title: Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.
abstract: The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Nadia
authors.initials: N
authors.lastname: Zaraî Jaouadi
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Bassem
authors.initials: B
authors.lastname: Jaouadi
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Hajer
authors.initials: H
authors.lastname: Ben Hlima
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Hatem
authors.initials: H
authors.lastname: Rekik
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Mouna
authors.initials: M
authors.lastname: Belhoul
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Maher
authors.initials: M
authors.lastname: Hmidi
authors.affiliation: National Leather and Shoe Center (CNCC), Mégrine, Ben Arous, Tunisia.
authors.forename: Houda Slimene
authors.initials: HS
authors.lastname: Ben Aicha
authors.affiliation: National Leather and Shoe Center (CNCC), Mégrine, Ben Arous, Tunisia.
authors.forename: Chiraz Gorgi
authors.initials: CG
authors.lastname: Hila
authors.affiliation: National Leather and Shoe Center (CNCC), Mégrine, Ben Arous, Tunisia.
authors.forename: Abdessatar
authors.initials: A
authors.lastname: Toumi
authors.affiliation: Laboratory for Biocrystallography and Structural Biology of Therapeutic Targets, Molecular and Structural Bases of Infectious Systems, UMR 5086-CNRS-University of Lyon 1, Institute for the Biology and Chemistry of Proteins (IBCP), FR3302, Lyon, France.
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.affiliation: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006041:Goats; D006197:Hair / Q000378:metabolism*; D006204:Hair Removal / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008855:Microscopy, Electron, Scanning; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012867:Skin / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003094:Collagen; D010450:Endopeptidases; D010447:Peptide Hydrolases; C035855:keratinase; C087822:alkaline protease