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

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 propeptide 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.

Engineered pro‐peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste

“…Subsequently, visual models of KerZ1 and 2-D12 were obtained through homology modeling to directly observe the amino acid changes of the best mutant (Figure S1). As the amino acid substitution of the pro-peptide occurs in the transition region of β-sheet and α-helix to random coils, this may lead to an increase in the flexibility of its secondary structure, and the flexible pro-peptide is more conducive to guiding the formation of mature enzyme (Su et al, 2019). The above results indicated that the catalytic performance had been enhanced on the basis of the increased activity of the mutants, which means that the effect of pro-peptide modification on enhancing the activity of mature enzyme was obvious (Rakestraw et al, 2009;Uehara et al, 2013).…”

“…Therefore, proper modification of the pro-peptide sequence can help to activate mature enzymes and increase the activity and yield of extracellular mature enzymes (Grimsby et al, 2010). Su et al (2019) has engineered the pro-peptide to increase the extracellular keratinase activity of mutant M7 by nearly 16 times. B.…”

Progress in Microbial Degradation of Feather Waste

“…As keratinases are able to cleave insoluble and recalcitrant keratins derived from keratin-rich wastes such as feathers, hair and wool, they have great potential of industrial applications. Accumulated studies have shown that keratinase can be used in several fields (Figure 2) including animal feed (Grazziotin et al, 2006), fertilizers, leather industries, biomedical fields, detergents, cosmetics and materials (Yue et al, 2011; Paul et al, 2016; Su et al, 2019). Keratinase produced by B. licheniformis PWD-1 was found to be able to degrade prions which are infectious agents and resistant to proteolytic and mild protein-destructive processes (Van de Wiel et al, 2003).…”

“…Mutations in the pro-peptide region can affect the folding rate of the protein, which leads to high enzyme production, conformational change of the mature enzyme and acceleration of enzyme maturation. Such studies have been carried out on proteases from several species (Takagi et al, 2001; Fang et al, 2010; Uehara et al, 2013; Su et al, 2019). Computational aided site-directed mutation was carried out for residues at different sites.…”

Biotransformation of keratin waste to amino acids and active peptides based on cell-free catalysis

“…Following a 60-h fermentation process, the resulting extracellular activity reached 1500 U/mL [18]. Further, Su et al employed a recombinant B. subtilis keratinase mutant M7 to obtain 3040 U/mL of extracellular keratinase by continuous fermentation for 32 h in a 15-L bioreactor [26]. To this end, we also constructed a recombinant Bacillus subtilis strain that can efficiently express keratinase and found that we were able to rapidly process large amounts of feathers with the highly active recombinant keratinase KerZ1, to obtain a considerable amount of free amino acids and active peptides.…”