Articles
ISOLATION, CHARACTERIZATION AND POTENTIAL APPLICATION OF DEOXYRIBONUCLEASE-FREE PHOSPHATASE FROM CASSAVA LEAVES
Article number
703_34
Pages
263 – 268
Language
English
Abstract
A deoxyribonuclease-free acid phosphatase from cassava leaves was prepared by ammonium sulfate precipitation, chromatofocusing and hydrophobic interaction chromatography with phenyl Sepharose.
The enzyme was purified 36-fold and had a specific activity of 16 U/ mg protein.
The enzyme preparation revealed a major phosphatase band of 77 kDa and three minor activity bands.
The pH and temperature optima for enzyme activity were 5.2 and 60°C, respectively.
The enzyme was inactivated by heating at 80°C for 15 minutes and could be stored at -20°C for up to two months.
The enzyme exhibited broad substrate specificity and had a Km (p-nitrophenyl phosphate) value of 1.7 mM. It was strongly inhibited by zinc and copper ions, molybdate and arsenate.
Aluminium ions, ferrous ions, dithiothreitol and mercaptoethanol stimulated enzyme activity within 0.5-5.0 mM range.
The enzyme partially dephosphorylated linearized pUC18 plasmid despite being an acid phosphatase and showed potential application as a substitute for alkaline phosphatase in the ELISA procedure.
The enzyme was purified 36-fold and had a specific activity of 16 U/ mg protein.
The enzyme preparation revealed a major phosphatase band of 77 kDa and three minor activity bands.
The pH and temperature optima for enzyme activity were 5.2 and 60°C, respectively.
The enzyme was inactivated by heating at 80°C for 15 minutes and could be stored at -20°C for up to two months.
The enzyme exhibited broad substrate specificity and had a Km (p-nitrophenyl phosphate) value of 1.7 mM. It was strongly inhibited by zinc and copper ions, molybdate and arsenate.
Aluminium ions, ferrous ions, dithiothreitol and mercaptoethanol stimulated enzyme activity within 0.5-5.0 mM range.
The enzyme partially dephosphorylated linearized pUC18 plasmid despite being an acid phosphatase and showed potential application as a substitute for alkaline phosphatase in the ELISA procedure.
Publication
Authors
S.C. Tham, S.H. Lim, H.H. Yeoh
Keywords
chromatofocusing, hydrophobic interaction, substrate specificity, inhibitors, dephosphorylation, ELISA
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