UDP-glucose: β-(1-3)-glucan (paramylon) synthase from Euglena gracilis
Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007.
The photosynthetic protist Euglena gracilis synthesizes a storage carbohydrate named paramylon, a glucan consisting only of β-(1-3)-glycosidic linkages. The enzyme that produces paramylon is a glycosyltransferase commonly known as paramylon synthase (EC 22.214.171.124; UDP-glucose: 1,3-β-D-glucan 3-β-D-glucosyl transferase). This enzyme uses UDP-glucose as its main substrate. In 2001, Bäumer et al. isolated and partially purified paramylon synthase, but never presented any sequence information. Hence, the main aim of this project was to isolate and characterize the gene(s) coding for the paramylon synthase. Different approaches were taken in order to isolate and characterize the gene(s). In the first part of the study molecular techniques were used to try and identify the gene. The two methods used were library screening and PCR amplification. Different libraries were screened using either functional staining or an affinity probe. The second method concentrated on the use of degenerate oligonucleotides, based on the amino acid sequences of conserved regions from known β-(1-3)-glucan synthase genes from various organisms, to PCR amplify the gene sequence from Euglena. These approaches were not successful in the isolation of the gene(s). In the second part of the study protein purification techniques were used in an attempt to obtain de novo protein sequence from the purified paramylon synthase enzyme. Several protein purification techniques were tried with the most successful being preparative ultra centrifugation followed either by sucrose density centrifugation or product entrapment (a type of affinity purification). These resulted in partial purification of the paramylon synthase protein. The partially purified proteins were separated using polyacrylamide gel electrophoresis, and the polypeptides able to bind the precursor, UDP-glucose, were identified using a radiolabeled isotope of UDP-glucose. These polypeptides were subjected to LC-MS-MS in order to obtain sequence information from them. One tryptic fragment showed high homology to β-(1,3)-glucan synthase genes from different yeasts.