Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-07-08
1999-06-01
Myers, Carla J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
536 2374, 536 2432, 435183, 435200, 435232, 530344, 530350, 530823, 935 11, 935 14, 935 68, C12N 1500, C12N 988, C07H 2104
Patent
active
059087609
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 same. 3401-3403 report on the production of 1,5-D-anhydrofructose ("AF") in Morchella vulganis by an apparent enzymatic reaction. The yield of production of AF is quite low. Despite a reference to a possible enzymatic 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. on the preparation of GL from red seaweed and its use to degrade .alpha.-1,4-glucan 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 culture of a fungus wherein the culture is substantially free of any other organism.
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 or derivatives thereof, preferably a cyclodextrin, more preferably beta-cyclodextrin.
According to the present invention there is also provided a GL enzyme prepared by the method of the present invention.
Preferably the fungus is Morchella costata or Morchella vulgaris.
Preferably the enzyme comprises the amino acid sequence SEQ. ID. No. 1 or SEQ. I.D. 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 an 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 enviroment (i.e. it 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 is made up of at least 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 in isolated form.
The present invention therefore relates to the isolation of the enzyme .alpha.-1,4-glucan lyase from a fungus. For ex
REFERENCES:
Yu, Shukun, et al.; Biochimica et Biophysuca Acta, 1156(1993) 313-320; a-1, 4-Glucan lyase, a new class of starch/glycogen degrading enzyme. I. Efficient purification and characterization from red seaweeds.
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Shukun Yu and Marianne Pedersen, Planta, "a-1,4, Glucan lyase, a new class of starch/glycogen-degrading enzyme," 191:137-143, (1993).
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Bojsen Kirsten
Christensen Tove
Kragh Karsten
Marcussen Jan
Yu Shukun
Danisco A/S
Myers Carla J.
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