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

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