title: Expression and characterization of an extremely thermostable β-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638.
abstract: Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This β-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both β-galactosidase and β-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-β-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable β-glycosidase may be useful for food and pharmaceutical applications.
authors.affiliation: Department of Food Science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada.
authors.forename: Sung H
authors.initials: SH
authors.lastname: Park
authors.forename: Kwan H
authors.initials: KH
authors.lastname: Park
authors.forename: Byung C
authors.initials: BC
authors.lastname: Oh
authors.forename: Inteaz
authors.initials: I
authors.lastname: Alli
authors.forename: Byong H
authors.initials: BH
authors.lastname: Lee
mesh_terms: D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal / Q000502:physiology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000737:chemistry; D008361:Mannosidases / Q000096:biosynthesis* / Q000235:genetics; D008362:Mannosides / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D019843:Archaeal Proteins; D008351:Mannans; D008362:Mannosides; D011994:Recombinant Proteins; C014647:mannobiose; C036119:4-nitrophenyl-beta-D-mannopyranoside; D008361:Mannosidases