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.
TY - JOUR
AU - Dojnov, Biljana
AU - Božić, Nataša
AU - Nenadović, V.
AU - Ivanović, J.
AU - Vujčić, Zoran
PY - 2008
UR - http://cer.ihtm.bg.ac.rs/handle/123456789/459
AB - 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.
PB - Elsevier Science Inc, New York
T2 - Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
T1 - Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae
VL - 149
IS - 1
SP - 153
EP - 160
DO - 10.1016/j.cbpb.2007.09.009
ER -
@article{
author = "Dojnov, Biljana and Božić, Nataša and Nenadović, V. and Ivanović, J. and Vujčić, Zoran",
year = "2008",
url = "http://cer.ihtm.bg.ac.rs/handle/123456789/459",
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 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.",
publisher = "Elsevier Science Inc, New York",
journal = "Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology",
title = "Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae",
volume = "149",
number = "1",
pages = "153-160",
doi = "10.1016/j.cbpb.2007.09.009"
}
Dojnov, B., Božić, N., Nenadović, V., Ivanović, J.,& Vujčić, Z. (2008). Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae.
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Elsevier Science Inc, New York., 149(1), 153-160.
https://doi.org/10.1016/j.cbpb.2007.09.009
Dojnov B, Božić N, Nenadović V, Ivanović J, Vujčić Z. Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology. 2008;149(1):153-160
Dojnov Biljana, Božić Nataša, Nenadović V., Ivanović J., Vujčić Zoran, "Purification and properties of midgut α-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae" Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 149, no. 1 (2008):153-160,
https://doi.org/10.1016/j.cbpb.2007.09.009 .
