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

• Published in: Process biochemistry (1991) 2014-04, Vol.49 (4), p.647-654
• Main Authors: Fang, Zhen, Zhang, Juan, Liu, Baihong, Jiang, Linghuo, Du, Guocheng, Chen, Jian
• Format: Article
• Language: English
• Subjects: Amino acids; Bacteria; Biodegradation; Cloning; Encoding; Escherichia coli; Genes; Heterologous expression; Keratinase; Mathematical models; Pre-peptidase C-terminal (PPC) domain; Protease; Recombinant; Stenotrophomonas maltophilia
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2014.01.009

•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 (48kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40kDa). KerSMD has a t1/2 of 90min at 50°C and 64min 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.