Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus.
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.
da Silva
Tony Marcio
TM
Departamento de Biologia, FFCLRP, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, Ribeirão Preto, SP 14.040-901, Brazil.
Michelin
Michele
M
Damásio
Andre Ricardo de Lima
AR
Maller
Alexandre
A
Almeida
Fausto Bruno Dos Reis
FB
Ruller
Roberto
R
Ward
Richard John
RJ
Rosa
José Cesar
JC
Jorge
João Atilio
JA
Terenzi
Héctor Francisco
HF
Polizeli
Maria de Lourdes Teixeira de Moraes
Mde L
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*
D005960:Glucosides; D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; C019502:4-nitrophenyl alpha-glucoside; D000520:alpha-Glucosidases