a PRODUCTION OF PROTEASE FROM BACILLUS SUBTILIS UNDER SSF AND EFFECT OF ORGANIC SOLVENTS ON LYOPHILIZED PROTEASE PREPARATIONS

Abstract

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.