Cleavage Specificities and Synergistic Catalytic Action of Brevibacillus gelatini -Derived Thermostable Keratinolytic Protease BrgM4 and Reductase BrgTrxR on Feather Keratin

Disulfide reductases can cleave disulfide bonds that interlock with the keratin structures to promote keratin biomass degradation by keratinolytic proteases. Yet, the synergy between purified keratinolytic proteases and disulfide reductases remains underexplored. The study identified a keratinolytic protease, BrgM4, and a thioredoxin reductase, BrgTrxR, from the keratinolytic Brevibacillus gelatini LD5. The BrgM4 exhibited high keratinolytic activity at 90 °C and pH 7.5 and had remarkable thermostability (t1/2 = 13.6 h at 65 °C). The BrgM4 and BrgTrxR showed synergism in degrading chicken feathers at 50 °C, pH 6.5. BrgM4 preferentially cleaves feather keratin at the C-terminus of Thr, Val, Pro, Arg, and Ala. Beyond the active site residues, the amino acids Asn106, Ala107, Trp109, Arg192, Asp215, His220, and Zn²⁺ are important for BrgM4-substrate binding. These findings provide insights into the BrgM4 function and synergistic catalytic activity of keratinolytic protease and reductase, guiding the design of enzyme cocktails for efficient keratin conversion.