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

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.

SUBMITTER: Rahimnahal S

PROVIDER: S-EPMC10206251 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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

Rahimnahal Somayyeh S Meimandipour Amir A Fayazi Jamal J Asghar Karkhane Ali A Shamsara Mehdi M Beigi Nassiri Mohammadtaghi M Mirzaei Hamed H Hamblin Michael R MR Tarrahimofrad Hossein H Bakherad Hamid H Zamani Javad J Mohammadi Yahya Y

Frontiers in microbiology 20230510

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 c ...[more]

PMID: 37234543

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Project description:Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Dataset Information

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

Publications

Biochemical and molecular characterization of novel keratinolytic protease from <i>Bacillus licheniformis</i> (KRLr1).

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