Journal Article10.1021/ACSSYNBIO.8B00442
Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase.
Su Chang,Jin-Song Gong,Yu Xin Sun,Jiufu Qin,Shen Zhai,Heng Li,Hui Li,Zhen Ming Lu,Zhenghong Xu,Jin-Song Shi +9 more
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TL;DR: 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, and provided a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.
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Abstract: 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.
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Citations
Progress in Microbial Degradation of Feather Waste
TL;DR: This review summarizes recent progress in microbial degradation of feathers, structures of keratinases, feather application, and microorganisms that are able to secrete keratinase and proposed the strategy that can be utilized for feather degradation.
Microbial keratinases: An overview of biochemical characterization and its eco-friendly approach for industrial applications
TL;DR: Keratin is resistant to degradation by common proteases and chemical catalysts due to high mechanical stability and cross-linked disulphide bonds present in their structure as mentioned in this paper.
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Biotransformation of keratin waste to amino acids and active peptides based on cell-free catalysis
TL;DR: A method based on the synergistic action of keratinase KerZ1 and sodium sulfite, to hydrolyze feathers into amino acids is proposed, which can rapidly degrade feather waste and produce amino acids and polypeptides.
Structure, Application, and Biochemistry of Microbial Keratinases.
TL;DR: In this article, a review of microbial keratinases and strategies to improve their efficiency in protein degradation is presented. But despite some progress made in isolating keratinase-producing microorganisms, structural studies of these enzymes, and biochemical characterization of their enzymes, effort is still required to expand the biotechnological application of Keratinase in diverse fields by identifying more and more active enzymes through molecular biology and protein engineering.
The tale of a versatile enzyme: Molecular insights into keratinase for its industrial dissemination.
TL;DR: This critical review systematically summarizes the application potential of keratinase, and in particular certain newly discovered catalytic capabilities, and reveals that the engineering of protein domains such as signal peptides and pro-peptides has become an important strategy to increase production of Keratinases.
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