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.
Highlights
Leather processing is one of the oldest industries known to mankind
The plasmid constructs pNZ7, pNZ8, pNZ9, pNZ10, pNZ11, and pNZ12 in which the sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, and sapB-T33S/N99Y genes were inserted between the restriction sites for EcoRI/ HindIII at the multiple cloning site of pBSMuL2 were prepared in E. coli, respectively
The findings revealed that the rSAPB and SAPB mutated proteases overexpressed in B. subtilis exhibited specific activities ranging from 24,950 U.mg21 to 45,500 U.mg21 when casein was used as a substrate
Summary
Leather processing is one of the oldest industries known to mankind. Despite its significant contributions to the socioeconomic development of several countries around the world, this industry has been the target of mounting criticism owing to the pollution it causes to the environment. Alkaline proteases produced from Bacillus can withstand high temperature, pH, chemical denaturing agents, and non-aqueous environments They have attracted considerable attention due to their promising potential for application in a wide range of industries, including the detergent, leather, and pharmaceutical industries. The keratinolytic activity of these enzymes has been of interest in several biotechnological processes, such as the management of waste from various food-processing industries, the recovery of silver from used X-ray and photographic films, and production of proteinaceous fodder from waste feathers or keratincontaining materials [4] Due to their efficiency, low cost, and eco-friendliness, microbial alkaline proteases have attracted increasing attention for application in enzymatic depilation as substitutes to chemical agents, such as sodium sulphide, lime, and chromate, which have long been used in conventional industrial depilation processes. The effects of L31I and T33S mutations on their animal hide depilation ability and the relationship between keratin/casein activity ratios were examined
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