Bendazzoli Claudia, Turroni Silvia, Gotti Roberto, Olmo Stefano, Brigidi Patrizia, Cavrini Vanni
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, Bologna 40126, Italy.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):350-6. doi: 10.1016/j.jchromb.2007.04.027. Epub 2007 May 1.
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.
草酰辅酶A脱羧酶（OXC）是高毒性草酸盐分解代谢中的关键酶，催化草酰辅酶A（Ox-CoA）脱羧形成甲酰辅酶A（For-CoA）。在本研究中，提出了一种毛细管电泳（CE）方法来评估来自两种细菌，即产甲酸草酸杆菌DSM 4420和嗜酸乳杆菌LA 14的重组OXC的活性。特别是，酶促反应中发生的底物Ox-CoA的降解可以通过离线CE方法进行监测。使用永久涂覆有聚乙烯亚胺（PEI）的毛细管，在中性背景电解质（pH 7.0的50 mM磷酸盐缓冲液）存在下，获得了电渗流的反转。在这些条件下，Ox-CoA（底物）和For-CoA（反应产物）发生阳极迁移，并在不到12分钟内完成分离。该CE方法在选择性、线性（Ox-CoA在0.005 - 0.650 mM范围内）、灵敏度（在254 nm检测波长下的检测限为1.5 microM）、精密度和准确度方面得到了验证。最终估算了两种细菌的OXC稳态动力学常数（V(max)、K(m)或k'），结果表明，尽管嗜酸乳杆菌LA 14的草酸盐分解能力低于产甲酸草酸杆菌DSM 4420，但它可能是预防与草酸盐相关疾病的潜在有用益生菌。
