Enhancing Keratinase Efficiency Through Computational Design and Site-Directed Mutagenesis for Keratin Waste Valorization

Information from the abstract

Keratinase-I from T. vulgaris TK1-21 was engineered through computationally guided site-directed mutagenesis to enhance keratin hydrolysis and keratin waste valorization. Recombinant keratinase-I (rK-I) was expressed in Bacillus subtilis RIK1285, and molecular modeling suggested Leu214, Leu222, Ala270, and Ser280 as potential residues within the predicted substrate binding pocket. Among the engineered variants, S280L exhibited the highest catalytic performance, with 16.94% and 19.52% increases in apparent kinetic parameters toward α- and β-keratin, respectively, compared with rK-I. S280L also enhanced the hydrolysis of hydrothermally treated chicken feathers, producing higher protein (5.59 mg/mL) and peptide (1.38 mg/mL) concentrations than rK-I (4.73 and 1.08 mg/mL, respectively). The resulting keratin hydrolysate exhibited higher DPPH and ABTS radical scavenging activities, reaching 23.85 and 1100 μmol TE/g protein, respectively. LC-MS/MS identified three peptides detected only in the S280L-derived hydrolysate: SEEGVPISSGGF, EGVPISSGGF, and HLPGHQF. Bioinformatic analysis predicted potential bioactivities of these peptides. Subsequent in vitro evaluation of synthetic peptides showed that HLPGHQF exhibited 79 ± 2.5% ACE inhibitory and 17 ± 0.5% antioxidant activities, whereas EGVPISSGGF showed no detectable activity. These findings suggest that engineering the rK-I predicted substrate binding pocket could enhance keratin hydrolysis and facilitate the generation of value-added bioactive peptides.

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Related topics: Enzyme Production and Characterization · Protein Hydrolysis and Bioactive Peptides · Enzyme Catalysis and Immobilization