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.
Palavras-chavea-Galactosidase
Thermostability
Talaromyces emersonii
Overexpression
Pichia pastoris
Data2010
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.
Tipoarticle
URIhttp://hdl.handle.net/1822/31863
DOI10.4014/jmb.1005.05043
ISSN1017-7825
e-ISSN1017-7825
Versão da editorahttp://www.springer.com/life+sciences/microbiology/journal/10061
Arbitragem científicayes
AcessoopenAccess
Aparece nas coleções:CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series

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