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

Abstract

Surface GRASP representation of closed forms and open forms of SSL and 4Ile-SSL. The catalytic serine is colored in red. The catalytic cavity is shown in yellow. The 4Ile residues are represented in dark blue. The lid domain is colored in green. ► The insertion of four residues Isoleucines at the N-terminus of SSL. ► Expression and purification of recombinant SSL and its mutant 4Ile-SSL. ► Comparative study on the biochemical properties of r-SSL and 4Ile-SSL. ► The importance of the N-terminus region in the biochemical properties of SSL. 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 ( k cat / K M 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.