Xia Wenli, Xie Jingcong, Zhao Linguo, Pei Jianjun
National Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, China; Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China.
National Key Laboratory for Development and Utilization of Forest Food Resources, Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, Nanjing 210042, China.
Bioorg Chem. 2025 Sep;164:108823. doi: 10.1016/j.bioorg.2025.108823. Epub 2025 Jul 31.
Thermophilic enzymes possess numerous favorable properties for biotransformation, including high catalytic activity, exceptional thermal stability, and ease of purification and preparation. However, the challenge arises from the lack of suitable enzyme immobilization materials that exhibit both thermal tolerance and reusability, thereby hindering the effective and recurrent utilization of thermophilic enzymes. In this study, a specially designed dual-network hybrid-gel consisting of acrylamide (AM) and sodium alginate (SA) was developed to serve as a thermotolerant and reusable carrier for the efficient immobilization of a thermotolerant α-rhamnosidase IagRha. The optimal amounts of AM and SA were determined to be 5 % and 2 %, respectively. Following the step of acrylamide crosslinking to establish the skeleton of hybrid-gel, a subsequent immersion in a Ca solution facilitated the formation of ionic bonds between SA and Ca, thereby adjusting the internal voids of the hydrogel, which effectively enhanced the thermal stability and enzyme immobilization efficiency of the hybrid-gel. This innovative approach resulted in the achievement of 93.65 % recovery of enzyme activity, along with remarkable enhancements in enzymatic properties. The immobilized α-rhamnosidase, AM-SA-IagRha, demonstrated a significant 1.34-fold increase in the V value, rising from 5.28 U/mg to 7.06 U/mg, compared to free IagRha. Furthermore, AM-SA-IagRha exhibited excellent thermostability, with a half-life of 99.0 min at 90 °C, which was 1.57-fold higher than that of free IagRha. After four cycles of reuse, the productivities of rutin and epmdin C by the immobilized enzyme were 1.91-fold and 2.53-fold that of the free enzyme, respectively. Additionally, the enzyme activity of AM-SA-IagRha remained at 90.16 % after storage at 4 °C for 30 days, and retained at 85.63 % after 20 cycles of reuse, demonstrating exceptional storage stability and reusability. The optimal expression conditions for IagRha were identified in TB medium incubated at 37 °C without IPTG, resulting in an enzyme activity of 6.32 U/mL. These significant findings underscore the potential of AM-SA-IagRha for industrial applications, particularly in high-temperature biotransformation.
嗜热酶具有许多有利于生物转化的特性，包括高催化活性、出色的热稳定性以及易于纯化和制备。然而，挑战在于缺乏既具有耐热性又具有可重复使用性的合适酶固定化材料，从而阻碍了嗜热酶的有效和反复利用。在本研究中，开发了一种由丙烯酰胺（AM）和海藻酸钠（SA）组成的特殊设计的双网络混合凝胶，用作耐热且可重复使用的载体，以高效固定耐热α-鼠李糖苷酶IagRha。确定AM和SA的最佳用量分别为5%和2%。在丙烯酰胺交联以建立混合凝胶骨架的步骤之后，随后浸入Ca溶液中促进了SA和Ca之间离子键的形成，从而调节了水凝胶的内部空隙，有效提高了混合凝胶的热稳定性和酶固定化效率。这种创新方法实现了93.65%的酶活回收率，同时酶学性质也有显著提高。与游离IagRha相比，固定化α-鼠李糖苷酶AM-SA-IagRha的V值显著增加了1.34倍，从5.28 U/mg升至7.06 U/mg。此外，AM-SA-IagRha表现出优异的热稳定性，在90°C下的半衰期为99.0分钟，比游离IagRha高1.57倍。经过四个循环的重复使用后，固定化酶生产芦丁和表儿茶素C的产率分别是游离酶的1.91倍和2.53倍。此外，AM-SA-IagRha在4°C储存30天后酶活仍保持在90.16%，在20个循环的重复使用后仍保留85.63%，显示出优异的储存稳定性和可重复使用性。IagRha的最佳表达条件是在37°C下于不含IPTG的TB培养基中培养，酶活为6.32 U/mL。这些重要发现突出了AM-SA-IagRha在工业应用中的潜力，特别是在高温生物转化方面。
