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Expression and crystallization of a bacterial glycoside hydrolase family 116 β‐glucosidase from Thermoanaerobacterium xylanolyticum
Author(s) -
Sansenya Sompong,
Mutoh Risa,
Charoenwattanasatien Ratana,
Kurisu Genji,
Ketudat Cairns James R.
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14025461
Subject(s) - glycoside hydrolase , crystallization , glycoside , chemistry , hydrolase , microbiology and biotechnology , biochemistry , stereochemistry , biology , enzyme , organic chemistry
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 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed the p ‐nitrophenyl ( p NP) glycosides p NP‐β‐D‐glucoside, p NP‐β‐D‐galactoside and p NP‐ N ‐acetyl‐β‐D‐glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris–HCl pH 7.0 by vapour diffusion with micro‐seeding to form crystals with maximum dimensions of 120 × 25 × 5 µm. The TxGH116 crystals diffracted X‐rays to 3.15 Å resolution and belonged to the monoclinic space group P 2 1 . Structure solution will allow a structural explanation of the effects of human GBA2 mutations.