Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Patent
1996-07-02
2000-01-11
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
435105, 4352523, 43525233, 4352572, 536 231, 536 232, 536 2374, 424 945, C12N 988
Patent
active
060135042
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an enzyme, in particular .alpha.-1,4-glucan lyase ("GL"). The present invention also relates to a method of extracting the same. The present invention also relates to nucleotide sequence(s) encoding for the same.
FR-A-2617502 and Baute et al in Phytochemistry [1988] vol. 27 No. 11 pp3401-3403 report on the production of 1,5-D-anhydrofructose ("AF") in Morchella vulgaris by an apparent enzymatic reaction. The yield of production of AF is quite low. Despite a reference to a possible enymatic reaction, neither of these two documents presents any amino acid sequence data for any enzyme, let alone any nucleotide sequence information. These documents say that AF can be a precursor for the preparation of the antibiotic pyrone microthecin.
Yu et al in Biochimica et Biophysica Acta [1993] vol 1156 pp313-320 report on the preparation of GL from red seaweed and its use to degrade .alpha.-1,4glucan to produce AF. The yield of production of AF is quite low. Despite a reference to the enzyme GL this document does not present any amino acid sequence data for that enzyme let alone any nucleotide sequence information coding for the same. This document also suggests that the source of GL is just algal.
According to the present invention there is provided a method of preparing the enzyme .alpha.-1,4-glucan lyase comprising isolating the enzyme from a fungally infected algae.
Preferably the enzyme is isolated and/or further purified using a gel that is not degraded by the enzyme.
Preferably the gel is based on dextrin, preferably beta-cyclodextrin, or derivatives thereof, preferably a cyclodextrin, more preferably beta-cyclo-dextrin.
According to the present invention there is also provided a GL enzyme prepared by the method of the present invention.
Preferably the enzyme comprises the amino acid sequence SEQ. ID. No. 1. or SEQ. ID. No. 2, or any variant thereof.
The term "any variant thereof" means any substitution of, variation of, modification of, replacement of, deletion of or addition of at least one amino acid from or to the sequence providing the resultant enzyme has lyase activity.
According to the present invention there is also provided a nucleotide sequence coding for the enzyme .alpha.-1,4-glucan lyase, preferably wherein the sequence is not in its natural environment (i.e. does not form part of the natural genome of a cellular organism expressing the enzyme).
Preferably the nucleotide sequence is a DNA sequence.
Preferably the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitution(s) for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4.
The expression "substantial homology" covers homology with respect to structure and/or nucleotide components and/or biological activity.
The expression "contains any suitable codon substitutions" covers any codon replacement or substitution with another codon coding for the same amino acid or any addition or removal thereof providing the resultant enzyme has lyase activity.
In other words, the present invention also covers a modified DNA sequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having the activity of a glucan lyase, preferably an enzyme having an increased lyase activity.
According to the present invention there is also provided a method of preparing the enzyme .alpha.-1,4-glucan lyase comprising expressing the nucleotide sequence of the present invention.
According to the present invention there is also provided the use of beta-cyclodextrin to purify an enzyme, preferably GL.
According to the present invention there is also provided a nucleotide sequence wherein the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4, preferably wherein the sequence is i
REFERENCES:
Ujhazi et al. Proc. of the fourth international symposium on cyclodextrin, Huber et al. (eds), pp. 497-501, 1988.
Baute et al. Bull. Soc. Pharm. Bordeaux. 128, 1-4, 9-18 English abstract, 1989.
Sanger, F., et al. "DNA sequencing with chain-terminating inhibitors" Proc. Natl. Acad. Sci. USA vol. 74, No. 12, pp. 5463-5467, Dec. 1977.
Baxten, F. P., et al. "Transformation of Aspergillus niger using the argb gene of Aspergillus nidulans" Gene vol. 37, pp. 207-214, 1985.
Collinge, D. B., et al. "Gene expression in Brassica campestris showing a hypersensitive . . . " Plant Molecular Biology vol. 8, pp. 405-414, 1987.
Frohman, M. A., et al. "Rapid production of full-length cDNAs from . . . " Proc. Natl. Acad. Sci. USA vol. 85, pp. 8998-9002, Dec. 1988.
Langdale, J.A., et al. "Cellular pattern of photosynthetic gene expression in developing maize . . . " Genes and Development vol. 2, pp. 106-115, 1988.
Pueschel, C. M., "An expanded survey of the ultrastructure of red algal pit plugs" J. Phycol vol. 25, pp. 625-636, 1989.
Punt, P. J., et al., "Intracellular and extracellular production of proteins in . . . " Journal of Biotechnology vol. 17, pp. 19-34, 1991.
Punt, P. J., et al., Transformation of Filamentous Fungi Based on Hygromycin . . . Methods in Enzymology vol. 216, pp. 447-457, 1992.
Archer, D. B., et al. "Proteolytic degradation of heterologous proteins expressed in aspergillus niger" Biotechnology Letters vol. 14, pp. 357-362.
Saunders, G. W., "Gel purification of red algal genomic DNA: An inexpensive and rapid method . . . " J. Phycol. vol. 29, pp. 251-254, 1993.
LeGendre, N., et al. "Purification of proteins . . . " A practical guide to protein and peptide purification for microsequencing 2nd Edit. pp. 74-101.
Pall, M. L., et al. "A series of six compact fungal transformations vectors containing polylinkers . . . " Fungal Genet Newslett vol. 40, pp. 59-62.
Yu, et al., BA Biochimica et Biohysica Acta, Elsevier Science, "a-1,4-Glucan lyase, a new class of starch/glycogen degrading enzyme," 1156:313-320, (1993).
Shukun Yu and Marianne Pedersen, Planta, "a-1,4, Glycan lyase, a new class of starch/glycogen-degrading enzyme," 191:137-143, (1993).
Plant Physiology, Biochemistry and Biophysics, Bio. Abstr. 57: AB-926, No. 52735, referencing Baute, et al., Phytochemistry, 27:3401-3403 (1988).
Baute, et al., Phytochemistry, "Fungal Enzymic Activiity Degrading 1,4-a-D-Glucans to 1,5-D-Anhydrofructose," 27:3401-3403 (1988).
Bojko Maja
Bojsen Kirsten
Kragh Karsten
Marcussen Jan
Nielsen John
Danisco A/S
Nashed Nashaat T.
Wax Robert A.
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