CONICET | Buscador de Institutos y Recursos Humanos

Euglena gracilis is a fresh water protist with a large metabolic capacity because it is able to grow photosynthetically or heterotrophically. E. gracilis is a microorganism of interest in biotechnology and biomedicine due to its ability to generate bioproducts such as polysaccharides, polyunsaturated fatty acids, vitamins, wax esters and other metabolites. Paramylon is the main reserve polymer of E. gracilis. It is a water-insoluble β-1,3-glucan with a high degree of polymerization. There is little information about the enzymes involved in the metabolism of paramylon. Recently, the presence of a protein in E. gracilis belonging to the family 149 of glycosyl hydrolases (EgGH149) was reported. GH149 is a new family of "carbohydrate active enzyme" (CAZyme) and is thought to group glycosyl phosphorylase. To obtain information about the function of this enzyme, we produced it recombinantly in a soluble and active form. Using gel filtration chromatography we found that the quaternary structure of this protein is homodimeric, in agreement with previous reports for the same enzyme from other sources. We study the kinetics in both directions of reaction (and for several substrates). EgGH149 catalyzed the partition of a disaccharide of glucose with β-1,3 bond (laminaribiose or Lam2) with inorganic phosphate (Kcat of 9.1 s-1 and Km values of 1.57 mM for inorganic phosphate and 1.24 mM for Lam2). We observed that the enzyme had no activity when testing other types of disaccharides. EgGH149 efficiency decreases with increasing degree of polymerization when testing different laminarisaccharides without activity towards paramylon. EgGH149 was able to catalyze in the sense of synthesis using glucose and glucose-1-Phosphate (Kcat 1.32 s-1 and Km 1.81 for the glucose). Also, show activity towards Lam2 with lower affinity; without enzymatic activity detect for several free sugars and sugar-1-Phosphate tested. Consistent with enzymatic assays, we did not observe protein affinity to paramylon and laminarin when performing binding assays. We use the recombinant protein to obtain specific serum against EgGH149. Through western blot assays we show the presence of the protein in cells grown under autotrophic and heterotrophic conditions. In order to obtain information about its intracellular location, we performed a confocal microscopy assay: we observed signal recognition in the cytosol, forming hotspots near the paramylon granules regardless of the culture condition analyzed. This work provides information about the kinetic and structural behavior of this enzyme, as well as its distribution pattern in E. gracilis cells.