Starch debranching enzymes

Details Starch debranching enzymes Starch debranching enzymes

2000-01-21

2002-10-22

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters carbohydrate production in the plant

C800S278000, C800S317200, C800S320000, C800S320100, C800S320200, C800S320300, C536S023600, C435S069100, C435S210000, C435S320100, C435S419000, C435S468000

Reexamination Certificate

active

06469230

ABSTRACT:

TECHNICAL FIELD
The present invention relates to enzymes having starch debranching activity. It further relates to nucleic acid encoding such enzymes, and methods of producing and using such enzymes and nucleic acid.
PRIOR ART
Starch is composed of highly branched (amylopectin), and lightly branched (amylose) glucan polymers arranged in a three-dimensional, semicrystalline structure, the starch granule. The degree of branching of amylopectin and the spatial organization of branches within the starch granule are very important in determining the physical properties of the starch and hence its value as a raw material for industry. The traditional view is that the branching pattern of amylopectin, and hence the way in which it is organised to form a granule, is determined by starch-branching enzymes which cleave short glucans from the non-reducing ends of chains and join them to residues within the same or an adjacent chain via &agr;(1-6) linkages to form branches. There is, however, increasing evidence that the branching pattern of amylopectin results from the combined actions of branching and debranching enzymes.
“Debranching enzymes” hydrolyse &agr;(1-6) glucosidic linkages in glucans. In plants, two quite different types have been described:
The “cullulanase” (EC 3.2.1.41) type is widely distributed in starch-degrading organs and in the chloroplasts of leaves. It is capable of the hydrolysis of the &agr;(1-6) linkages of pullulan, amylopectin and &agr;-limit dextrins, but usually cannot hydrolyse glycogen.
The second type of debranching enzyme, the “isoamylase” (EC 3.2.1.68) type, has been described only in potato tubers and maize endosperm, but this is probably because there is, at the moment, no specific assay for isoamylase activity in crude extracts (i.e. where other hydrolysing enzymes may be present). It can hydrolyse the &agr;(1-6) linkages of amylopectin, glycogen and &agr;-limit dextrins, but not pullulan.
Evidence that debranching enzymes may be involved in determining amylopectin structure comes from analysis of the sugary (su 1) mutant of maize (Pan and Nelson 1984, James et al. 1995), the sugary mutant of rice (Nakamura et al. 1996a) and the STA 7 mutant of Chlamydomonas (Mouille et al. 1996). All three mutations reduce or eliminate synthesis of conventional starch and cause the accumulation of a highly-branched, water-soluble glucan known as phytoglycogen. This change is accompanied by a reduction in the activity of debranching enzymes. In both maize and rice endosperm the activity of the pullulanase type of debranching enzyme is decreased, and in Chlamydomonas the activity of a debranching enzyme of unknown type disappears. In general terms, therefore, these phenotypes suggest that debranching enzyme is involved in determining the structure of amylopectin. However, understanding of the mutant phenotypes is far from complete.
Before the priority date of the present application, the sul locus from maize had been shown to encode a polypeptide which is very similar in amino-acid sequence to the bacterial isoamylase type of debranching enzyme, and not to pullulanases (James et al. 1995). Note, though, that the 5′ end of the sequence was not necessarily complete in this publication. No effect of the mutation on isoamylase activity in the endosperm was reported. The way in which the mutation brings about a decrease in pullulanase activity, and the relationship between this decrease and the accumulation of phytoglycogen were also not known.
After the priority date of the present application, nearly full-length maize SU1 was expressed in
E. coli
and purified. The recombinant enzyme was classified as an isoamylase (Rahman et al, 1998 Plant Physiol 117: 425-435).
Neither the rice nor the Chlamydomonas mutations have been fully characterised. In the former case, it has been established that the gene at the sugary locus does not encode the pullulanase that decreases in activity in the mutant endosperm (Nakamura et al. 1996b). In the latter case, the nature of the gene at the STA7 locus is not known.
The general effects of these mutations form the basis for a new model to explain the synthesis of amylopectin and its organisation to form a granule (Ball et al. 1996). Briefly, it is proposed that debranching enzyme acts to “trim” a highly-branched phytoglycogen-like structure synthesised at the periphery of the growing granule. This creates the branching pattern typical of amylopectin which, unlike the branching pattern of phytoglycogen, allows the polymer to pack in an organised manner to form the semi-crystalline matrix of the granule.
A critical assessment of the validity of this model is not yet possible, in part because of the lack of understanding of the mutations on which it is based, and in part because of the lack of information about debranching enzymes generally, and in starch-synthesising organs in particular. The nature, number and intracellular location of proteins with debranching activity is not known for any starch-synthesising organ, and sequences have been reported for only one plant isoamylase (the sul gene product) and a very few pullulanases. It is not known whether either isoamylase or pullulanase actually have the properties and specificities required by the Ball model.
Regardless of the validity of the Ball model, it seems highly likely that debranching enzymes play an important role in determining amylopectin structure, and hence in determining the physical properties of starch. The fact that the sul gene encodes an isoamylase suggests that this type of enzyme in particular may be involved. The decrease in pullulanase activity in the sul and sugary mutants also implicates this type of enzyme, and it has been reported (J. Kossmann and colleagues, MPI-MPP, Golm, Germany; verbal reports at open meetings) that modification of pullulanase activity in potato tubers brings about changes in the physical properties of the tuber starch.
Patent application WO 95/04826 [Kossmann et al] relates to a debranching enzyme obtained from potato. From the purification procedure used to obtain the amino acid sequence information it would appear that this relates to a single enzyme of the pullulanase type.
Patent application WO 95/03513 [Barry et al] relates to an isoamylase obtained from flavobacterium spp. The application does not disclose any corresponding enzymes or sequences from plants.
It can thus be seen that novel starch debranching enzymes, particularly those from plants, and particularly isoamylases, may provide a useful contribution to the art.
SUMMARY OF THE INVENTION
In a first aspect of the invention there is disclosed an isolated nucleic acid which comprises a nucleotide sequence which encodes a polypeptide which has the properties of an isoamylase, and is obtainable from
Solanum tuberosum.
Preferably the nucleic acid molecule has the sequence shown in any of Seq ID Nos 1 to 3 or is degeneratively equivalent or complementary thereto.
Seq ID Nos 1 to 3 (
FIGS. 1
to
3
) represent nucleotide sequences derived by the present inventors from cDNA clones (designated 21, 15 and 9 respectively) from potato tubers and minitubers. Clone 15 came from a minituber library; clone 9 from a tuber library and clone 21 was found in both types of library. Each of these clones encodes all or part of an independent novel starch debranching enzyme.
The amino acid sequences for clones 21, 15 and 9 are given as Seq ID Nos 4-6 (
FIGS. 4-6
) respectively
The original nucleotide sequences for clones 21, 15 and 9 which were determined initially by the inventors are given as Seq ID Nos 10-12 (
FIGS. 10-12
) respectively. Owing to very minor variations in the sequencing process these differ at a very few positions from the sequences above: however in the case of clones 21 and 15 there is in excess of 99.5% identity between new and old sequences. Clone 9 has also been extended at its 3′ terminus (still in excess of 99% identity). Corresponding amino acid sequences are at Seq ID Nos 13-15 (
FIGS. 13-15
) respectively.
TABLE 1
Similarity
Identity
sul
C

Affiliated with

Also associated with

Rating

Starch debranching enzymes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Starch debranching enzymes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Starch debranching enzymes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2919377