Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp keratinase KerSMD by domain exchange with KerSMF

In this study, we enhanced the catalytic efficiency and thermostability of keratinase KerSMD by replacing its N/C-terminal domains with those from a homologous protease, KerSMF, to degrade feather waste. Replacement of the N-terminal domain generated a mutant protein with more than twofold increased catalytic activity towards casein. Replacement of the C-terminal domain obviously improved keratinolytic activity and increased the k(cat)/K-m value on a synthetic peptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, by 54.5%. Replacement of both the N-and C-terminal domains generated a more stable mutant protein, with a T-m value of 64.60 +/- 0.65 degrees C and a half-life of 244.6 +/- 2 min at 60 degrees C, while deletion of the C-terminal domain from KerSMD or KerSMF resulted in mutant proteins exhibiting high activity under mesophilic conditions. These findings indicate that the prepeptidase C-terminal domain and N-propeptide are not only important for substrate specificity, correct folding and thermostability but also support the ability of the enzyme to convert feather waste into feed additives.