Fazria Rizqi Maharani, Suharti Suharti
Keratinase is an enzyme capable of degrading keratin, exhibiting two activities: protease activity, which cleaves peptide bonds, and disulfide reductase activity, which reduces disulfide bonds (-S-S-). However, keratinase activity is often measured using casein as a substrate, even though casein lacks disulfide bonds and is therefore less suitable. The limited availability of effective keratin substrates highlights the need to develop a more appropriate alternative. This study aimed to extract keratin from chicken feathers, characterize the extract-focusing on peptide and disulfide bonds-and evaluate its effectiveness as a keratinase substrate using keratinase produced by Bacillus sp. MD24. The research was conducted in three stages: (1) extraction of keratin from chicken feathers, (2) production of keratinase, and (3) evaluation and optimization of chicken feather keratin as a substrate using Plackett-Burman Design (PBD) and Central Composite Design- Response Surface Methodology (CCD-RSM). Keratin was extracted with DMSO, and ATR-FTIR and EDX analyses confirmed the presence of characteristic functional groups (- C- O, - N- H, - C- N, - C- H, - C- S, - S- H, and - S- S), indicating successful extraction. Keratinase production was optimized using the One-Factor-at-a-Time (OFAT) method, with optimal conditions identified at a temperature of 37 °C and pH 8. Keratinase activity was then tested with casein, chicken feather keratin, and azure keratin. CCD-RSM analysis predicted the following optimal conditions: 56.64 °C and pH 8.02 for casein, 63.07 °C and pH 8.14 for chicken feather keratin, and 53.07 °C and pH 7.97 for azure keratin. Although additional data are needed in PBD to address the lack of fit and in CCD-RSM to evaluate the significance of temperature and pH interactions, the findings demonstrate that keratin extracted from chicken feathers can serve as a viable substrate for keratinase activity assays. © 2026 The Authors, published by EDP Sciences.
Department of Chemistry, Faculty of Mathematics and Natural Science, State University of Malang, Indonesia
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