Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-17
2003-10-21
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S210000, C435S252300, C435S320100, C536S023200, C536S102000, C530S300000, C530S350000
Reexamination Certificate
active
06635454
ABSTRACT:
The present invention relates to DNA molecules coding for proteins from plants having the enzymatic activity of a debranching enzyme (R enzyme). The invention furthermore relates to a process for modifying the branching degree of amylopectin synthesized in plants, and to plants and plant cells in which an amylopectin having a modified branching degree is synthesized due to the expression of an additional debranching enzyme activity or the inhibition of an endogenous debranching enzyme activity, as well as to the starch obtainable from said plant cells and plants.
Starch plays an important role both as storage substance in a variety of plants and as reproductive, commercially useful raw material and is gaining significance. For the industrial application of starch it is necessary that the starch meets the requirements of the manufacturing industry in terms of its structure, form and/or other physico-chemical parameters. For the starch to be useful in as many fields of application as possible it is furthermore necessary that it is obtainable in as many forms as possible.
While the polysaccharide starch is composed of chemically uniform components, the glucose molecules, it is a complex mixture of different molecule forms that exhibit differences as regards their polymerization degree and the presence of branches. One distinguishes the amylose starch, an essentially unbranched polymer of &agr;-1,4 glycosidically linked glucose molecules from the amylopectin starch, a branched polymer, the branches of which are the result of additional &agr;-1,6 glycosidic bonds.
In plants typically used for starch production, such as maize or potato, both starch forms are present in a ratio of about 25 parts of amylose to 75 parts of amylopectin. In addition to amylopectin, maize, for example, exhibits another branched polysaccharide, the so-called phytoglycogen which differs from the amylopectin by a higher branching degree and a differing solubility (see, e.g., Lee et al., Arch. Biochem. Biophys. 143 (1971), 365-374; Pan and Nelson, Plant Physiol. 74 (1984), 324-328). In the present application the term amylopectin is intended to comprise phytoglycogen.
With a view to the uniformity of the basic compound starch for its industrial application starch-producing plants are required that contain, e.g., either only the component amylopectin or only the component amylose. For other applications plants are required that synthesize forms of amylopectin of different degrees of branching.
Such plants can be generated, e.g., by breeding or mutagenesis techniques. It is known of certain plant species, e.g., maize, that mutagenesis can be used to generate varieties producing only amylopectin. For potato, a genotype was generated by chemical mutagenesis for a haploid line that does not produce amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221). Haploid lines, however, or the homozygous diploid or tetraploid lines derived thereof are not useful for agricultural purposes. Mutagenesis techniques, however, cannot be applied to the tetraploid lines that are interesting for agriculture since due to the presence of four different genotypes inactivation of all copies of a gene is not technically feasible. Therefore, in the case of potato, one must fall back on other techniques, e.g., the specific genetically engineered modification of plants.
For example it is known from Visser et al. (Mol. Gen. Genet. 225 (1991), 289) and WO 92/11376 that varieties can be generated by anti-sense inhibition of the gene for the starch granule-bound starch synthase in potato that synthesize substantially pure amylopectin starch.
WO 92/14827 discloses DNA sequences coding for a branching enzyme (Q enzyme) that introduces &agr;-1,6 branches into amylopectin starch. With these DNA sequences it should be possible to generate transgenic plants which exhibit a modified amylose/amylopectin ratio of the starch.
In order to furthermore specifically modify the branching degree of starch synthesized in plants by using genetic engineering it is still necessary to identify DNA sequences coding for enzymes that are involved in starch metabolism, particularly in the branching of starch molecules.
In addition to the Q enzymes that are capable of introducing branches into starch molecules plants comprise enzymes that are capable of dissolving branching. These enzymes are referred to as debranching enzymes and are divided into three groups in terms of their substrate specificity:
(a) Pullulanases that use amylopectin as substrate in addition to pullulan can be found in microorganisms such as Klebsiella and in plants. In plants these enzymes are often referred to as R enzymes.
(b) Isoamylases that do not use pullulan but glycogen and amylopectin as substrate can likewise be found in microorganisms and plants. Isoamylases have been described, e.g., for maize (Manners and Rowe, Carbohydr. Res. 9 (1969), 107) and potato (Ishizaki et al., Agric. Biol. Chem. 47 (1983), 771-779).
(c) Amylo-1,6-glucosidases have been described for mammals and yeasts and use limit dextrins as substrates.
Li et al. (Plant Physiol. 98 (1992), 1277-1284) succeeded in detecting in sugar beet only one debranching enzyme of the pullulanase type in addition to five endoamylases and two exoamylases. Having a size of about 100 kD and a pH optimum of 5.5, this enzyme is localized in the chloroplasts. For spinach, too, a debranching enzyme has been described that uses pullulan as substrate. Both the debranching enzyme of spinach and that of sugar beet possess an activity that is lower by a factor of 5 when reacting it with amylopectin as substrate instead of pullulan as substrate (Ludwig et al., Plant Physiol. 74 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284).
The activity of a debranching enzyme was examined by Hobson et al. (J. Chem. Soc. (1951), 1451) for potato which is a starch-storing cultivated plant, that is important from the agricultural point of view. They succeeded in proving that the corresponding enzyme—in contrast to the Q enzyme—does not possess chain-extending activity but merely hydrolyzes &agr;-1,6 glycosidic bonds. However, it has not been possible so far to characterize the enzyme in more detail.
For potato processes for the purification of the debranching enzyme as well as partial peptide sequences of the purified protein have been proposed (German patent application P 43 27 165.0 and PCT/EP94/026239). In principle, it should be possible to identify DNA molecules coding for the respective proteins by means of known peptide sequences when using degenerate oligonucleotide probes. However, in practice, often the problem arose that the degree of degeneration of the probe is too high or the probes are too short to specifically identify sequences coding for the desired protein.
Despite the knowledge of the proposed peptide sequences of the debranching enzyme of potato researchers so far have not been, able to isolate DNA molecules coding for debranching enzymes of plants by hybridization to degenerate oligonucleotides or by other genetic or immunological approaches such as proposed in German patent application P 43 27 165.0.
For spinach, too, for which the purification of the debranching enzyme has been described by Ludwig et al. (Plant Physiol. 74 (1984), 856-861), researchers have not been able to either determine peptide sequences or to identify DNA molecules coding for said protein.
The problem underlying the present invention is therefore to provide DNA molecules coding for proteins of plants having the enzymatic activity of a debranching enzyme and allowing to generate transgenic plant cells and plants having an increased or reduced activity of a debranching enzyme.
The problem is solved by the provision of the embodiments described in the claims.
The present invention therefore relates to DNA molecules coding for proteins of plants having the biological activity of a debranching enzyme, or a biologically active fragment thereof.
Such a DNA molecule preferably codes for a debranching enzyme of plants comprising the amino acid sequ
Emmermann Michael
Kossmann Jens
Renz Andreas
Virgin Ivar
Fish & Neave
Haley Jr. James F.
Kalinowski Grant
Saidha Tekchand
LandOfFree
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