Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation

Abstract

Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive. Graphical Abstract

Fabrication of biodegradable chicken feathers into ecofriendly-functionalized biomaterials: characterization and bio-assessment study

“…Notable, fibers of BC produced by EDCFs medium diameters were found to be an average of 50–100 nm, whereas diameters of bacterial cellulose produced by HS medium were found to be between 100–150 nm. The current SEM investigation is almost consistent with SEM investigations as previously reported by Tsouko et al 53 and Zhang et al 54 .

Figure 6 SEM image of BC obtained from HS medium ( A ) and from EDCFs based medium ( B ) at magnification power 20,000X.

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Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste

“…Keratinases (EC 3.4.-.-) are a type of proteases with unique hydrolytic properties. They enable the degradation of hard keratin waste into high value-added products such as soluble proteins, amino acids, and bioactive peptides. , To date, research on keratinases has mainly focused on the mining of new keratinases and the modification of enzyme properties. − Previously, the keratinase gene of Bacillus licheniformis was successfully expressed in Bacillus subtilis to obtain the highly active keratinase enzyme KerZ1 . Recently, Peng et al achieved improved KerZ1 expression and 188.1-fold increased activity compared with that of the wild enzyme by optimizing the promoter sequence and ribosome binding site (RBS) .…”

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production

“…subtilis 168 also increased the expression of β-galactosidase and nattokinase . Knockout of autolysis-related genes lytC and sigD effectively increased exogenous protein production and delayed autolysis by 10–15 days. − It is worth noting that not all deletions of autolysis genes are positive. For example, when the peptide chain endopeptidase genes lytE and lytF were individually deleted, the mutants exhibited growth division defects but were not effective in inhibiting cellular autolysis .…”

“…18,19 Researchers have done extensive studies on the efficient heterologous expression of keratinase and found that B. subtilis is an excellent expression host. 15,20,21 We have successfully expressed keratinase derived from B. licheniformis BBE11-1 in B. subtilis WB600 and enhanced the expression of keratinase by promoter optimization, RBS sequence screening, and lead peptide engineering strategies. 22−24 It is worth noting that the low titer of recombinant keratinase in B. subtilis remains the main problem limiting its industrial application, and cellular autolysis may be one of the important reasons hindering the enhancement of enzyme protein titer.…”

“…Keratinase is a kind of subtilisin that can specifically degrade keratin and hydrolyze keratin waste to produce nutrients such as soluble proteins, amino acids, and peptides. , Researchers have done extensive studies on the efficient heterologous expression of keratinase and found that B. subtilis is an excellent expression host. ,, We have successfully expressed keratinase derived from B. licheniformis BBE11-1 in B.…”