T Santos

Purification and partial characterization of a developmentally regulated 1, 3-β-D-glucanase from Penicillium italicum.

Summary: 1,3-β-Glucanase I is one of the three 1,3-β-glucanase enzymes which are developmentally regulated in Penicillium italicum. On incubation of the fungus in derepressing medium (limited in glucose), 1,3-β-glucanases II and III are produced first, followed by 1,3-β-glucanase I. We have purified the latter enzyme 290-fold from cell-free extracts of derepressed mycelium. The purification involved Sephadex G-100 chromatography, adsorption to DEAE-Sephadex, concanavalin A-Sepharose and preparative isoelectric focusing. The purified enzyme appeared homogeneous on SDS-PAGE. The apparent molecular weights estimated by Sephadex G-100 filtration and SDS-PAGE were 65000 and 68000 respectively. The enzyme appears to be an acidic glycoprotein (pI 4.7) with an endo-splitting mode of action, clearly different from 1,3-β-glucanases II and III. The Km on a standard, slightly branched 1,3-β-glucan (laminarin) was 0.04 mg ml-1, at least ten times lower than the average Km for β-glucanases. It showed almost no activity on heavily branched substrates (yeast wall glucan). An evaluation of the physicochemical properties and other characteristics suggests a metabolic role for this endo-1,3-β-glucanase.

5-Azacytidine Induces Heritable Biochemical and Developmental Changes in the Fungus Aspergillus niger

SUMMARY: Transient exposure of mycelia from Aspergillus niger to the cytidine analogue 5-azacytidine, at concentrations which do not affect the growth rate of the fungus on nearly minimal media, result in a dose-dependent, heritable change in the timing of the conidiation programme as well as heritable over-production of adaptive enzymes (glycosidases and phosphatases) and modification in the control properties of acid phosphatase. These heritable changes are induced by 5-azacytidine in a non-random way since the new phenotypes are exhibited not only by isolated clones but also by mixed populations of mycelia several life cycles (thousands of mitoses) after exposure to the drug.

Purification and partial characterization of a new, sporulation specific, exo-β-glucanase from Saccharomyces cerevisiae

Abstract A new exo-β-glucanase, sporulation-specific, was purified from sporulating S. cerevisiae ( AP 1 , a α ). Characterization of this new activity shows that the enzyme is a glycoprotein with substrate specificities, kinetic parameters and aminoacid composition clearly different from those of its vegetative counterpart.

Characterization and synthesis regulation of Penicillium italicum 1,6-β-glucanase

The filamentous fungus Penicillium italicum when grown in a synthetic medium, produced and secreted 1,6-β-glucanase into the culture medium. This enzyme has been partially purified by gel filtration. After this step the active fractions were free of 1,3-β-glucanase, α-amylase and β-glucosidase activities. Only four proteins, one associated with the enzyme, were found by polyacrylamide gel electrophoresis under non denaturing conditions. The enzyme behaves as an acidic protein (pI 4.65) with an optimum pH of 5 and an endohydrolytic mode of action. The activity was lost at pHs greater than 7. The enzyme was also found associated with the mycelium. Its synthesis was repressed by glucose or growth-promoting sugars. Derepression in low glucose containing medium required protein synthesis. 8-Hydroxyquinoline, an RNA synthesis inhibitor, added during the derepression period did permit some increase in the specific activity but prevented it when added at the beginning of that period.