Publications by Year: 2011

Four sampling sites were selected in the area of Velouhi mountain, Greece in order to screen for Pseudomonas syringae isolates with high ice nucleation activity from a ski resort environment. Bacterial isolates (n=147) were obtained from soil and phyllosphere samples. Seven isolates exhibited morphological, biochemical and physiological profile similar to P. syringae. Phylogenetic relationships of the seven isolates were determined by 16S rRNA gene sequencing. Two isolates were phylogenetically affiliated to P. syringae, three to P. viridiflava, one to P. avellanae, and one Pseudomonas strain could not be assigned to a known species. The seven isolates were examined for their ice-nucleation activity properties. Three out of the seven studied isolates exhibited ice nucleation activity from –4.67 to –4.35 ice nuclei per cell, values similar to those obtained from a known ice-nucleation protein producer P. syringae strain and therefore could be used for the production of artificial snow in ski resort areas with short snow periods.

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 Co2+. The apparent Km for Co2+ was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn2+, Mg2+, Ni2+ and Ca2+ but to a lesser extent. The following kinetic constants were determined: vmax, 0.74 μmol mgprotein−1 min−1; kcat, 44.2 min−1; Km(G1P), 0.10 mM;Km(G1,6diP), 1.03 μM; kcat/Km(G1P), 443 mM−1 min−1 and kcat/Km(G1,6diP), 42,860 mM−1 min−1. The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.

In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-β-1,4-xylanase 2 gene was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for further study due to their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5–10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translational level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants’ fermentation broths that were maintained 2–2.5-fold higher compared to the wild type.