Utilize este identificador para referenciar este registo: http://hdl.handle.net/1822/31863

TítuloCloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris
Autor(es)Similä, Janika
Gernig, Anita
Murray, Patrick
Fernandes, Sara
Tuohy, Maria G.
Talaromyces emersonii
Pichia pastoris
EditoraSpringer Verlag
RevistaJournal of Microbiology and Biotechnology
CitaçãoJanika, S.; Gernig, A.; Murray, P.; Fernandes, Sara; Tuohy, M. G., Cloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris. Journal of Microbiology and Biotechnology, 20(12), 1653-1663, 2010
Resumo(s)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.
Versão da editorahttp://www.springer.com/life+sciences/microbiology/journal/10061
Arbitragem científicayes
Aparece nas coleções:CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series

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