Boroša Vilim Marijan, Koštan Kristian, Vičević Renata, Cingesar Ivan Karlo, Vrsaljko Domagoj, Zelić Bruno, Jurinjak Tušek Ana, Šalić Anita
University of Zagreb Faculty of Chemical Engineering and Technology, Marulićev Trg 19, HR-10000 Zagreb, Croatia.
Department of Packaging, Recycling and Environmental Protection, University North, Trg dr. Žarka Dolinara 1, HR-48000 Koprivnica, Croatia.
Micromachines (Basel). 2024 Dec 20;15(12):1514. doi: 10.3390/mi15121514.
Enzymatic reactions play an important role in numerous industrial processes, e.g., in food production, pharmaceuticals and the production of biofuels. However, a major challenge when using enzymes in industrial applications is maintaining their stability and activity, especially under harsh operating conditions. To solve this problem, enzyme immobilization techniques have been developed. Immobilization involves fixing the enzymes on solid supports, which increases their stability, enables their reusability and facilitates the easy separation of reaction mixtures. In addition, immobilized enzymes are ideal for continuous flow systems such as millireactors, where they allow better control of reaction conditions, improving efficiency and product consistency. Glucose dehydrogenase is an important enzyme in biotechnology, particularly in biosensors and the production of biofuels, as it catalyzes the oxidation of glucose to gluconolactone, reducing NAD to NADH. However, like many other enzymes, it tends to lose activity over time. The immobilization of glucose dehydrogenase in a millireactor provides a controlled environment that increases the stability and activity of the enzyme. The aim of this study was to investigate the effects of different immobilization strategies on the performance of glucose dehydrogenase in a 3D printed millireactor. The enzyme was immobilized in alginate gel in three immobilization strategies: as beads, on the bottom surface, and on both the top and bottom surfaces of the millireactor. The results showed that the application of the enzyme on both surfaces improved the glucose conversion two-fold compared to immobilization in beads and four-fold compared to immobilization only on the bottom surface. The dual-surface enzyme immobilization strategy showed the highest efficiency, achieving the highest conversion of 95.76 ± 1.01% ( = 131 min) and NADH productivity of 0.166 ± 0.01 mmol/(L·min) ( = 7.11 min) combined with operational stability over five days. Effective diffusion rates comparable to those of aqueous solutions confirmed the suitability of alginate gels for biocatalysis. These advancements highlight the potential of this modular and scalable platform for various biotechnological applications.
酶促反应在众多工业过程中发挥着重要作用，例如在食品生产、制药和生物燃料生产中。然而，在工业应用中使用酶时的一个主要挑战是保持其稳定性和活性，特别是在恶劣的操作条件下。为了解决这个问题，人们开发了酶固定化技术。固定化包括将酶固定在固体载体上，这增加了它们的稳定性，使其能够重复使用，并便于反应混合物的轻松分离。此外，固定化酶对于诸如微反应器之类的连续流动系统是理想的，在这些系统中，它们可以更好地控制反应条件，提高效率和产品一致性。葡萄糖脱氢酶是生物技术中的一种重要酶，特别是在生物传感器和生物燃料生产中，因为它催化葡萄糖氧化为葡萄糖酸内酯，将NAD还原为NADH。然而，与许多其他酶一样，它往往会随着时间的推移而失去活性。将葡萄糖脱氢酶固定在微反应器中提供了一个可控的环境，提高了酶的稳定性和活性。本研究的目的是研究不同固定化策略对3D打印微反应器中葡萄糖脱氢酶性能的影响。酶通过三种固定化策略固定在藻酸盐凝胶中：制成珠子、固定在微反应器的底面以及固定在微反应器的顶面和底面。结果表明，与制成珠子固定化相比，酶在两个表面上的应用使葡萄糖转化率提高了两倍，与仅固定在底面相比提高了四倍。双面酶固定化策略显示出最高的效率，实现了95.76±1.01%（ = 131分钟）的最高转化率和0.166±0.01 mmol/（L·分钟）（ = 7.11分钟）的NADH生产率，并在五天内具有操作稳定性。与水溶液相当的有效扩散速率证实了藻酸盐凝胶适用于生物催化。这些进展突出了这个模块化和可扩展平台在各种生物技术应用中的潜力。
