Production and characterization of bacillus halodurans c-125 alkaline serine protease and investigation of using as detergent additive

Microbial proteases are among the most important hydrolytic enzymes and have commercial value and multiple applications in various industries. Two-thirds of commercial proteases worldwide originate from microorganisms. The major producers of commercial alkaline proteases are bacteria, especially Bacillus species. The purpose of this study was to clone BH0855 gene encoding alkaline serine protease (BHASP) from B. halodurans C-125 strain, to enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases, and characterize its physicochemical properties. BH0855 gene is 1083 nucleotides, encoding a 361 amino acid pre-pro-protein enzyme (24-aa pre-signal peptide, 69-aa pro-peptide and 268-aa mature protein). It has a predicted molecular mass of 27.67 kDa and pI 9.47). The recombinant gene was successfully cloned into a shuttle vector PMA091 under the control of cat promoter and signal peptide LipA and extracellularly expressed in Bacillus subtilis WB800 strain. The deduced amino acid sequence has the highest homology of 65.4% with B. lentus protease (PDB ID:1GCI). The enzyme showed activity in a broad range of pH values and temperatures, with an optimum pH of 12 and a temperature of 60 °C. This enzyme retained nearly 60% of its initial activity after preincubation for 1h at 60 °C. The enzyme showed good stability at high pH, maintaining more than 93% of the initial activity after 24 hours pre-incubation at 37 ° C and pH 12. The Km and Vm values of the enzyme were calculated as 0,2899 mg/mL and 76.12 U/ml, respectively. Due to the extreme properties of the protease, its usability has been demonstrated in various fields, especially in the detergent industry.