Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase

• Published in: ACS synthetic biology Vol. 8; no. 2; pp. 425 - 433
• Main Authors: Su, Chang, Gong, Jin-Song, Sun, Yu-Xin, Qin, Jiufu, Zhai, Shen, Li, Heng, Li, Hui, Lu, Zhen-Ming, Xu, Zheng-Hong, Shi, Jin-Song
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.02.2019
• Subjects: Amino Acid Substitution; Bacillus - enzymology; Biotechnology - methods; Catalysis; Hydrogen-Ion Concentration; Mutagenesis; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Substrate Specificity; keratinase; expression; pro-peptide engineering; saturation mutagenesis; modification
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.8b00442

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.