Glucan lyase producing 1,5-anhydrofructose

Details Glucan lyase producing 1,5-anhydrofructose Glucan lyase producing 1,5-anhydrofructose

1995-04-18

1997-12-09

Wax, Robert A.

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

435232, 43525233, 4352523, 4352572, 536 232, C12P 1902, C12N 988, C12N 120

Patent

active

056959702

DESCRIPTION:

BRIEF SUMMARY
This is the national stage application of PCT/SE93/00854, filed Oct. 19, 1993.
The present invention relates to a new enzyme and applications thereof. More precisely, it relates to an enzyme, exo-.alpha.-1,4-glucan lyase, which is capable of successively cleaving the terminal .alpha.-1,4-D-glucosidic bonds from the non-reducing ends of an .alpha.-1,4-glucan and a method of producing the same. The main degradation product is 1,5-anhydrofructose. It has now been shown that 1,5-anhydrofructose is useful as an oxygen radical scavenger or an anti-oxidant and as a substitute for other sugars and their derivatives.


BACKGROUND

It is well known that the degradation of .alpha.-glucans (maltosaccharides, starches and glycogen) is catalyzed by two groups of enzymes, hydrolases and phosphorylases, yielding the degradation products glucose and glucose 1-phosphate. In studying metabolites in algae a new enzyme that can degrade .alpha.-glucans was suprisingly found. Research efforts resulted in characterization of the enzyme and the finding that the enzyme degrades starch yielding one major product which was analyzed to be 1,5-anhydrofructose.
The compound 1,5-anhydrofructose (AF) was first synthesized chemically by Lichtenthaler F. W., et al in 1980 (Tetrahedron Letters Vol. 21, pp. 1429-1432). It was proposed to be useful in the synthesis of other 1,5-anhydroketoses. Baute M-A., et al (Phytochemistry, Vol. 27, No. 11, pp. 3401-3403, 1988) reported that a crude preparation of an enzyme activity from fungi could degrade starch to AF. Their pure AF could be prepared from starch in 40-50 % yield by removing glucose with baker yeast and maltose by gel filtration. The enzyme was not purified and the authors disclosed that the fungi that had been tested possesses an enzyme (or an enzyme system), and they disclose in the discussion part that the presence of glucose and maltose among the products of the reaction might result either from minor activity of the enzyme or from its contamination by one or several conventional amylolytic enzymes. Further, Baute, M-A. et al (Phytochemistry 1987, 26 (5), 1391-3 and 1991, 30 (5), 1419-25, respectively) disclosed that their enzymic activity degrades 1,4-.alpha.-D-glucans to 1,5-D-anhydrofructose and then converts this sugar to the antibiotics microthecin and pentenomycin.
There is no disclosure in the prior art that 1,5-anhydrofructose would be an oxygen radical scavenger or anti-oxidant and as a substitute for other sugars and their derivatives.


DESCRIPTION OF THE INVENTION

The new enzyme, exo-.alpha.-1,4-glucan lyase, can be obtained from algae, specifically red algae of the order of Gigartinales. Such an enzyme, having a molecular weight of approximately 54 000 Da, has been obtained from Phyllophora truncata. However, the experiments disclosed in this specification was conducted with such an enzyme which was isolated from the two red seaweeds Gracilariopsis lemaneiformis and Gracilaria verrucosa. The reaction catalyzed is cleavage of the terminal .alpha.-1,4-D-glucosidic bonds of an .alpha.-1,4-glucan successively from the non-reducing ends of the chains with the formation of 1,5-anhydrofructose.
1. When a linear .alpha.-1,4-glucan, such as maltose, maltosaccharides and amylose, is used as a substrate: yield of glucose (%)=1
.times.100. n indicates the polymerization number of this .alpha.-glucan. For example, if the glucan has 100 glucose units, the yield of AF is 99%, while the yield of glucose is 1%.
2. When a branched glucan (.alpha.-1,4-glucan with .alpha.-1,6-branches), such as amylopectin and glycogen, is used as a substrate
The products are AF and a neo-limit dextrin. The enzyme will release AF from the non-reducing ends and the degradation stops when a .alpha.-1,6-linked glucose unit is met. Therefore the yield of AF depends on the chain length with non-reducing ends in the whole .alpha.-glucan molecule.
The proposed naming of this enzyme is: Exo-.alpha.-1,4-glucan lyase; .alpha.-1,4-glucan 1,5-anhydrofructose eliminase; .alpha.-1,4-glucan exolyase; systematic name: .al

REFERENCES:
Yu et al. "alpha-1,4 Glucan lyase, a new class . . . I. Efficient purification and characterization from red seaweeds" Biochimica et biophysica Acta 1156, 313-320, Mar. 1993.
Yu et al. "alpha-1,4 Glucan lyase, a new class, . . . II. Subcellular localization and partial amino-acid sequence." Planta 191, 137-142, 1993.
Baute et al. "Fungal bioconversions yielding unusual antibiotic pyrones from carbohydrates. XV-Biogenesis . . . " Bull. Soc. Pharm. Bordeaux 128, 9-18, 1989.
Phytochemistry, vol. 27, No. 11, 1988, M-A Baute et al, "Fungal enzymatic activity degrading 1,4-alfa-D-glucan to 1,5-D-anhydrofructose" pp. 3401-3403.
Dialog Information Services, file 154, Medline, Dialog acc.No. 05922849, Nakamura T. et al: "Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes", J. Biochem (Tokyo) Mar. 1986, 99 (3) pp. 607-613.
Phytochemistry, vol. 30, No. 5, 1991, M-A Baute et al, "Fungal enzymatic activity degrading 1, 4-alfa-D-glucans to echinosporin (5-epipentenomycin I)" pp. 1419-1423.

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