Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae.
Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.
Dojnov
Biljana
B
Institute of Chemistry, Technology and Metallurgy, Department of Chemistry, Studentski trg 12-16, Belgrade, Serbia. bdojnov@chem.bg.ac.yu
Bozić
Natasa
N
Nenadović
Vera
V
Ivanović
Jelisaveta
J
Vujcić
Zoran
Z
D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D007422:Intestines / Q000201:enzymology*; D007814:Larva / Q000201:enzymology; D013213:Starch / Q000737:chemistry; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; D019476:Insect Proteins; D013213:Starch; D000516:alpha-Amylases