Site‐directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis

Abstract

A fragment of Staphylococcus epidermidis lipase gene (Lys-303 to Lys-688) was inserted into plasmid pET-20b(+). The resulting C-terminal His-tagged recombinant protein (43 kDa) was overexpressed in Escherichia coli BL21(DE3) as a highly active lipase and was purified with nickel-coupled resin. Putative catalytic sites were determined by site-directed mutagenesis. Mutant enzymes (S418C and H648K) lost enzyme activities, which strongly suggests that the proposed residues of Ser-418 and His-648 are involved in catalysis. Site-directed mutagenesis showed that in comparison with wild-type enzyme, the M419A and V649I enzymes showed a 2.0-and 4.0-fold increase in the k cat /K m , respectively, but the M419l, M419Q, V649A, and V649L variants lost enzyme activities. The wildtype enzyme and the V649I mutant favored the hydrolysis of pnitrophenyl esters of butyrate, but the M419A favored decanoate. The results suggested that the amino acid residues (Met-419 and Val-649), following the catalytic triad, could affect the substrate specificity and/or catalytic efficiency.

Synthesis of Fatty Acid Esters by Recombinant Staphylococcus epidermidis Lipases in Aqueous Environment

“…The S. epidermidis lipase gene has been subjected to increased lipase activity and improved substrate specificity by site-directed mutagenesis. In comparison with wild-type enzyme, the mutants (M419A and V649I) showed a 2.0 and 4.0-fold increase in the catalytic efficiency (k cat /K m ), respectively (15). In the present work, we found that both the wild-type and mutant lipases (M419A and V649I) could efficiently synthesize various flavor esters in aqueous media.…”

Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase

“…A lipase from S. saprophytics presented a K m value of 1.47 mM for the same substrate [31] and a recombinant lipase from S. epidermidis presented 0.9 mM [37]. The k cat value presented by the recombinant lipase from S. xylosus (0.0078 s -1 ) was lower than recombinant lipase of S. epidermidis (25.1 s -1 ) [37] as well as catalytic efficiency (0.0154 and 28.2 s -1 mM -1 , respectively).…”

“…Three pH values (7.0, 8.0, and 9.0) and three temperatures (25,37, and 42°C) were tested with pNPC 4 as substrate in order to determine the pH and temperature dependence on the activity of recombinant S. xylosus lipase (Fig. 4).…”

Crystallographic analysis of the Staphylococcus epidermidis lipase involved in esterification in aqueous solution

“…The GehC gene was first cloned and characterized by Farrell et al (1993). Structure-function studies of GehC have shown that aminoacid residues Met419 and Val649, which follow the catalytic triad, affect substrate specificity and catalytic efficiency, and that Ser418 is the active site (Chang et al, 2000). GehC exhibits a higher esterification yield with medium-chain fatty acids (from C8 to C14), with an optimal yield for lauric acid (Chang et al, 2001).…”

“…The C-terminal regions of these proteins are closely related: 49% of the 386 residues are identical and 60% of the differences reflect conservative changes. The significant differences in substrate specificity and enzyme catalytic efficiency between these two enzymes suggest that dissimilar amino-acid residues, particularly around the active site, are important for their biochemical properties (Chang et al, 2000). To clarify the mechanism by which rGehC catalyzes esterification in aqueous solvents, rGehC was crystallized and modelled using ShLip (PDB entry 2hih) as the initial model.…”