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Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

Author:
da Silva, Tony Marcio, Michelin, Michele, de Lima Damásio, Andre Ricardo, Maller, Alexandre, Almeida, Fausto Bruno Dos Reis, Ruller, Roberto, Ward, Richard John, Rosa, José Cesar, Jorge, João Atilio, Terenzi, Héctor Francisco, de Lourdes Teixeira de Moraes Polizeli, Maria
Source:
Antonie van Leeuwenhoek 2009 v.96 no.4 pp. 569-578
ISSN:
0003-6072
Subject:
Aspergillus fumigatus, Hypocrea jecorina, amylose, crystal structure, filtration, glucan 1,4-alpha-glucosidase, glucose, glycosylation, high performance liquid chromatography, hydrolysis, ion exchange chromatography, isoelectric point, isomaltose, mass spectrometry, molecular weight, pH, peptides, polyacrylamide gel electrophoresis, proteins, starch products, sucrose, temperature, thin layer chromatography
Abstract:
An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.
Agid:
472405