One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite.
Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+)) and stimulated by 50mM Fe(2+). Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.
Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia.
Aleksandra
A
Dimitrijević
Dušan
D
Veličković
Filip
F
Bihelović
Dejan
D
Bezbradica
Ratko
R
Jankov
Nenad
N
Milosavić
D002175:Candida / Q000201:enzymology*; D017886:Durapatite / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol
D017830:Octoxynol; D017886:Durapatite; D008049:Lipase