High-Yield Protease Production by a Gram-Negative Soil Bacterium Under Optimized Conditions

Abstract

Proteases are vital enzymes that catalyze the hydrolysis of peptide bonds and are extensively used in diverse industrial sectors including food, pharmaceuticals, leather, and detergents. This study aimed to isolate and characterize potent protease-producing bacteria from soil samples collected from Eco Park, Bhopal. A total of 11 bacterial isolates were screened for protease activity using skim milk agar, of which isolate 4 showed the highest zone of clearance (+++), indicating strong protease production. Morphological analysis revealed isolate 4 as a greyish, circular, raised colony with an entire margin. Gram staining indicated it was a Gram-negative rod-shaped bacterium. Biochemical characterization showed the isolate to be catalase, nitrate reduction, ONPG, indole, and glucose positive, aligning with selective metabolic traits favourable for protease synthesis.
Optimization studies revealed that isolate 4 exhibited maximum protease activity at pH 7 and 30°C, suggesting neutral and moderate thermal conditions support optimal enzyme production. Among the carbon sources tested, glucose significantly enhanced protease activity, followed by sucrose and starch. Similarly, among nitrogen sources, peptone supported the highest enzyme activity, with yeast extract and tryptone also showing favorable results. These findings underscore the potential of isolate 4 as a robust microbial candidate for industrial-scale protease production due to its high enzymatic yield under optimized culture conditions. Further molecular identification is recommended to determine its species and explore genetic pathways involved in enzyme regulation.