Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

• Two keratinase genes were isolated from Stenotrophomonas maltophilia by TAIL-PCR.
• Extracellular expression of keratinases in E. coli was succeeded by pelB leader.
• Two keratinases belong to serine protease with PPC domain.
• KerSMD has better thermostability, substrate specificity, and surfactant tolerance.
• The predicted model of KerSMD indicates the special function of PPC domain.

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.