Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves.
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 Km is 6.3 microM and Vmax 9.8 nKat.
Campos
Alexandre
A
Lab Biotechnologia de Celulas Vegetais, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av Republica, Apt. 127, 2781 - 901 Oeiras, Portugal.
Rijo-Johansen
Maria J
MJ
Carneiro
Maria F
MF
Fevereiro
Pedro
P
D040503:Coffea / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology*
D009699:N-Glycosyl Hydrolases; C019599:adenosine nucleosidase