Characterization of Alkaline Protease from Thermoactinomyces sp.

Abstract

Thermoactinomyces sp. was isolated from fermented composed sample. The bacterial isolate was identified according to the cultural, microbiological and biochemical characteristics. The bacterial cells secreted the extracellular protease in alkaline agar medium that contained 1% Hammerstein casein and liquid culture. The enzyme was partially purified 40-fold from culture filtrate by sequential steps including salting out with Ammonium sulfate precipitation (80% saturation) subsequently ion exchange chromatography with DEAE-cellulose column and then CM- Cellulose column which increased the specific activity of the enzyme up to 1880 U/mg proteins with 72% recovery.
Characterization study of the partially purified enzyme was revealed that the enzyme had an optimum activity at pH of 10 and the activity was stable in the alkaline pH range from 8 to 12 for 30 min. Enzyme activity toward casein increased with increasing temperature and the maximal activity at 60 C. The enzyme was stable at temperature under 55˚C and approximately 75% of the activity abolished by incubation of the enzyme at 75  C for 30 min. Alkaline protease activity was completely inhibited by PMSF and DFP; while EDTA showed no inhibition on protease activity that is suggested that the enzyme is a serine protease.