Isolation of Protease-Producing Bacteria and Optimization of Protease Production Using Box-Behnken Design

Abstract

Proteases are important hydrolytic enzymes with broad applications in pharmaceutical, medical, food, detergent, leather, and other industrial processes. The present study focused on the isolation, screening, and optimization of protease-producing bacterial strains for enhanced enzyme production. Soil samples were collected and screened for proteolytic activity using gelatin agar as a selective screening medium. Among the bacterial isolates obtained, three promising strains, designated DK1, DK2, and DK3, demonstrated significant proteolytic activity based on clear-zone formation. Protease production was further evaluated under different cultivation conditions to identify factors influencing enzyme yield. Optimization studies were performed by investigating key parameters, including pH, temperature, and incubation time. A Box–Behnken experimental design was applied to evaluate the combined effects of these variables and determine conditions that favored maximum protease production. The optimized conditions resulted in improved enzyme production compared with the initial cultivation conditions. The findings demonstrate the potential of the selected bacterial isolates as sources of extracellular protease and provide useful information for optimizing microbial enzyme production. The study may contribute to the development of efficient and economical approaches for producing proteases with potential applications in pharmaceutical and other industrial processes.