Expression, crystallization and preliminary X-ray crystallographic analysis of cellobiose 2-epimerase from Dictyoglomus turgidum DSM 6724

Abstract

Cellobiose 2-epimerase epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, β=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Keywords: Dictyoglomus turgidum; cellobiose 2-epimerase.

Figure 1

Purified His-tagged DT_epimerase and untagged DT_epimerase are shown on a 10% SDS–PAGE gel. Lane M1, His-tagged DT_epimerase (∼48 kDa); lane M2, untagged DT_epimerase (∼46 kDa); lane P, Prestained Protein Ladder (Fermentas).

Figure 2

Crystals of His-tagged and untagged DT_epimerase. (a) The optimized crystal of His-tagged DT_epimerase obtained using the reservoir condition 23%(w/v) PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M bis-tris pH 6.0 and (b) the optimized crystal of untagged DT_epimerase obtained using the reservoir condition 26.0%(v/v) 2-propanol, 10.0%(w/v) PEG 8000, 0.1 M imidazole–HCl pH 7.0.

Figure 3

Diffraction images of (a) His-tagged and (b) untagged DT_epimerase. Diffraction resolutions are marked as circles.