The importance of microbial lipases like the staphylococcal ones, results not only from their significance implication in the bacterial lipid metabolism but also their involvement in some pathogenic processes. The aminoacid sequence of the mature Staphylococcus xylosus lipase (SXL) shows 99.7% identity with the mature aminoacid sequence of Staphylococcus simulans lipase (SSL) with one substitution of Gly3l I by Val. In spite of this high homology, the two lipases present significant differences in the specific activities and in the optimal temperature of action. In order to check the importance of the residue 311, we substituted, using site-directed mutagenesis, the Gly3l I of S. xylosus lipase (SXL) by Len, Trp, Asp, or Lys. The mutants SXL-G31 I L, SXL-G31 I W, SXL-G31 I D, and SXL-G31 I K were expressed in Escherichia coli BL21 (DE3) and purified to homogeneity. A comparative study of the biochemical properties of the wild-type SXL (SXL-WT) and the mutants was performed and show that an increase in the size of the 311 aminoacid side chain residue was accompanied by a decrease of lipase activity. The absence of lipase activity observed with the mutant SXL-G31 I W was probably due to the inaccessibility of substrate to interact with the catalytic serine. On the other hand, the SXL-G31 I L mutant, which presents a significant decrease on the lipase activity, displays a novel action towards phospholipids. Furthermore, the mutant SXL-G31 I L becomes also stable to extreme pH and displays a half life of 280 min after incubation at 50 degrees C. The purified SXL-G31 I D and SXL-G31 I K mutants show distinct pH profiles. The SXL-G31 I D mutant is optimally active at pH 6.5, whereas, the SXL-G31 I K mutant is active at pH 9.5. In addition, these two mutants show an important decrease in the specific activities that generates a decrease in the catalytic efficiency (k(cat)/K(m)app.). We can essentially notice that the SXL-G311 K mutant displays a 33- or 44-fold decrease in k(cat)/K(m)app. values compared to the SXL-WT, when using TC4 or TC8 as substrate, respectively. (C) 2007 Elsevier Inc. All rights reserved.