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

Abstract

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303-Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Å with an overall R_merge value of 7.3%. The crystals were orthorhombic and belonged to space group P2₁2₁2₁, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å, α = β = γ = 90°. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.

Keywords: SeLip; Staphylococcus epidermidis; aqueous media; catalytic mechanism; lipase.

Figure 1

SDS–PAGE analysis of recombinant lipase (rGehC) cloned in pET-28a and expressed in E. coli Tuner (DE3) cells. Lane M, PageRuler Prestained Protein Ladder (labelled in kDa); lane 1, crude soluble extract; lane 2, pass-through from nickel column; lane 3, wash; lane 4, purified rGehC.

Figure 2

Crystals of lipase (rGehC) obtained using the sitting-drop vapour-diffusion method. The crystals shown are approximately 305 × 55 × 18 µm in size.

Figure 3

Typical X-ray diffraction pattern of S. epidermidis lipase (rGehC). The arrow indicates 1.9 Å resolution.