Production, characterization, and immobilization of protease from the yeast Rhodotorula oryzicola.

The protease was produced extracellularly in submerged fermentation by the yeast Rhodotorula oryzicola using different sources of nitrogen and maximum activity (6.54 × 10-3 U/mg) was obtained in medium containing 2% casein (w/v). Purification of the protease by gel filtration chromatography resulted in a 3.07-fold increase of specific protease activity. The optimal pH and temperature for enzyme activity were 6.51 and 63.04 °C, respectively. Incubation in the presence of some salts enhanced enzyme activity, which peaked under 0.01 M BaCl2 . The enzyme retained about 90% of enzymatic activity at temperatures 50-60 °C. The commercially available enzyme carriers evaluated, silica gel, Celite 545, and chitosan effectively immobilized the protease. The enzyme immobilized in Celite 545 retained 73.53% of the initial activity after 15 reuse cycles. These results are quite promising for large-scale production and immobilization of protease from R. oryzicola, as the high operational stability of the immobilized enzyme lowers production costs in biotechnological applications that require high enzymatic activity and stability under high temperatures.

Characterization and immobilization of protease secreted by the fungus Moorella speciosa.

Protease was extracellularly produced in submerged fermentation by the fungus Moorella speciosa with maximum activity of 8.6 × 103 U/mL. The optimal pH and temperature for enzyme activity were 6.78 and 60.88 °C, respectively. The enzyme was incubated in the presence of several ions at concentrations of 0.1 M and 0.01 M to address the effect on enzyme activity. Enzyme activity was increased by 56% and 130% in the presence of 0.1 M BaCl2 and of 0.01 M Na2SO4, respectively. The V max and K m values were 0.01474 U/min/mg protein and 0.04190 mg/mL, respectively. The enzyme retained about 90% of enzymatic activity at 90 °C. Among the methods tested for enzyme immobilization, adsorption onto MAT540 carrier led to the most promising results, since after 15 reuse cycles up to 60% of the initial catalytic activity was retained. Entrapment in calcium alginate matrix allowed to retain up to 51% of the initial catalytic activity after 8 reuse cycles. This protease from M. speciosa, in either free or immobilized form, can be foreseen as a useful biocatalytic tool in process design by reducing operating costs, decreasing the use of chemical processing and, consequently, meeting the global demand for clean technologies.