Design, development and characterization of a chimeric protein with disulfide reductase and protease domain showing keratinase activity.

Autor: Kumari P; Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India., Abhinand CS; Center for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore 575018, India., Kumari R; Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India., Upadhyay A; Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India., Satheeshkumar PK; Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India. Electronic address: Satheesh.bot@bhu.ac.in.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Oct; Vol. 278 (Pt 4), pp. 135025. Date of Electronic Publication: 2024 Aug 24.
DOI: 10.1016/j.ijbiomac.2024.135025
Abstrakt: Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni 2+ -NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni 2+ , Mg 2+ , and Na + inhibited the keratinolytic activity, Cu 2+ , Ca 2+ , and Mn 2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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