Characterization of a thermostable protease from Bacillus subtilis BSP strain

Authors: MAJEED, TANVEER - Lahore College For Women University; Lee, Charles; Orts, William; ALI SHAH, TAWAF - University Of Beijing; BIN JARDAN, YOUSEF - King Saud University; DAWOUD, TURKI - Ibn Zohr University; BOURHIA, MOHAMMED - King Saud University

Submitted to: BMC Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2024
Publication Date: 7/15/2024
Citation: Majeed, T., Lee, C.C., Orts, W.J., Ali Shah, T., Bin Jardan, Y.A., Dawoud, T.M., Bourhia, M. 2024. Characterization of a thermostable protease from Bacillus subtilis BSP strain. BMC Biotechnology. 24. Article 49. https://doi.org/10.1186/s12896-024-00870-5.
DOI: https://doi.org/10.1186/s12896-024-00870-5

Interpretive Summary: A new and novel strain of thermophilic Bacillus subtilis BSP strain was recently identified that produces high levels of extracellular thermostable protease. Proteases are used in cheese production (milk coagulation), meat tenderizing, leather conditioning and in laundry detergents. Thermostable enzymes provide multiple advantages that expand their commercial appeal, such as improving their efficacy in laundry applicaitons.Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to maximize protease yields. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to by 30% to 295 U/mL. The protease was purified and characterized biochemically. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Technical Abstract: 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 confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.