Determination of oxalyl-coenzyme A decarboxylase activity in Oxalobacter formigenes and Lactobacillus acidophilus by capillary electrophoresis.
Oxalyl-coenzyme A decarboxylase (OXC) is a key enzyme in the catabolism of the highly toxic oxalate, catalysing the decarboxylation of oxalyl-coenzyme A (Ox-CoA) to formyl-coenzyme A (For-CoA). In the present study, a capillary electrophoretic (CE) method was proposed for the assessment of the activity of recombinant OXC from two bacteria, namely Oxalobacter formigenes DSM 4420 and Lactobacillus acidophilus LA 14. In particular, the degradation of the substrate Ox-CoA occurring in the enzymatic reaction could be monitored by the off-line CE method. A capillary permanently coated with polyethylenimine (PEI) was used and in the presence of a neutral background electrolyte (50 mM phosphate buffer at pH 7.0), a reversal of the electroosmotic flow was obtained. Under these conditions, the anodic migration of Ox-CoA (substrate) and For-CoA (reaction product) occurred and their separation was accomplished in less than 12 min. The CE method was validated for selectivity, linearity (range of Ox-CoA within 0.005-0.650 mM), sensitivity (LOD of 1.5 microM at the detection wavelength of 254 nm), precision and accuracy. Steady state kinetic constants (V(max), K(m) or k') of OXC were finally estimated for both the bacteria showing that although L. acidophilus LA 14 provided a lower oxalate breakdown than O. formigenes DSM 4420, it could be a potentially useful probiotic in the prevention of diseases related to oxalate.
Bendazzoli
Claudia
C
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, Bologna 40126, Italy.
Turroni
Silvia
S
Gotti
Roberto
R
Olmo
Stefano
S
Brigidi
Patrizia
P
Cavrini
Vanni
V
D002138:Calibration; D002262:Carboxy-Lyases / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D020624:Oxalobacter formigenes / Q000201:enzymology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002262:Carboxy-Lyases; C059418:oxalyl CoA decarboxylase