Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 149; no. 1; pp. 153 - 160
• Main Authors: Dojnov, Biljana, Božić, Nataša, Nenadović, Vera, Ivanović, Jelisaveta, Vujčić, Zoran
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 2008
• Subjects: alpha-Amylases - antagonists & inhibitors; alpha-Amylases - chemistry; alpha-Amylases - isolation & purification; Animals; Cerambycid beetle; Coleoptera - enzymology; Enzyme Activation; Enzyme Inhibitors - chemistry; Insect Proteins - antagonists & inhibitors; Insect Proteins - chemistry; Insect Proteins - isolation & purification; Intestines - enzymology; Isoform; Larva - enzymology; Midgut α-amylase; Morimus funereus; Starch - chemistry; Xylophagous larvae; Isoform; Cerambycid beetle; Xylophagous larvae; Midgut α-amylase; Morimus funereus
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2007.09.009

Using soluble starch as a substrate five isoforms of α-amylase were identified in a crude extract of Morimus funereus larvae. The main α-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 °C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 °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 α-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.