Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

• Published in: Journal of industrial microbiology & biotechnology Vol. 35; no. 1; pp. 17 - 25
• Main Authors: Michelin, Michele, Ruller, Roberto, Ward, Richard J, Moraes, Luiz Alberto B, Jorge, João A, Terenzi, Héctor F, Polizeli, Maria de Lourdes T. M
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 2008; Springer-Verlag; Springer; Oxford University Press
• Subjects: Amino Acid Sequence; Amino acids; Amylopectin; Amylose; Aspergillus oryzae; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Cellular biology; Cellulose; Chromatography; Circular Dichroism; Culture Media; Cyclodextrins; Dichroism; enzyme activity; Enzyme Stability; Enzymes; Fundamental and applied biological sciences. Psychology; Fungi; Gel electrophoresis; Gel filtration; Genetic Engineering; Glucan; glucan 1,4-alpha-glucosidase; Glucan 1,4-alpha-Glucosidase - chemistry; Glucan 1,4-alpha-Glucosidase - isolation & purification; Glucan 1,4-alpha-Glucosidase - metabolism; Glucoamylase; Glucose; Glycogen; Glycogens; guavas; Hot Temperature; Hydrogen-Ion Concentration; Inorganic Chemistry; Ion exchange; Kinetics; Laboratories; Life Sciences; Manganese; Mass spectrometry; Metal ions; Microbiology; Molecular Sequence Data; Original Paper; Paecilomyces; Paecilomyces - classification; Paecilomyces - enzymology; Paecilomyces - growth & development; Paecilomyces - physiology; Paecilomyces variotii; Peptides; Proteins; Psidium guajava; Scientific imaging; Sodium; Sodium lauryl sulfate; Starch; Studies; Substrates; Sucrose; Talaromyces; Thermal denaturation; thermal stability; Thin layer chromatography; Trehalose; β-Cyclodextrin; Thermostable; Glucoamylase; Purification; Glucan 1,4-α-glucosidase; Paecilomyces variotii; Enzyme; Extracellular; Thermal stability; Glycosylases; Characterization; Fungi; Hydrolases; Fungi Imperfecti; Thermotolerance
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-007-0261-1

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).