Isolation of SU1, a starch debranching enzyme, the product...

Details Isolation of SU1, a starch debranching enzyme, the product... Isolation of SU1, a starch debranching enzyme, the product...

1999-02-24

2002-06-25

Smith, Lynette R. F. (Department: 1649)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024100, C435S468000, C435S419000, C800S295000, C800S298000

Reexamination Certificate

active

06410716

ABSTRACT:

BACKGROUND OF THE INVENTION
Starch is the major storage carbohydrate in higher plants. The biochemical mechanisms of starch biosynthesis are of interest for understanding fundamental aspects of plant physiology and also for their potential utility in manipulating plant growth for practical purposes. Not only is starch a critical primary source of dietary carbohydrates, but it is also used extensively for various industrial purposes ranging from formation of packaging materials to ethanol production. Despite its wide availability in nature and many industrial applications, the mechanisms by which starch is formed in plant endosperm tissue are not well understood.
Starch consists essentially of a mixture of the homopolysaccharides amylose and amylopectin. Amylose is a linear chain of glucosyl units joined by &agr;-(1→4) glycosidic bond and normally constitutes about 25% of the total endosperm starch in maize (
Zea mays
). Amylopectin comprises many linear chains of glucosyl monomers joined by &agr;-(1→4) linkages and constitutes approximately 75% of the starch. The chains of amylopectin are joined to each other by &agr;-(1→6) glycosidic bonds, often referred to as branch linkages.
Sugary1 (su1) is one of the oldest known mutations of maize and has been utilized as a sweet corn variety in North America since the 1700s. Phenotypically, immature mutant kernels with the su1 gene mutations accumulate sucrose and other simple sugars, including phytoglycogen (Black et al., 1966; Evensen and Boyer, 1986), which gives corn its desirable sweetness. Specific efforts to improve particular varieties of sweet corn date back to the middle of the nineteenth century. More recently, Sumner and Somers reported in 1944 that the principal polysaccharide storage product in su1 endosperm was a high molecular weight polysaccharide they called phytoglycogen. In 1958, Erlander proposed that phytoglycogen was a normal intermediate in the process of starch synthesis and that a debranching enzyme removed some of the branches by hydrolyzing the &agr;-1,6 branch points.
Phytoglycogen resembles amylopectin in the respect that &agr;-(1→4)-linked chains are joined by &agr;-(1→6) branch linkages, but the ratio of &agr;-(1→6) to &agr;-(1→4) linkages is significantly higher in phytoglycogen than it is in amylopectin (Manners, 1985). Although it has been suggested that the su1 gene codes for a starch debranching enzyme (Pan and Nelson, 1984), three different protein isoforms, each with a different level of glycosidase activity, were observed. It is not clear whether this observation was due to differential posttranslational modifications of the proteins, or whether the active enzyme is a multimer which requires combination with products of other gene loci. Further investigation into the mechanisms of starch biosynthesis in plants would be desirable.
BRIEF SUMMARY OF THE INVENTION
The invention is based on the discovery of a maize endosperm cDNA that encodes a novel starch debranching enzyme, termed SU1. The su1 gene produces a mRNA transcript of approximately 2.8 kb in kernels which includes a continuous open reading frame of 789 codons. The amino acid sequence deduced from the nucleotide sequence is significantly similar to that of bacterial isoamylases, enzymes that hydrolyze &agr;-(1→6) glycosidic bonds. Amino acid sequence similarity establishes SU1 as a member of the &agr;-amylase superfamily of starch hydrolytic enzymes, and indicates that SU1 is a starch debranching enzyme active in maize endosperm.
cDNA sequences encoding the SU1 protein or portions thereof can be incorporated into replicable expression vectors and the vectors transfected into an appropriate host (e.g., bacterial, yeast, eucaryotic cell culture). Alternatively, genomic DNA fragments encoding the SU1 protein can be utilized in situ. The expressed SU1 protein can be used as a replacement for the bacterial and fungal enzymes currently used in the starch processing industry. Also, the expressed products can be employed as immunogens in order to raise antibodies against SU1. Antibodies reactive with the SU1 protein show that SU1 is expressed in wild type maize endosperm tissue.
Thus, the invention generally features nucleic acid isolates encoding starch debranching enzyme, SU1, or portions thereof; the encoded SU1 protein or portions thereof; methods of producing SU1 or portions thereof; cells transformed with a recombinant vector containing the su1 gene; antibodies to the SU1 protein or fragment thereof and methods to produce such antibodies; transgenic plants containing the su1 gene, and methods to produce the transgenic plants.
The invention features a nucleic acid isolate able to hybridize under stringent conditions to the complement of a nucleic acid sequence encoding the SU1 protein, and the protein or polypeptide fragment, e.g., immunogenic fragment, thereof encoded by the nucleic acid isolate. The invention also features a recombinant expression vector comprising a nucleic acid isolate able to hybridize under stringent conditions to the complement of a sequence encoding the SU1 protein, and cells transformed with the recombinant expression vector, and methods of expressing the SU1 protein or polypeptide fragment encoded within the recombinant expression vector.
Also featured is a method of producing SU1 protein or polypeptide fragment thereof, comprising transforming a host cell with a nucleic acid able to hybridize under stringent conditions to a sequence encoding the SU1 protein having the amino acid sequence shown in FIG.
1
and linked to a nucleic acid sequence under the control of an inducible promotor, and inducing the inducible promotor to form a fusion protein comprising the SU1 protein.
The invention also features methods of producing antibodies to an SU1 fusion protein, and antibodies produced by such method. Also featured are transgenic plants containing the su1 gene, and methods of making the transgenic plants.
As used herein, the term “mutate” and “mutation” refers to a nucleic acid sequence that possess one or more base pair insertions, deletions, or changes. As used herein, the term “identify” is intended to include other activities that require identification of an entity, such as isolation or purification. The terms “isolated” or “purified” refer to a nucleic acid or protein sequence that has been separated or isolated from the environment in which it was prepared or in which it naturally occurs. Such nucleic acid or protein sequences may be in the form of chimeric hybrids or fusions, useful for combining the function of the nucleic acid or protein sequences of the invention with other species and also include recombinant forms. The term “determinant” as used herein includes the site on an antigen at which a given antibody molecule binds. The term “immunogenic fragment” refers to a fragment of SU1 protein that reacts with antibodies specific for a determinant of Su1.
The SU1 protein can be used as an alternative hydrolase, including bacterial and fungal starch hydrolases and debranching enzymes, that are utilized in industrial starch processing applications. Su1 cDNA, Su1 genomic DNA, or portions thereof may be utilized as markers for the identification of specific corn varieties, and for the development of corn varieties with starch properties tailored for specific industrial applications. Su1 cDNA or genomic DNA fragments, can be used to produce these proteins or peptide fragments or as probes to identify nucleic acid molecules encoding related proteins or polypeptides (e.g., homologous polypeptides from related species and heterologous molecules from the same species). Assays for SU1 function, production or expression by cells are made possible by the development of antibodies reactive with the SU1 protein.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.

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