College of Food Science and Light Industry, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
World J Microbiol Biotechnol. 2023 Jun 7;39(8):222. doi: 10.1007/s11274-023-03668-3.
κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104 bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30 kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35 °C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35 °C for 60 min after enzymatic inactivation by boiling for 10 min. K, Na, and EDTA had an activating effect on the enzyme activity, while Ni, Cu, and Zn inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.
κ-卡拉胶寡糖具有多种生物活性。κ-卡拉胶酶降解 κ-卡拉胶导致聚合度（DP）不同的降解产物。从 Colwellia echini 中克隆出一种新型基因（CecgkA），该基因编码一种新的 κ-卡拉胶酶，并在大肠杆菌 BL21（DE3）中异源表达。该酶长 1104bp，编码 367 个氨基酸残基，分子量为 41.30kDa。多重比对分析表明，CeCgkA 属于糖苷水解酶（GH16）家族，与 Rhodopirellula maiorica SM1 的 κ-卡拉胶酶具有最高的同源性，同源性为 58%。CeCgkA 在 pH8.0 和 35°C 时表现出最大活性（453.15U/mg）。生化特性测定表明，CeCgkA 是一种热恢复酶，在酶失活后立即在 35°C 下放置 60min，可使样品中 51.6%的初始酶活性得到恢复，而在 10min 煮沸失活后。K、Na 和 EDTA 对酶活性有激活作用，而 Ni、Cu 和 Zn 则抑制酶活性。此外，TLC 和 ESI-MS 分析表明，CecgkA 的最大识别单元为十糖，主要降解产物为二糖、四糖和六糖，表明该酶为内切型卡拉胶酶。
