Purification and characterization of alpha-amylase from Aspergillus flavus.
Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an alpha-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5 +/- 2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55 degrees C and it was stable for 1 h up to 50 degrees C. The Km and V for gelatinized tapioca starch were 0.5 g/L and 108.67 mumol reducing sugars per mg protein per min, respectively.
Khoo
S L
SL
School of Biological Sciences, Universiti Sains Malaysia, Penang.
Amirul
A A
AA
Kamaruzaman
M
M
Nazalan
N
N
Azizan
M N
MN
D001231:Aspergillus flavus / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D008670:Metals; D000516:alpha-Amylases