Zhu Chunhua, Mou Mingjing, Yang Leilei, Jiang Zedong, Zheng Mingjing, Li Zhipeng, Hong Tao, Ni Hui, Li Qingbiao, Yang Yuanfan, Zhu Yanbing
College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China.
College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China; Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China.
Int J Biol Macromol. 2023 Dec 1;252:126401. doi: 10.1016/j.ijbiomac.2023.126401. Epub 2023 Aug 18.
κ-Carrageenase can degrade κ-carrageenan to produce bioactive κ-carrageenan oligosaccharides (KCOs) that have potential applications in pharmaceutical, food, agricultural, and cosmetics industries. Immobilized enzymes gain their popularity due to their good reusability, enhanced stability, and tunability. In this study, the previously characterized catalytic domain of Pseudoalteromonas purpurea κ-carrageenase was covalently immobilized on the synthesized amine-modified zeolitic imidazolate framework-8 nanoparticles with the formation of cross-linked enzyme aggregates, and the immobilized κ-carrageenase was further characterized. The immobilized κ-carrageenase demonstrated excellent pH stability and good reusability, and exhibited higher optimal reaction temperature, better thermostability, and extended storage stability compared with the free enzyme. The KCOs produced by the immobilized κ-carrageenase could significantly decrease the TC, TG, and LDL-C levels in HepG2 cells, increase the HDL-C level in HepG2 cells, and reduce the free fatty acids level in Caco-2 cells. Biochemical assays showed that the KCOs could activate AMPK activity, increase the ratios of p-AMPK/AMPK and p-ACC/ACC, and downregulate the expression of the lipid metabolism related proteins including SREBP1 and HMGCR in the hyperlipidemic HepG2 cells. This study provides a novel and effective method for immobilization of κ-carrageenase, and the KCOs produced by the immobilized enzyme could be a potential therapeutic agent to prevent hyperlipidemia.
κ-卡拉胶酶可降解κ-卡拉胶以产生具有生物活性的κ-卡拉胶寡糖（KCOs），这些寡糖在制药、食品、农业和化妆品行业具有潜在应用。固定化酶因其良好的可重复使用性、增强的稳定性和可调性而受到欢迎。在本研究中，将先前表征的紫色假交替单胞菌κ-卡拉胶酶的催化结构域共价固定在合成的胺修饰沸石咪唑酯骨架-8纳米颗粒上，形成交联酶聚集体，并对固定化的κ-卡拉胶酶进行进一步表征。固定化的κ-卡拉胶酶表现出优异的pH稳定性和良好的可重复使用性，与游离酶相比，具有更高的最佳反应温度、更好的热稳定性和更长的储存稳定性。固定化κ-卡拉胶酶产生的KCOs可显著降低HepG2细胞中的总胆固醇（TC）、甘油三酯（TG）和低密度脂蛋白胆固醇（LDL-C）水平，提高HepG2细胞中的高密度脂蛋白胆固醇（HDL-C）水平，并降低Caco-2细胞中的游离脂肪酸水平。生化分析表明，KCOs可激活高脂血症HepG2细胞中的AMPK活性，增加p-AMPK/AMPK和p-ACC/ACC的比率，并下调包括SREBP1和HMGCR在内的脂质代谢相关蛋白的表达。本研究为κ-卡拉胶酶的固定化提供了一种新颖有效的方法，固定化酶产生的KCOs可能是预防高脂血症的潜在治疗剂。
