Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

• Published in: Phytochemistry (Oxford) Vol. 66; no. 2; pp. 147 - 151
• Main Authors: Campos, Alexandre, Rijo-Johansen, Maria J., Carneiro, Maria F., Fevereiro, Pedro
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 2005; Elsevier
• Subjects: Adenosine; Adenosine nucleosidase; Biological and medical sciences; Coffea - enzymology; Coffea arabica; Coffea arabica L; Coffee; coffee (beverage); enzyme activity; enzyme kinetics; Enzyme purification; Enzymes; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Metabolism; N-glycoside hydrolases; N-Glycosyl Hydrolases - isolation & purification; N-Glycosyl Hydrolases - metabolism; Plant Leaves - enzymology; Plant physiology and development; plant proteins; Purine metabolism; Rubiaceae; Adenosine nucleosidase; Adenosine; Enzyme purification; Rubiaceae; Coffea arabica L; Coffee; Purine metabolism; Purification; Coffea arabica; Enzyme; Stimulant plant; Plant leaf; Characterization; Enzymatic activity; Dicotyledones; Glycosidases; Angiospermae; Hydrolases; N-Glycosidases; Spermatophyta; Purine; Kinetics
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2004.11.018

The purification and preliminary characterization of an adenosine nucleosidase (ANase) (EC 3.2.2.7) of Coffea arabica L. cv. Catimor is reported. An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg −1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS–PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated K m is 6.3 μM and V max 9.8 nKat.