Properties of a purified thermostable glucoamylase from Aspergillus niveus.
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.
da Silva
Tony Marcio
TM
Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Monte Alegre, Ribeirão Preto, São Paulo, Brazil.
Maller
Alexandre
A
Damásio
André Ricardo de Lima
AR
Michelin
Michele
M
Ward
Richard John
RJ
Hirata
Izaura Yoshico
IY
Jorge
João Atilio
JA
Terenzi
Héctor Francisco
HF
de Polizeli
Maria Lourdes T M
ML
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight
D005087:Glucan 1,4-alpha-Glucosidase
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