Abstract
An isolation of extracellular lipases from the endemic fungal strain Geotrichum candidum-M2 was performed by vacuum evaporation, precipitation with cold acetone and vacuum drying. The lipases wereproduced by submerged fermentation on a medium containing sunflower oil waste in a concentration of 10 mL/L. The isolation of lipases was performed at the 48th h of fermentation when the process of cell autolysis has already been started. Almost 17 fold increase of the total enzyme activity and 2 fold increaseof the specific activity was achieved when concentrating the enzyme solution from 10.74 mg proteins per liter (in the supernatant) to 106.01 mg proteins per liter (in buffered solution of the previously precipitated enzyme). The crude enzyme preparation was further purified by an ion exchange chromatography using the Q-Sepharose FF resin and the hydrophobic interactive resin Phenyl-Sepharose Cl-4B. When purifying the enzyme preparation with the anion exchange resin Q-Sepharose FF, a lipolytic activity was detected only in the fractions representing the last two peaks with values of 0.27 and 0.20 U/mL. When those two collected fractions were further purified on the hydrophobic interactive resin Phenyl-Sepharose Cl-4B, an enzyme activity was detected only in the fractions representing the second peak, with a value of 0.22 U/mL. The factor of purification that characterized the whole purification procedure was 11.27. The purified enzyme preparation was stable in a broad temperature interval from 30-80 °C showing highest activity at 50 °C. At 80 °C, the enzyme preparation derived from solution with pH=9, showed activity of 0.15 U/mL. Those properties indicate that isolated lipases are especially suitable for usage in bioremediation processes and in detergent formulations.
