Optimization of Conditions for the Higher Level Production of Protease: Characterization of Protease from Geobacillus SBS-4S

  • Waheed Ahmad
  • Muhammad TayyabEmail author
  • Muhammad Nauman Aftab
  • Abu Saeed Hashmi
  • Mansoorud Din Ahmad
  • Sehrish Firyal
  • Muhammad Wasim
  • Ali Raza Awan
Original Paper


Current study was planned keeping in mind the importance of proteases and their role in industry. In the present study protease was produced using Luria Bertani medium. The LB medium was supplemented with various carbon and nitrogen sources separately for the enhancement of growth of Geobacillus SBS-4S and for protease production. The optimization studies demonstrated the increase in protease production from 10.6 to 24.4 U/mL or 34.5 U/mL when the medium was supplemented with additional 2% yeast extract or 5% wheat bran respectively. Under the optimal conditions we could produce 46.2 U/mL of protease. The protein was purified by column chromatography and the purified protein was utilized for characterization studies. SDS-PAGE analysis confirmed the molecular weight of protease as 37 kDa. The enzyme exhibited its maximal activity at 60 °C and pH 9.0. Presence of Ca2+ and Mn2+ at a final concentration of I mM in the activity assay mixture enhanced protease activity from 100% to 133 and 150% respectively. Protease activity was slightly reduced (92%) in the presence of SDS whereas the presence of non-ionic detergents Triton X-100, Tween-20 and Tween-80 reduced the enzyme activity to 87, 82 and 69% respectively. Thermostability studies demonstrated that the protein was stable with 50% residual activity after an incubation of 2 h and 25 min at 60 °C in the presence of 1 mM Mn2+. The kinetic studies demonstrated the Km and Vmax values of 16.67 mg/mL and 143 U/mL respectively. The stability of protease at wide range of pH and temperature makes this enzyme suitable for its utilization in detergent and poultry feed industry.

Graphic Abstract


Protease Geobacillus SBS-4S Optimization Carbon sources Thermostability Chromatography Azo-casein 


Compliance with ethical standards

Conflict of interest

There is no conflict of interest from authors.


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© Springer Nature B.V. 2020

Authors and Affiliations

  • Waheed Ahmad
    • 1
  • Muhammad Tayyab
    • 1
    Email author
  • Muhammad Nauman Aftab
    • 2
  • Abu Saeed Hashmi
    • 1
  • Mansoorud Din Ahmad
    • 3
  • Sehrish Firyal
    • 1
  • Muhammad Wasim
    • 1
  • Ali Raza Awan
    • 1
  1. 1.Institute of Biochemistry and BiotechnologyUniversity of Veterinary and Animal SciencesLahorePakistan
  2. 2.Institute of Industrial BiotechnologyGovernment College UniversityLahorePakistan
  3. 3.Department of Epidemiology and Public HealthUniversity of Veterinary and Animal SciencesLahorePakistan

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