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Title: Isolation of disproportionating enzyme (EC from potato and investigation of its role in starch metabolism
Author: Takaha, Takeshi
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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D-enzyme, disproportionating enzyme, or 4-α-glucanotransferase (EC catalyses glucan transfer from one α-1,4-glucan molecule to another, or to glucose. From the wide distribution of D-enzyme in starch accumulating organs of plants, it is believed that D-enzyme is involved in starch metabolism, but the function in vivo is not known. In the present work, D-enzyme was purified from potato tubers, and cDNA and genomic DNA clones for potato D-enzyme were isolated. The biochemical analysis of purified recombinant D-enzyme suggested that high molecular weight starch (amylose and amylopectin) can serve as donor and acceptor, and very long α-1,4-glucans or even highly branched glucans can be transferred by the enzyme. It was also discovered that D-enzyme catalyses an intra-molecular transglycosylation (cyclisation) reaction on amylose and amylopectin, as well as the well studied inter-molecular transglycosylation (disporportionation) reaction. Analysis of D-enzyme gene expression was carried out by northern and western blot analysis, and in transgenic potato plants using the GUS-reporter gene fusion system. These results suggested that D-enzyme mRNA accumulates under circumstances when starch biosynthesis is most active but declines in amount under conditions when starch is broken down. These observations appear to contradict the widely held view that D-enzyme is involved in starch breakdown, and may suggest a function in starch synthesis. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the CaMV35S promoter, and various phenotypic changes were observed. These plants grew slower than wild type, produced less leaves, less mass of tubers and the apical meristems suffered necrosis. Furthermore, tubers from these plants sprouted later and the growth of sprouts was slower than wild type. However no significant difference was found in starch produced in tubers, either in its quantity or quality. From all these results and the available information about starch metabolism, possible roles of D-enzyme in starch metabolism are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available