Proteases make up two-thirds of all enzymes used in various industries. This study aimed to optimize the production of alkaline protease enzyme under stress conditions in Bacillus cereus strain zk2 and Bacillus thuringiensis strain ILBB224 isolated from dairy sludge. Protease activity was measured quantitatively based on released tyrosine during casein hydrolysis. Response surface methodology was used for the optimization of enzyme production. Results revealed that the optimal enzyme production yield can be achieved at 5 C, 1 M KCl (the incubation medium is skimmed milk with 1 M KCl), and 20 kHz frequency of ultrasound for 1 min. Under optimal conditions, protease activities of zk2 and ILBB224 strains were 2.8 and 3.2 times higher, respectively, compared with the suboptimal conditions tested. Therefore, low temperatures, high concentrations of KCl, and short-time ultrasound stress appear to increase alkaline protease production by these strains.

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