Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1)

Somayyeh Rahimnahal; Amir Meimandipour; Jamal Fayazi; Ali Asghar Karkhane; Mehdi Shamsara; Mohammadtaghi Beigi Nassiri; Hamed Mirzaei; Michael R Hamblin; Hossein Tarrahimofrad; Hamid Bakherad; Javad Zamani; Yahya Mohammadi

doi:10.3389/fmicb.2023.1132760

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1)

Front Microbiol. 2023 May 10:14:1132760. doi: 10.3389/fmicb.2023.1132760. eCollection 2023.

Authors

Affiliations

¹ Department of Animal Science and Food Technology, Agriculture Science and Natural Resources University Khouzestan, Ahwaz, Iran.
² Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, (NIGEB), Tehran, Iran.
³ Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran.
⁴ Faculty of Health Science, Laser Research Centre, University of Johannesburg, Johannesburg, South Africa.
⁵ Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
⁶ Department of Animal Science, Ilam University, Ilam, Iran.

Abstract

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca²⁺ and Mg²⁺ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as K_m 14.54 mM, k_cat 912.7 × 10^-3 (S^-1), and k_cat/K_m 62.77 (M^-1 S^-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Keywords: HPLC; cloning; keratinase; molecular dynamics; thermodynamics.