Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-29
2003-10-14
Monshipouri, Maryam (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S252300, C435S006120, C536S123130
Reexamination Certificate
active
06632643
ABSTRACT:
The present invention also relates to the use of an enzyme, in particular &agr;-1,4-glucan lyase (“GL”), to prepare 1,5-D-anhydrofructose (“AF”) from substrates based on &agr;-1,4-glucan.
The present invention also relates to the use of a sugar, in particular 1,5-D-anhydrofructose (“AF”), as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.
The present invention relates to the use of 1,5-D-anhydrofructose (“AF”) as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.
FR-A-2617502 and Baute et al in Phytochemistry [1988] vol. 27 No. 11 pp3401-3403 report on the production of AF in
Morchella vulgaris
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.
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 &agr;-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.
A typical &agr;-1,4-glucan based substrate is starch. Today, starches have found wide uses in industry mainly because they are cheap raw materials.
Starch degrading enzymes can be grouped into various categories. The starch hydrolases produce glucose or glucose-oligomers. A second group of starch degrading enzymes are phosphorylases that produce glucose-1-phosphate from starch in the presence of inorganic phosphate.
AF has also been chemically synthesised—see the work of Lichtenthaler in Tetrahedron Letters Vol 21 pp 1429-1432. However, this chemical synthesis involves a large number of steps and does not yield large quantities of AF.
The chemical synthetic route for producing AF is therefore very expensive.
There is therefore a need for a process that can prepare AF in a cheap and easy manner and also in a way that enables large quantities of AF to be made.
Furthermore, anti-oxidants are typically used to prevent oxygen having any deleterious effect on a substance such as a foodstuff. Two commonly used anti-oxidants are GRINDOX 142 and GRINDOX 1029. These anti-oxidants contain many components and are quite expensive to make.
There is therefore a need to have a simpler and cheaper form of anti-oxidant.
Furthermore, sweeteners are often used in the preparation of foodstuffs and beverages. However, many sweeteners are expensive and complex to prepare.
There is therefore a need to have a simpler and cheaper form of sweetener.
According to the present invention there is provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an &agr;-1,4-glucan with the enzyme &agr;-1,4-glucan lyase characterised in that enzyme is used in substantially pure form.
Preferably if the glucan contains links other than and in addition to the &agr;-1,4-links the &agr;-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other links—such as a hydrolase—preferably glucanohydrolase.
Preferably the glucan is starch or a starch fraction prepared chemically or enzymatically. If prepared enzymatically the reaction can be performed before the addition of the &agr;-1,4-glucan lyase or the reactions can be performed simultaneously. The suitable reagent can be an auxiliary enzyme. Preferred auxiliary enzymes are alpha- or beta-amylases. Preferably a debranching enzyme is used. More preferably the auxiliary enzyme is at least one of pullanase or isoamylase.
Preferably the &agr;-1,4-glucan lyase either is bound to a support or, more preferably, is in a dissolved form.
Preferably the enzyme is isolated from either a fungus, preferably
Morchella costata
or
Morchella vulgaris,
or from a fungally infected algae, preferably
Gracilariopsis lemaneiformis,
or from algae lone, preferably
Gracilariopsis lemaneiformis.
Preferably the enzyme is isolated and/or further purified from the fungus or from the fungally infected algae or algae alone using a gel that is not degraded by the enzyme.
Preferably the gel is based on dextrin or derivatives thereof.
Preferably the gel is a cyclodextrin—more preferably beta-cyclodextrin. Preferably the enzyme comprises the amino acid sequence SEQ. I.D. No. 1. or the amino acid sequence SEQ. I.D. No. 2 or the amino acid sequence SEQ. ID. No. 5 or the amino acid SEQ. I.D. No. 6, or any variant thereof.
In an alternative preferable embodiment, the enzyme comprises any one of the amino acid sequences shown in SEQ. I.D. No.s 9-11, 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.
Preferably the enzyme is used in combination with amylopectin or dextrin.
Preferably, the enzyme is obtained from the expression of a nucleotide sequence coding for 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 substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8.
In an alternative preferable embodiment, the DNA sequence comprises any one of the sequences that are the same as, or are complementary to, or have substantial homology with, or contain any suitable codon substitutions as shown as SEQ. ID. No.s 12-14.
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 having an increased lyase activity.
Preferably the starch is used in high concentration—such as up to about 25% solution.
Preferably the substrate is treated with the enzyme in the presence of a buffer.
More preferably the substrate is treated with the enzyme in the presence of substantially pure water.
Preferably the substrate is treated with the enzyme in the absence of a co-factor.
According to the present invention there is also provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an &agr;-1,4-glucan with the enzyme &agr;-1,4-glucan lyase characterised in that enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino acid sequence SEQ. ID. No. 6, or any one of the amino acid sequences SEQ. I.D. No.s 9-11, or any variant thereof.
According to the present invention there is also provided the sugar 1,5-D-anhydrofructose when prepared by the method of the present invention.
AF prepared by the present method was confirmed and characterised by
13
C NMR.
One of key advantages of the present method is that the sugar 1,5-D-anhydrofructose can be prepared in much larger quantities than before and by a method that is relatively easier and cheaper than the known processes. For example the sugar can now be prepared in amounts of for example greater than 100 g—such as 500 g—compared to the prior art methods when only much smaller amounts wer
Bojko Maja
Bojsen Kirsten
Christensen Tove
Kragh Karsten
Marcussen Jan
Danisco A/S
Monshipouri Maryam
Nixon & Vanderhye P.C.
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