Ghéczy Nicolas, Xu Weina, Szymańska Katarzyna, Jarzębski Andrzej B, Walde Peter
Laboratory for Multifunctional Materials, Department of Materials, ETH Zürich, Vladimir-Prelog-Weg 5, Zürich 8093, Switzerland.
Department of Chemical Engineering and Process Design, Silesian University of Technology, Księdza Marcina Strzody 7, Gliwice 44-100, Poland.
ACS Omega. 2022 Jul 21;7(30):26610-26631. doi: 10.1021/acsomega.2c02815. eCollection 2022 Aug 2.
Although many different methods are known for the immobilization of enzymes on solid supports for use in flow-through applications as enzyme reactors, the reproducible immobilization of predetermined amounts of catalytically active enzyme molecules remains challenging. This challenge was tackled using a macro- and mesoporous silica monolith as a support and dendronized polymer-enzyme conjugates. The conjugates were first prepared in an aqueous solution by covalently linking enzyme molecules and either horseradish peroxidase (HRP) or bovine carbonic anhydrase (BCA) along the chains of a water-soluble second-generation dendronized polymer using an established procedure. The obtained conjugates are stable biohybrid structures in which the linking unit between the dendronized polymer and each enzyme molecule is a bisaryl hydrazone (BAH) bond. Quantitative and reproducible enzyme immobilization inside the monolith is possible by simply adding a defined volume of a conjugate solution of a defined enzyme concentration to a dry monolith piece of the desired size. In that way, (i) the entire volume of the conjugate solution is taken up by the monolith piece due to capillary forces and (ii) all conjugates of the added conjugate solution remain stably adsorbed (immobilized) noncovalently without detectable leakage from the monolith piece. The observed flow-through activity of the resulting enzyme reactors was directly proportional to the amount of conjugate used for the reactor preparation. With conjugate solutions consisting of defined amounts of both types of conjugates, the controlled coimmobilization of the two enzymes, namely, BCA and HRP, was shown to be possible in a simple way. Different stability tests of the enzyme reactors were carried out. Finally, the enzyme reactors were applied to the catalysis of a two-enzyme cascade reaction in two types of enzymatic flow-through reactor systems with either coimmobilized or sequentially immobilized BCA and HRP. Depending on the composition of the substrate solution that was pumped through the two types of enzyme reactor systems, the coimmobilized enzymes performed significantly better than the sequentially immobilized ones. This difference, however, is not due to a molecular proximity effect with regard to the enzymes but rather originates from the kinetic features of the cascade reaction used. Overall, the method developed for the controllable and reproducible immobilization of enzymes in the macro- and mesoporous silica monolith offers many possibilities for systematic investigations of immobilized enzymes in enzymatic flow-through reactors, potentially for any type of enzyme.
尽管已知有许多不同方法可将酶固定在固体载体上，用于作为酶反应器的流通应用，但可重复地固定预定量的具有催化活性的酶分子仍然具有挑战性。使用大孔和介孔二氧化硅整体柱作为载体以及树枝状聚合物 - 酶共轭物解决了这一挑战。共轭物首先在水溶液中通过既定程序沿着水溶性第二代树枝状聚合物的链共价连接酶分子与辣根过氧化物酶（HRP）或牛碳酸酐酶（BCA）来制备。所得到的共轭物是稳定的生物杂化结构，其中树枝状聚合物与每个酶分子之间的连接单元是双芳基腙（BAH）键。通过简单地将确定体积的具有确定酶浓度的共轭物溶液添加到所需尺寸的干燥整体柱片上，就可以在整体柱内实现定量且可重复的酶固定。通过这种方式，（i）由于毛细作用力，共轭物溶液的整个体积被整体柱片吸收，并且（ii）添加的共轭物溶液中的所有共轭物保持稳定地非共价吸附（固定），没有可检测到的从整体柱片泄漏的情况。所得到的酶反应器的观察到的流通活性与用于反应器制备的共轭物的量直接成比例。对于由确定量的两种共轭物组成的共轭物溶液，已表明可以以简单的方式实现两种酶即BCA和HRP的可控共固定。对酶反应器进行了不同的稳定性测试。最后，将酶反应器应用于两种类型的酶流通反应器系统中两种酶级联反应的催化，这两种系统中BCA和HRP要么是共固定的，要么是顺序固定的。根据泵入两种类型酶反应器系统的底物溶液的组成，共固定的酶的性能明显优于顺序固定的酶。然而，这种差异不是由于酶的分子邻近效应，而是源于所使用的级联反应的动力学特征。总体而言，所开发的用于在大孔和介孔二氧化硅整体柱中可控且可重复地固定酶的方法为在酶流通反应器中系统研究固定化酶提供了许多可能性，可能适用于任何类型的酶。
