Cloning, expression, and characterization of a surfactant-stable alkaline serine protease (KNBSSP1) from Bacillus safensis PRN1 with remarkable applications in laundry and leather industries

Abstract

Proteases represent an important category of proteolytic enzymes with vast biotechnological applications in various industries like detergent, leather, agriculture, poultry, pharmaceutical, food, etc. The present study proposes an eco-friendly enzyme-mediated approach as opposed to conventional chemical approaches in detergent and laundry industries that are hazardous to human health and the environment. Accordingly, the knbsSP1gene, which encodes the trypsin-like serine protease KNBSSP1, from Bacillus safensis PRN1 was isolated, cloned into an expression vector (PET-28a), and expressed in the Escherichia coli cells BL21 (DE3). A single Ni-NTA affinity chromatography step was utilized to purify the recombinant protease (rKNBSSP1). The molecular weight of the rKNBSSP1 was approximately ∼33 kDa determined in Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The rKNBSSP1 showed optimal activity at 60 °C and pH 8. It retained above 70% of its enzyme activity at pH 10 and 70 °C. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate suggesting its inclusion in the serine family of protease. The rKNBSSP1 demonstrated outstanding compatibility and stability with metal ions, commercial detergents, and surfactants, exhibiting 97.8 ± 2.5% stability with Surf Excel, a commercial laundry detergent, and 96.9 ± 2.0% stability with SDS. Interestingly, the protease demonstrated blood-stained removal and goat skin dehairing (8 h) properties. All these remarkable characteristics make this protease a potential eco-friendly candidate in the detergent formulation and leather processing industries.