The insertion of four residues Isoleucines at the N-terminus of Staphylococcus simulans lipase affects its catalytic and biochemical properties

For the sake of evaluating the effect of the hydrophobic residues insertion in the N-terminus of Staphylococcus simulans lipase (SSL), four residues of Isoleucines have been inserted at the N-terminus of this enzyme. The recombinant Staphylococcus simulans lipase (r-SSL) and its constructed mutant (4Ile-SSL) were expressed in Escherichia coli BL21 (DE3) and purified to homogeneity using classical chromatographic techniques. We have performed, then, a comparative study on the biochemical properties of the two enzymes. Due to the insertion of 4Ile at the N-terminus of Staphylococcus simulans lipase (SSL), some important differences in the biochemical properties between r-SSL and 4Ile-SSL have been found. We can essentially notice that, when using short chain triacylglycerols (tributyrin) as substrate, the insertion of four Isoleucines residues (4Ile) was accompanied by an increase in the specific activity (3 fold) as well as in the catalytic efficiency (kcat/KM app.) (2 fold), as compared to the recombinant SSL. Furthermore, our results indicate that the presence of 4Ile at the N-terminus of SSL has greatly affected the pH stability of the enzyme and considerably increased its thermostability.