Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain; Federal University of Ceará, Food Engineering Department, Campus do Pici, Bloco 858, Fortaleza, CE CEP 60440-900, Brazil.
Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain.
Int J Biol Macromol. 2021 Nov 30;191:79-91. doi: 10.1016/j.ijbiomac.2021.09.061. Epub 2021 Sep 16.
Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.
脂肪酶从Thermomyces lanuginosus（TLL）已共价固定在异功能辛基-乙烯基琼脂糖上。以这种方式，固定化酶将具有相同的取向。然后，用己胺（HEX）、乙二胺（EDA）、甘氨酸和天冬氨酸进行封闭。不同生物催化剂的初始活性/稳定性有很大差异，用甘氨酸封闭的生物催化剂最稳定。这些生物催化剂已被用于分析酶活性是否会因不同的热失活过程而不同，具体取决于所用的底物（即，在使用 4 种不同的底物失活过程中，酶的特异性是否会发生改变，以确定其活性），以及这种改变是否可以通过封闭剂的性质和失活条件来改变（我们使用 pH 5、7 和 9）。结果表明，在失活过程中，酶的特异性发生了很大变化（例如，三醋酸甘油酯的活性比其他底物的活性下降得更快），以及固定化方案和失活条件如何对此进行调节。荧光研究证实了固定化和失活引起的变化之间的差异。也就是说，部分失活固定化酶的功能和结构分析表明，它们的失活途径强烈依赖于载体的特征和失活条件。
