Ghéczy Nicolas, Sasaki Kai, Yoshimoto Makoto, Pour-Esmaeil Sajad, Kröger Martin, Stano Pasquale, Walde Peter
Laboratory for Multifunctional Materials, Department of Materials, ETH Zürich Vladimir-Prelog-Weg 5 CH-8093 Zürich Switzerland
Department of Applied Chemistry, Yamaguchi University Tokiwadai 2-16-1 Ube 755-8611 Japan.
RSC Adv. 2020 May 18;10(32):18655-18676. doi: 10.1039/d0ra01204a. eCollection 2020 May 14.
Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) - with hydrogen peroxide (HO) as HRP "activator" - was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2',7'-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH-DA/HO substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).
目前，固定化酶在流通系统中的酶催化级联反应在探索其在生物合成和生物分析应用方面的潜力时备受关注。该领域的基础研究通常旨在了解固定化酶的稳定性及其催化性能，例如，根据所需反应产物的产率、分析物检测限、酶稳定性或反应重现性。在本文介绍的工作中，首先在pH = 7.2的本体溶液中对涉及两种酶——牛碳酸酐酶（BCA）和辣根过氧化物酶（HRP）——的级联反应进行了详细研究，其中过氧化氢（HO）作为HRP的“激活剂”。所研究的反应是2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）水解并氧化为2',7'-二氯荧光素（DCF），发现该反应沿着两条反应途径进行。然后将这种双酶级联反应应用于分析固定在玻璃纤维滤器中的BCA和HRP的性能，这些滤器放置在一个滤器支架装置内，通过该装置泵送DCFH-DA/HO底物溶液。对（i）共固定化和（ii）顺序固定化的酶（先固定BCA，后固定HRP）进行了比较。基于在本体溶液中进行的定量紫外/可见吸收测量，可以解释两种排列在测量产物产率（DCF）方面的显著差异。我们发现，顺序固定化酶时观察到的较低DCF产率源于由于酶分离导致的两条可能反应途径之一的变化，而共固定化的酶（或在本体溶液实验中同时存在的酶）则并非如此。共固定化酶时观察到的较高DCF产率并非源于分子邻近效应（与顺序固定化相比，氧化没有增加）。
