title: Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum.
abstract: The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.
authors: Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Zografou, Greece.
authors: Elisavet
authors: E
authors: Kourtoglou
authors: George E
authors: GE
authors: Anasontzis
authors: Diomi
authors: D
authors: Mamma
authors: Evangelos
authors: E
authors: Topakas
authors: Dimitris G
authors: DG
authors: Hatzinikolaou
authors: Paul
authors: P
authors: Christakopoulos
mesh_terms: D001709:Biotechnology; D002384:Catalysis; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010733:Phosphoglucomutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D013696:Temperature
chemical_list: D010733:Phosphoglucomutase