Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A

• Published in: International journal of biological macromolecules Vol. 114; pp. 1033 - 1048
• Main Authors: Hadjidj, Raziqa, Badis, Abdelmalek, Mechri, Sondes, Eddouaouda, Kamel, Khelouia, Lamia, Annane, Rachid, El Hattab, Mohamed, Jaouadi, Bassem
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-07-2018
• Subjects: Alkaline protease; Bacillus licheniformis; Detergent formulation; Expression; Peptide biocatalysis; Recombinant enzyme; Detergent formulation; Peptide biocatalysis; Recombinant enzyme; Bacillus licheniformis; Expression; Alkaline protease
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.03.167

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.
•A new extracellular B. licheniformis protease was purified (SAPHM) and characterized.•SAPHM was a serine protease and a monomer with a molecular mass of 30,325.12 Da.•Optimum pH and temperature values for activity were pH 10 and 70 °C, respectively.•The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in E. coli.•SAPHM is a potential candidate for peptide synthesis and detergent formulations.