Abstract
Billions of tons of keratin bio-wastes are generated by poultry industry annually but discarded that result in serious environmental pollution. Keratinase is a broad spectrum protease with the unique ability to degrade keratin, providing an eco-friendly way to convert keratin wastes to valuable amino acids. In this report, a feather-degrading thermophilic bacterium, Meiothermus taiwanensis WR-220, was investigated due to its ability to apparently complete feather decay at 65 °C in two days. By genomics, proteomics, and biochemical approaches, the extracellular heat-stable keratinase (MtaKer) from M. taiwanensis WR-220 was identified. The recombinant MtaKer (rMtaKer) possesses keratinolytic activities at temperatures ranging from 25 to 75 °C and pH from 4 to 11, with a maximum keratinolytic activity at 65 °C and pH 10. The phylogenetic and structural analysis revealed that MtaKer shares low sequence identity but high structural similarity with known keratinases. Accordingly, our findings have enabled the discovery of more keratinases from other extremophiles, Thermus and Deinococcus. Proteins encoded in the extremophiles shall be evolved to be functional in the extreme conditions. Hence, our study expands the current boundary of hunting keratinases that can tolerate extreme conditions for keratin wastes biorecycle and other industrial applications.
Highlights
Over one billion tons of animal-derived keratinous wastes, such as poultry feathers, bristles, wools, and horns, are generated annually, but lack proper applications and are discarded[1, 2]
We identified a thermostable keratinase (MtaKer) from the thermophile strain, Meiothermus taiwanensis WR-220, which grows at 55–65 °C11
After two days of culture, the feather degradation indicated that M. taiwanensis produced an extracellular keratinase to degrade feather keratin as a nutrient source (Fig. 1a)
Summary
The entire amino acid sequence of MtaKer is 402 residues with the expected molecular weight of 41.3 kDa (Supplementary Table S1), but we observed a protease of 28 kDa in our zymogram analysis (Fig. 2c) This molecular weight discrepancy is due to the 100 residues of the N-terminal pro-peptide that are removed to form the mature and active MtaKer by autoproteolysis, which is a known maturation process in the subtilisin family[28, 29]. We observed that the C-terminal His-tag was removed, and the remaining C-terminal resides (Tyr278-Glu279-Asn280-Leu281-Tyr282) were still buried in the active site groove of the adjacent rMtaKer molecule in our crystal structure (Fig. 5e and Supplementary Fig. S7), indicating that the tag can be digested via intermolecular autoproteolysis This is consistent with our finding that the mature rMtaKer that is purified by Ni-affinity chromatography can no longer be captured by Ni-resin after long store at 4 °C. Our results revealed that rMtaKer has potential as a less expensive and environmentally-friendly technology to degrade keratinous wastes, and our methodology and sequence data provide new insight into the identification or design of highly efficient keratinases
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