title: Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus.
abstract: An extracellular a-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 alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-D-glucopyranoside and presented an optimum temperature and pH of 65 degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60 degrees C, presenting t(50) of 90 min at 65 degrees C. Hydrolysis of polysaccharide substrates by alpha-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 beta-ciclodextrin were poor substrates, and sucrose and alpha-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 a-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.
authors.affiliation: Departamento de Biologia, FFCLRP, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, Ribeirão Preto, SP 14.040-901, Brazil.
authors.forename: Tony Marcio
authors.initials: TM
authors.lastname: da Silva
authors.forename: Michele
authors.initials: M
authors.lastname: Michelin
authors.forename: Andre Ricardo de Lima
authors.initials: AR
authors.lastname: Damásio
authors.forename: Alexandre
authors.initials: A
authors.lastname: Maller
authors.forename: Fausto Bruno Dos Reis
authors.initials: FB
authors.lastname: Almeida
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
authors.forename: José Cesar
authors.initials: JC
authors.lastname: Rosa
authors.forename: João Atilio
authors.initials: JA
authors.lastname: Jorge
authors.forename: Héctor Francisco
authors.initials: HF
authors.lastname: Terenzi
authors.forename: Maria de Lourdes Teixeira de Moraes
authors.initials: Mde L
authors.lastname: Polizeli
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism; D004795:Enzyme Stability; D005960:Glucosides / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D035901:Hypocrea; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D005960:Glucosides; D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; C019502:4-nitrophenyl alpha-glucoside; D000520:alpha-Glucosidases