biostudies-literatureUnknown24(1)Majeed TThis study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca²⁺ and Fe²⁺ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confBMC biotechnology49https://www.ebi.ac.uk/biostudies/studies/S-EPMC11247832biostudies-literatureCharacterization of a thermostable protease from Bacillus subtilis BSP strain.PMC11247832Bourhia MJardan YABShah TAMajeed TDawoud TMLee CCTabassum ROrts WJfalseCharacterization of a thermostable protease from Bacillus subtilis BSP strain.This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca²⁺ and Fe²⁺ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was conf2024-01-01T00:00:00Z2024 Jul2026-06-01T19:33:32.269Z2025-04-06T11:29:49.053ZS-EPMC112478323901000410.1186/s12896-024-00870-5