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

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2008
Authors
Dojnov, Biljana
Božić, Nataša
Nenadović, V.
Ivanović, J.
Vujčić, Zoran
Article (Published version)
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Abstract
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 C...aCl2, 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.

Keywords:
Cerambycid beetle / Isoform / Midgut a-amylase / Morimus funereus / Xylophagous larvae
Source:
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 2008, 149, 1, 153-160
Publisher:
  • Elsevier Science Inc, New York
Projects:
  • Interakcije prirodnih proizvoda i njihovih analoga sa proteinima i nukleinskim kiselinama (RS-142026)

DOI: 10.1016/j.cbpb.2007.09.009

ISSN: 1096-4959

PubMed: 17942357

WoS: 000254219600016

Scopus: 2-s2.0-36849076255
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URI
http://cer.ihtm.bg.ac.rs/handle/123456789/459
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