Cloning, Expression, And Structural Modeling Of Two Alkaline Serine Protease Genes From Extremophilic Bacillaceae -related Species: Application In Valorization Of Invasive Crustaceans

Two novel protease genes sapA and sapN from the thermophilic Anoxybacillus kamchatkensis M1V and Melghiribacillus thermohalophilus Nari2AT strains respectively, encoding a polypeptide of 381 and 379 residues, were identified, cloned and successfully heterologously expressed in Escherichia coli BL21(DE3)pLysS. The deduced putative amino-acid residues of SAPA and SAPN enzymes evinced identity with proteases from Bacillus strains. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. While, the highest sequence identity (97.10%) of SAPN was observed with Bacillus licheniformis MP1 protease, but with 10 difference residues. rSAPA and rSAPN enzymes were purified until homogeneity, characterized, and compared to wild-type proteases.  The purified recombinant enzymes rSAPA and rSAPN were two monomers of about 28 and 30 kDa, correspondingly. rSAPA displayed the highest activity at pH 11 and 70°C. While, rSAPN displayed the highest activity at pH 10 and 75°C. To initiate structure-function relationships, a 3D-model of the Pro-SAPA and Pro-SAPN proteins were thereafter built based on the available structures of common proteases. The comparative molecular modeling studies with the less thermostable protease, revealed extra charged residues at the surface of SAPA and SAPN potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability.