Production and Characterization of Neutral and Alkaline Protease from Different Bacillus subtilis Strains

Abstract

An extracellular neutral and alkaline protease from two different Bacillus subtilis strains were studied. The optimal activity occured when the pH level was 7.0 and 10.5 at a temperature of 35 and 45 ºC for neutral protease and alkaline protease, respectively. When neutral protease was stable in the temperature range 35-60 ºC, alkaline protease was found stable between 35-55 ºC for 0.5 h. Divalent cations, especially Ca²⁺ increased enzymes activity and were inhibited by Mn²⁺,Ni²⁺, Cu²⁺, Fe²⁺, Co²⁺, Cd²⁺ and Hg²⁺ for neutral protease and by Zn²⁺, Cd²⁺,Co²⁺, Cu²⁺ and Hg²⁺ for alkaline protease. The neutral protease was also inhibited by ethylenediaminetetraacetic acid, 1,10 phenanthroline and dithiothreitol whereas alkaline protease was inhibited with phenylmethyl-sulfonyl fluoride. The obtained K_m values were 2.28 × 10^-3 ± 3.65 × 10^-4, 2.28 × 10^-4 ± 4.21 × 10^-5 and 1.70 × 10^-4 ± 5.18 × 10^-5 M for azocasein, BSA and casein for neutral protease, respectively. The K_m values with azocasein, BSA, casein, N-suc-Ala-Ala- Ala-pNA and N-cbz-Ala-Ala-Leu-pNA were 2.02 × 10^-3 ± 7.3 × 10^-5, 2.13 × 10^-4 ± 6.04 × 10^-5, 8.75 × 10^-4 ± 1.36 × 10^-4, 1.71 × 10^-3 ± 3.93 × 10^-4 and 2.64 × 10^-4 ± 4.93 × 10^-5 for alkaline protease.