title: Cloning and expression of a thermostable alpha-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris.
abstract: The first gene (alpha-gal1) encoding an extracellular alpha-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The alpha-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal alpha-galactosidases belonging to glycosyl hydrolase family 27. The alpha-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant alpha-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70 degrees C, pH 4.5, and lost no activity over 10 days at 50 degrees C. alpha-Gal1 followed Michaelis-Menten kinetics (Vmax of 240.3 micronM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km obs of 0.57 mM, Ki of 2.77 mM). The recombinant T. emersonii alpha-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the alpha-galactosidic linkage. Owing to its substrate preference and noteworthy stability, alpha-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.
authors.affiliation: Molecular Glycobiotechnology Group, Biochemistry, School of Natural Sciences, National University of Ireland, University Road, Galway, Ireland.
authors.forename: Janika
authors.initials: J
authors.lastname: Simila
authors.forename: Anita
authors.initials: A
authors.lastname: Gernig
authors.forename: Patrick
authors.initials: P
authors.lastname: Murray
authors.forename: Sara
authors.initials: S
authors.lastname: Fernandes
authors.forename: Maria G
authors.initials: MG
authors.lastname: Tuohy
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010843:Pichia; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000519:alpha-Galactosidase