“Screening and Characterization of Alkaline Protease Producing Bacteria from Patagonia Arid Soils, Argentina”

OLIVERA N., SEQUEIROS C., AND BRECCIA J.D.

Bariloche, Río Negro, Argentina

Congreso; XXXIX Reunión Anual de SAIB y I Simposio de Proteinas; 2003

SAIB

A screening program for alkaliphilic bacteria producers of extracellular proteases was performed using samples from Patagonia arid soils. Isolate PAT 05, a gram-positive sporulating bacterium, was selected for proteolytic activity/stability properties. Its 16S rDNA gene sequence had the closest match (96 % homology) with that of Bacillus clausii. This isolate grew at pH between 7 and 10 with an optimum around pH 9-10, and with up to 20 % (w/v) NaCl at pH 10. The range of temperature for growth was from 5 to 40°C, with an optimum around 20°C. Optimal proteolytic activity was detected in the range of pH between 9-13 and the optimal temperature showed to be around 60°C. SDS-PAGE and casein zymogram analyses confirmed two protein active bands. Purification steps included supernatant protein precipitation with ammonium sulfate (70 %) and desalting by Sephadex G-25 column chromatography. According to the pI value of the active fraction (>9.3), the desalted eluate was partially purified by cation exchange chromatography (CM-Sepharose Fast Flow equilibrated with 20 mM Tris-HCl buffer, pH 8.0, containing NaCl).