Abstract
BackgroundKeratin 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.ResultsHerein, 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.ConclusionsThe 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.
Highlights
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
Keratinase is a proteolytic enzyme that degrades hard keratin and is widespread in fungi and bacteria, which can break down keratin materials, converting them into soluble proteins, peptides, and amino acids [13, 14]
The activity of extracellular keratinase expressed by Bacillus subtilis WB600-pMA5-ker was 2514.4 U/mL, while that of Bacillus subtilis WB600pP43NMK-ker reached 6659.0 U/mL
Summary
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. Bacillus subtilis NRC3 was reported to show higher keratinase activity under extreme conditions, such as high salinity and high temperature, completely degrading feathers within 24 h [17], while the recombinant Bacillus amyloliquefaciens showed enhanced feather degradation properties and shortened reaction time within 12 h [18]. These keratinases exhibit minimal effects after they become detached from the host bacteria, posing a major obstacle to the commercialization of keratinase
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