Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.
Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga 680001, Colombia.
Molecules. 2014 Jun 10;19(6):7629-45. doi: 10.3390/molecules19067629.
A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL® CHP20P compared to octyl-Sepharose® (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase® Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose® immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose® immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL® CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones.
一种商业的、疏水性很强的苯乙烯-二乙烯基苯基质 MCI GEL® CHP20P，与辛基-Sepharose®珠相比，被用作固定三种不同的酶：从Thermomyces lanuginosus（TLL）和Rhizomucor miehie（RML）中提取的脂肪酶，以及 Lecitase® Ultra，一种商业人工磷脂酶。两种载体上的固定化机制相似：酶的界面活化与载体的疏水性表面相对应。与辛基-Sepharose®相比，MCI GEL® CHP20P 的固定化速度和负载能力要高得多（使用 TLL 时为 87.2mg 蛋白/g 载体，使用 RML 时为 310mg/g，使用 Lecitase® Ultra 时为 180mg/g）。所有新制备物的热稳定性都远低于标准的辛基-Sepharose®固定化制备物，而在存在有机共溶剂的情况下进行失活时则相反。关于水解活性，结果强烈依赖于底物和测量的 pH 值。与 p-NPB 相比，辛基-Sepharose®固定化的酶比固定在 MCI GEL® CHP20P 上的酶更活跃，而 RML 对甲基苯基乙酸酯的活性降低了 700 倍。因此，需要对这种基质上的脂肪酶的固定化进行经验评估，因为在某些情况下它可能会产生非常积极的效果，而在其他情况下它可能会产生非常消极的效果。
