• SELMIHAN SAHIN Suleyman Demirel University, Arts and Sciences Faculty, Department of Chemistry, Cunur, Isparta, 32260, Turkey
  • YASEMİN DEMIR Suleyman Demirel University, Arts and Sciences Faculty, Department of Chemistry, Cunur, Isparta, 32260, Turkey
  • ISMAIL OZMEN Suleyman Demirel University, Arts and Sciences Faculty, Department of Chemistry, Cunur, Isparta, 32260, Turkey


Objective: In the present work, protease was produced from Bacillus subtilis under solid-state fermentation (SSF). The effect of lyophilization with different additives on the activity of protease in an organic solvent and kineteci properties was investigated.

Methods: Production conditions of protease (fermentation time, moisture level, initial pH, temperature) were optimized. After production, it was partially purified and then, lyophilized with different additives from an aqueous buffer solution containing 98% (w/w) of different additives (pumice, KCl, without additive) for 72 h after freezing in liquid nitrogen. After that, the effect of organic solvents (2.5% and 5% of DCM, ethanol, hexane, toluene) on these lyophilized protease preparations was determined and their kinetic properties were determined.

Results: Optimum protease production was obtained with 40% of moisture level, at pH 7.5, 37 °C after 24 h fermentation. It was partially purified by using ammonium sulphate precipitation (20-80%) with 5.8-fold and specific activity of 38 U/mg and then dialysed with 6.4-fold and a specific activity of 35 U/mg. Co-lyophilization of protease with pumice and KCl was increased activity of an enzyme in aqueous organic solvents when compared lyophilized protease without additive. Used solvents, except DCM, were increased activity of lyophilized protease with pumice/KCl. It was found that the lyophilization with pumice and KCl resulted in an increasing in the catalytic efficiency, while it was decreased in Km and Vmax values.

Conclusion: The obtained findings demonstrated that protease from B. subtilis can effectively be produced under SSF by using wheat bran and used in industrial applications because of showing improved activity in an organic solvent by co-lyophilization with pumice/KCl.

Keywords: Bacillus Subtilis, Lyophilization, Organic Solvent, Pumice, Protease


Download data is not yet available.


1. Singh S, Bajaj BK. Bioprocess optimization for production of term alkali-stable protease from bacillus subtilis K-1 under solid-state fermentation. Prep Biochem Biotechnol 2016;46:717-24.
2. Contesini FJ, Melo RR, Sato HH. An overview of bacillus proteases: from production to application. Crit Rev Biotechnol 2018;38:321-34.
3. Gulmez C, Atakisi O, Dalginli KY, Atakisi E. A novel detergent additive: Organic solvent-and thermo-alkaline-stable recombinant subtilisin. Int J Biol Macromol 2018;108:436-43.
4. Ramakrishnan V, Thambidurai Y, Rajasekharan SK, Mohanvel SK. Partial characterization and cloning of protease from bacillus. Asian J Pharm Clin Res 2017;10:187.
5. Saba I, Hamid M, Ikram ulH. Production of alkaline protease by bacillus subtilis using solid-state fermentation. Afr J Microbiol Res 2013;7:1558-68.
6. Prakasham RS, Rao Ch S, Sarma PN. Green gram husk--an inexpensive substrate for alkaline protease production by bacillus sp. in solid-state fermentation. Bioresour Technol 2006;97:1449-54.
7. Mukherjee AK, Adhikari H, Rai SK. Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using imperata cylindrica grass and potato peel as a low-cost medium: characterization and application of enzyme in detergent formulation. Biochem Eng J 2008;39:353-61.
8. Pant G, Prakash A, Pavani JVP, Bera S, Deviram GVNS, Kumar A, et al. Production, optimization and partial purification of protease from Bacillus subtilis. J Taibah Univ Sci 2018;9:50-5.
9. Pandey A. Solid-state fermentation. Biochem Eng J 2003;13:81-4.
10. Gupta MN, Roy I. Enzymes in organic media. Forms, functions and applications. Eur J Biochem 2004;271:2575-83.
11. Zhang L, Li Y, Yuan Y, Jiang Y, Guo Y, Li M, Pu X. Molecular mechanism of carbon nanotube to activate subtilisin carlsberg in polar and non-polar organic media. Sci Rep 2016;6:36838.
12. Pfromm PH, Rezac ME, Würges K, Czermak P. Fumed silica activated subtilisin Carlsberg in hexane in a packed-bed reactor. AIChE J 2007;53:237-42.
13. Cruz JC, Pfromm PH, Rezac ME. Immobilization of candida antarctica lipase b on fumed silica. Process Biochem 2009;44:62-9.
14. Khmelnitsky YL, Welch SH, Clark DS, Dordick JS. Salts dramatically enhance the activity of enzymes suspended in organic solvents. J Am Chem Soc 1994;116:2647-8.
15. Ru MT, Hirokane SY, Lo AS, Dordick JS, Reimer JA, Clark DS. On the salt-induced activation of lyophilized enzymes in organic solvents: effect of salt kosmotropicity on enzyme activity. J Am Chem Soc 2000;122:1555-71.
16. Lindsay JP, Clark DS, Dordick JS. Combinatorial formulation of biocatalyst preparations for increased activity in organic solvents: salt activation of penicillin amidase. Biotechnol Bioeng 2004;85:553-60.
17. Khmelnitsky YL, Budde C, Arnold JM, Usyatinsky A, Clark DS, Dordick JS. Synthesis of water-soluble paclitaxel derivatives by enzymatic acylation. J Am Chem Soc 1997;119:11554-5.
18. Altreuter DH, S DJ, Clark DS. Nonaqueous biocatalytic synthesis of new cytotoxic doxorubicin derivatives: exploiting unexpected differences in the regioselectivity of salt-activated and solubilized subtilisin. J Am Chem Soc 2002;24:1871-6.
19. Lindsay JP, Clark DS, Dordick JS. Penicillin amidase is activated for use in nonaqueous media by lyophilizing in the presence of potassium chloride. Enzyme Microb Technol 2002;31:193–7.
20. Würges K, Pfromm PH, Rezac ME, Czermak P. Activation of subtilisin carlsberg in hexane by lyophilization in the presence of fumed silica. J Mol Catal B: Enzym 2005;34:18-24.
21. Sahin S, Ozmen I, Kir E. Purification, immobilization, and characterization of protease from local Bacillus subtilis M-11. Asia Pac J Chem Eng 2015;10:241-7.
22. Cupp Enyard C. Sigma's non-specific protease activity assay-casein as a substrate. J Vis Exp 2008;17:pii899.
23. Chutmanop J, Chuichulcherm S, Chisti Y, Srinophakun P. Protease production by aspergillus oryzae in solid-state fermentation using agroindustrial substrates. J Chem Technol Biotechnol 2008;83:1012-8.
24. Saminathan D, J SN. Optimization and production of alkaline protease from Bacillus subtilis IAS01 using agro-industrial by-product under SSF. Int Res J Biol Sci 2015;4:60-4.
25. Badgujar SB, Mahajan RT. Characterization of thermo-and detergent stable antigenic glycosylated cysteine protease of Euphorbia nivulia Buch.-Ham. and evaluation of its eco-friendly applications. Sci World J 2013;716545.
26. Hermanova S, Zarevucka M, Bousa D, Pumera M, Sofer Z. Graphene oxide immobilized enzymes show high thermal and solvent stability. Nanoscale 2015;7:5852-8.
27. Ru MT, Wu KC, Lindsay JP, Dordick JS, Reimer JA, Clark DS. Towards more active biocatalysts in organic media: increasing the activity of salt-activated enzymes. Biotechnol Bioeng 2001;75:187–96.
425 Views | 352 Downloads
How to Cite
SAHIN, S., Y. DEMIR, and I. OZMEN. “PRODUCTION OF PROTEASE FROM BACILLUS SUBTILIS UNDER SSF AND EFFECT OF ORGANIC SOLVENTS ON LYOPHILIZED PROTEASE PREPARATIONS”. International Journal of Chemistry Research, Vol. 4, no. 2, Mar. 2020, pp. 1-6, doi:10.22159/ijcr.2020v4i2.104.
Research Article