Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae
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- 作者：Biljana Dojnov ; Nataš ; a Bož ; ić ; Vera Nenadović ; Jelisaveta Ivanović ; ; Zoran Vujč ; ić
- 关键词：Midgut α ; -amylase ; Isoform ; Cerambycid beetle ; Morimus funereus ; Xylophagous larvae
- 刊名：Comparative Biochemistry and Physiology, Part B
- 出版年：2008
- 出版时间：January 2008
- 年：2008
- 卷：149
- 期：1
- 页码：153-160
- 全文大小：535 K
文摘
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 Km value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl2, 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.