College of Biological Science and Engineering, Fujian Provincial Key Laboratory of Marine Enzyme Engineering, Fuzhou University, China, 350108.
College of Biological Science and Engineering, Fujian Provincial Key Laboratory of Marine Enzyme Engineering, Fuzhou University, China, 350108.
Int J Biol Macromol. 2019 Jun 1;130:958-968. doi: 10.1016/j.ijbiomac.2019.02.109. Epub 2019 Feb 19.
Carrageenase is useful for preparation of carrageenan oligosaccharides, which have significant bioactivity. We expressed a κ‑carrageenase gene from Zobellia sp. ZL-4 in full-length (κ-ZL-4) or after truncation of the carbohydrate binding module and the Type-IX secretion module (κ-ZL-4-GH16). κ-ZL-4-GH16 showed a specific activity (134.22 U/mg) 1.93 times higher than that of κ-ZL-4, and its thermal and pH stability also increased. The best activity of κ-ZL-4-GH16 was presented at pH 3.0-6.0, which was lower than the optimal pH of reported κ-carrageenases. The enzyme-substrate affinity of κ-ZL-4-GH16 was higher than that of κ-ZL-4, demonstrated by its lower Michaelis-Menten constant (0.704 mg/mL at pH 6.0). Importantly, κ-ZL-4-GH16 released 10-fold more κ-carrageenan disaccharides than κ-ZL-4. The κ-carrageenan tetrose and hexose produced by the two enzymes were purified and structurally identified. Molecular docking with κ-carrageenan hexose suggested that the efficiency improvement after truncation might be attributed to the conformation differences of the two enzymes.
卡拉胶酶可用于制备具有显著生物活性的卡拉胶低聚糖。我们在全长（κ-ZL-4）或截短碳水化合物结合模块和 Type-IX 分泌模块后（κ-ZL-4-GH16）表达了来自 Zobellia sp. ZL-4 的 κ-卡拉胶酶基因。κ-ZL-4-GH16 的比活（134.22 U/mg）比 κ-ZL-4 高 1.93 倍，其热稳定性和 pH 稳定性也有所提高。κ-ZL-4-GH16 的最佳活性出现在 pH 3.0-6.0 之间，低于报道的 κ-卡拉胶酶的最适 pH。κ-ZL-4-GH16 的酶-底物亲和力高于 κ-ZL-4，其米氏常数（在 pH 6.0 时为 0.704 mg/mL）较低。重要的是，κ-ZL-4-GH16 释放的 κ-卡拉胶二糖是 κ-ZL-4 的 10 倍。两种酶产生的 κ-卡拉胶四糖和六糖被纯化并进行了结构鉴定。与 κ-卡拉胶六糖的分子对接表明，截短后效率提高可能归因于两种酶的构象差异。
