasaccharide may be separated from the reaction mixture by chromatograph
Deck Randall E.
Fado John D.
Maier Leigh C.
The United States of America as represented by the Secretary of
Wilson James O.
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