El Alaoui Meddy, Soulère Laurent, Noiriel Alexandre, Queneau Yves, Abousalham Abdelkarim
Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, UMR 5246, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM(2)), 43, Bd du 11 novembre 1918, F-69622 Villeurbanne cedex, France; Univ Lyon, Université Lyon 1, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, UMR 5246, Chimie Organique et Bioorganique, Bâtiment Jules Verne, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France.
Univ Lyon, Université Lyon 1, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, UMR 5246, Chimie Organique et Bioorganique, Bâtiment Jules Verne, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France.
Chem Phys Lipids. 2017 Aug;206:43-52. doi: 10.1016/j.chemphyslip.2017.06.005. Epub 2017 Jun 16.
Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases.
脂肪酶本质上被描述为具有sn-1和sn-3区域选择性。实际上，很少有方法可用于测量这种脂肪酶的区域选择性，而且这些方法需要手性色谱分析或特定衍生化，既不连续又耗时。在本研究中，我们描述了一种新的、便捷、灵敏且连续的分光光度法，用于使用含有α-桐酸（9Z, 11E, 13E-十八碳三烯酸）的合成甘油三酯（TG）来筛选脂肪酶的区域选择性，该α-桐酸位于sn-1位[1-α-桐酰基-2,3-十八烷基-sn-甘油（sn-EOO）]或sn-3位[1,2-十八烷基-3-α-桐酰基-sn-甘油（sn-OOE）]，并包被在微量滴定板的孔中。在其他sn位引入了一个不可水解且具有非紫外吸收烷基链的醚键，以防止在甘油三酯合成或脂解过程中酰基链迁移。含α-桐酸甘油三酯的合成从S-缩水甘油开始，分六步得到sn-EOO，分五步得到sn-OOE。α-桐酸共轭三烯构成一种固有发色团，因此赋予了这种游离脂肪酸以及含有它的甘油三酯强烈的紫外吸收特性。通过α-桐酸从吸附态转变为可溶态导致的在272nm处吸光度的增加来测量包被的sn-EOO或sn-OOE上的脂肪酶活性。人胰腺脂肪酶、猪胰腺脂肪酶、豚鼠胰腺脂肪酶相关蛋白2、嗜热栖热菌脂肪酶、南极假丝酵母脂肪酶A和南极假丝酵母脂肪酶B均用于验证该测定法。这种连续的高通量筛选方法可以在脂解后直接测定各种脂肪酶的区域选择性，无需任何处理。
