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

Abstract

Background: Keratin is the primary constituent of the vertebrate epidermis and epidermal appendages, as well as the main waste product generated during poultry processing from feathers, hair, scales, nails, etc. Keratin is generally hard, stubborn and difficult to hydrolyze; however, it is also inexpensive and contains more than 85% protein. Currently, tens of millions of tons of keratin waste are produced each year worldwide; however, no effective methods for the recovery of keratin waste have been reported thus far, making such research urgent. Keratinase has been reported to be useful for keratin waste recovery; however, nearly all keratinases are unable to hydrolyze keratin after they are detached from living cell systems. This may be due to low keratinase activity and lack of synergistic factors. Results: Herein, the keratinase gene from Bacillus licheniformis BBE11-1 was successfully expressed in Bacillus subtilis WB600, allowing for improved activity of the recombinant keratinase KerZ1 to 45.14 KU/mL via promoter substitution and screening of the ribosome-binding sites. Further, real-time control of temperature, pH, dissolved oxygen, and feed strategy allowed the activity of KerZ1 to reach 426.60 KU/mL in a 15-L fermenter, accounting for a 3552-fold increase compared to the wild-type keratinase (120.1 U/mL). Most importantly, we proposed a method based on the synergistic action of keratinase KerZ1 and sodium sulfite, to hydrolyze feathers into amino acids. In specific, 100 g/L of feather waste can be successfully converted into 56.6% amino acids within 12 h, while supporting the production of dozens of bioactive peptides. Conclusions: The activity of recombinant keratinase can be greatly enhanced via transcription and translational regulation in Bacillus subtilis. The synergistic action of keratinase and sulfite can rapidly degrade feather waste and produce amino acids and polypeptides.

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

“…The remaining operations were the same as in the experimental group. In this study, one unit of keratinase activity was defined as an absorbance value increase of 0.001 units per minute at 660 nm (Peng et al, 2020).…”

“…In previous studies, we expressed the keratinase gene Ker from Bacillus licheniformis BBE11-1 in B. subtilis WB600. By optimizing the promoter and ribosome binding sequence, the recombinant B. subtilis WB600-Ker can secrete 127.6 KU/mg mature enzyme extracellularly (Peng et al, 2020). The zymogen of KerZ1 was composed of a signal peptide, pro-peptide, and mature peptide, containing 29, 76, and 274 amino acids, respectively.…”

“…In our previous studies, we have reported that keratinase and sodium sulfite work synergistically to rapidly hydrolyze feathers (Peng et al, 2020). To study the feather degradability of 2-D12, we optimized the temperature, initial pH, feather content, sulfite content and hydrolysis time of the hydrolysis system based on single factor experiments.…”

“…Recombinant keratinase KerZ1 has shown a strong ability to hydrolyze feather keratin with the help of sulfite. However, when the feather concentration reached 100 g/L, the activity of keratinase seemed insufficient (Peng et al, 2020). Here, pro-peptide engineering will be used to increase the activity of the mature enzyme to enhance its feather handling capabilities.…”

Microbial keratinase and the bio-economy: a three-decade meta-analysis of research exploit

“…The prospects of keratinases as suitable enzymes for industrial and biotechnological processes include their robustness in withstanding extreme conditions on like the classical proteases (Verma et al 2017). Microbial keratinases possess unique properties that bestows a potential suitable for use in a green economy including applications in the formulation of functional feed, detergent formulation, bio-decontamination, bating and tanning processes, personal care product formulation, and nanotechnology (Mitsuiki et al 2006;Paul et al 2014b;Gupta et al 2015;Sanghvi et al 2016;Kalaikumari et al 2019;Peng et al 2020). A continuum in the discovery of keratinases with novel properties and function would, significantly, revolutionize the bio-economy landscape, and the prospects presented by proteolytic enzymes.…”