Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii.
An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).
Michelin
Michele
M
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, Ribeirão Preto-SP, 14040-901, Brazil.
Silva
Tony M
TM
Benassi
Vivian M
VM
Peixoto-Nogueira
Simone C
SC
Moraes
Luiz Alberto B
LA
Leão
Juliana M
JM
Jorge
João A
JA
Terenzi
Héctor F
HF
Polizeli
Maria de Lourdes T M
Mde L
D002482:Cellulose / Q000737:chemistry; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D004983:Ethanolamines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D010143:Paecilomyces / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D004983:Ethanolamines; D002482:Cellulose; D000516:alpha-Amylases; C007369:2-diethylaminoethanol