Biosynthesis, Purification, and Comprehensive Characterization of a Novel Alkaline Protease from Pseudomonas aeruginosa Isolated from Hydrocarbon-Polluted Soils

Alkaline proteases are produced by a wide range of bacteria, making them highly versatile tools across various industrial and biotechnological processes. In this work, proteolytic Pseudomonas species were identified from soil samples obtained from an oil field. Following the screening of 25 isolates, P. aeruginosa 16 was identified based on its genetic and biochemical characteristics. A two-stage protocol was employed to purify the extracellular protease, starting with ammonium sulfate fractionated precipitation followed by chromatographic separation using an ÄKTA Pure 25 system. A relatively low molecular weight of 37 kDa was recorded for the enzyme, as the SDS-PAGE revealed. Purification of the protease enzyme was carried out 14.9-fold with a relative activity of 696.170 %. Protease activity was exposed to be optimal at an alkaline pH of 8, with an activity average of 599.77 U/ml, whereas the optimal temperature for protease activity was 35°C, which corresponded to 680.18 U/ml. To determine ion dependency, the protease was tested against several cations. At 1 mM concentration, the enzyme's activity remained entirely unaffected by the addition of Mg2+, Na+, Ca2+, or Zn2. However, at 5 mM, the minerals (Mg+2, Na+, Ca+2, and Zn+2) triggered the enzyme activity to decline by 10, 5, 5, and 8%, respectively.