A recombinant subtilisin with keratinolytic and fibrin(ogen)olytic activity

• A recombinant subtilisin displaying keratinolytic and fibrin(ogen)olytic activity.
• A subtilisin E homologous keratinase posses fibrinogen(olytic) activity.
• The effect of mutations known as increasing thermal stability in subtilins seems to be neutralized by a His-tag tail.
• A His-tag tail at C-terminal improves thermal stability in a subtilisin.

KerS14 is a keratinase with great potential in tannery, since it degrades keratin without damaging collagen, a feature suitable for various industrial uses. The enzyme was previously characterized and described as a serino endopeptidase belonging to the subtilisin group. However, KerS14 low thermal stability impairs its biotechnological potential. The present work presents several attempts to improve KerS14 thermal stability. KerS14 ORF was cloned into pET-5a vector with a His-tag at C-terminal, and four different mutants enzymes (G61C, S98C, P239R and G61C-S98C) were produced by site-direct mutagenesis. The recombinant enzyme and four mutants were expressed, purified and characterized regarding their thermal stability, optimum temperature and pH. The presence of a His-tag was shown to increase the KerS14 thermal stability, and to decrease the thermal stability of mutant enzymes. In addition, the recombinant enzyme has a remarkable fibrin(ogen)olytic activity. This indicates that the enzyme has a potential for application in cardiovascular diseases, besides its use in tanning as a dehairing agent.