Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum

Abstract

The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-β-d-glucoside, pNP-β-d-galactoside and pNP-N-acetyl-β-d-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6M sodium citrate tribasic, 0.1M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120 × 25 × 5μm. The TxGH116 crystals diffracted X-rays to 3.15Å resolution and belonged to the monoclinic space group P21. Structure solution will allow a structural explanation of the effects of human GBA2 mutations.