Novel substrate specificity of a membrane‐bound β‐glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

[摘要]

A β-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85°C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100°C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90°C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k _cat/K _m values high enough for hydrolysis of β-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of β-linked glucose dimer more hydrophilic than aryl- or alkyl-β-D-Glcp.

[学科分类] 生物化学/生物物理

[关键词] β-Glycosidase;Thermophilic archaeon;Membrane protein;Thermostable enzyme;Pyrococcus horikoshii;BGPh;β-glycosidase from Pyrococcus horikoshii;BMPh;a β-mannosidase gene homolog from P. horikoshii;BGPf;β-glucosidase from Pyrococcus furiosus;BMPf;β-mannosidase from P. furiosus;Sβ-gly;β-glycosidase from Sulfolobus solfataricus;IPTG;isopropyl-β-D-thiogalactopyranoside;His-BGPh;BGPh with His-tag;SDS–PAGE;sodium dodecyl sulfate–polyacrylamide gel electrophoresis;X-Glu;5-bromo-4-chloro-3-indolyl-β-glucopyranoside;p-Nph-β-D-Glcp;p-nitrophenyl-β-D-glucopyranoside;LA-β-D-Glcp;β-D-glucopyranosides with long alkyl chains