Publicação
Cloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris
| Resumo: | 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. |
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| Autores principais: | Similä, Janika |
| Outros Autores: | Gernig, Anita; Murray, Patrick; Fernandes, Sara; Tuohy, Maria G. |
| Assunto: | a-Galactosidase Thermostability Talaromyces emersonii Overexpression Pichia pastoris |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | artigo |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | 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. |
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