Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum.
An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Brazil.
A C M M
AC
Aquino
J A
JA
Jorge
H F
HF
Terenzi
M L T M
ML
Polizeli
D001203:Ascomycota / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D020536:Enzyme Activators; D008670:Metals; D013213:Starch; D000516:alpha-Amylases