Statistical optimization of production and tannery applications of a keratinolytic serine protease from Bacillus subtilis P13

Bacillus subtilis P13, an isolate from Vajreshwari hot springs near Mumbai, India, produces an extracellular serine protease that catalyses dehairing of hides as well as feather degradation. Present work reports the optimization of production medium at shake flask level, resulting in a 3.2 fold increase in enzyme activity with a concomitant reduction in submerged culturing time by 24 h. Soaking, dehairing and bating of hides were efficiently carried using enzyme from optimized medium. The growth pattern in the optimized medium was different from that in the unoptimized medium, showing entry into stationary phase 4 h earlier. Zymogram profiling of the extracellular proteases initially showed the appearance of a major ∼31 kDa protease, but at later growth stages additional proteases of ∼66, 43 and 20 kDa were prominent. By correlating the caseinolytic activity, feather degrading activity and zymogram patterns in different media combinations used for the statistical optimization, the 31 kDa protease could be identified to be responsible for feather disintegration. The purified 31 kDa protease is a PMSF-sensitive serine protease with keratinase and sulfitolysis activity and is proficient at feather degradation and hide depilation. Its N-terminal amino acid sequence is similar to that of subtilisins of other B. subtilis strains.