Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that pro-peptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.
- Pro-peptide engineering
- Saturation mutagenesis
Su, C., Gong, J. S., Sun, Y. X., Qin, J., Zhai, S., Li, H., Li, H., Lu, Z. M., Xu, Z. H., & Shi, J. S. (2019). Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis to Improve the Catalytic Potential of Keratinase. A C S Synthetic Biology, 8(2), 425-433. https://doi.org/10.1021/acssynbio.8b00442