School of Life Science and Technology, Dalian University, Dalian 116622, China; Key Laboratory of Marine Biotechnology of Dalian City, Dalian 116622, China; Key Laboratory of Glycolipid Metabolism of Liaoning Province, Dalian 116622, China; School of Life Science and Technology, Dalian University,10 Xuefu Street, Dalian Economic Technological Development Zone, Dalian, Liaoning, 116622, China.
School of Life Science and Technology, Dalian University, Dalian 116622, China; Key Laboratory of Marine Biotechnology of Dalian City, Dalian 116622, China; Key Laboratory of Glycolipid Metabolism of Liaoning Province, Dalian 116622, China; School of Life Science and Technology, Dalian University,10 Xuefu Street, Dalian Economic Technological Development Zone, Dalian, Liaoning, 116622, China.
Int J Biol Macromol. 2017 Dec;105(Pt 1):509-515. doi: 10.1016/j.ijbiomac.2017.07.071. Epub 2017 Jul 20.
A novel κ-carrageenase gene (Cly-κ-car) was cloned and heterologously expressed from marine bacterium Cellulophaga lytica strain N5-2. The gene comprised an open reading frame of 1488bp encoding 495 amino acid residues. The deduced protein had a calculated molecular weight of 55.24kDa with an estimated isoelectric point of 9.90. Multiple alignment analysis revealed that Cly-κ-CAR shared identity with κ-carrageenases from Zobellia sp. M-2 (46%), Zobellia galactanivorans (42%) and Rhodopirellula islandica (38%). Recombinant Cly-κ-CAR (R-Cly-κ-CAR) had maximum specific activity of 620.08U/mg at 35°C, pH 7.0, 0.7% κ-carrageenan and in the presence of 0.6% NaCl. It retained >75% of its initial activity after heat treatment below 35°C for 2h. More than 50% of its activity was maintained after incubation at pH 5.0-8.0 and 4°C for 6h. The K and V values for κ-carrageenan were 0.94mg/ml and 13.42mM/min/mg, respectively. Thin layer chromatographic analysis of the R-Cly-κ-CAR hydrolysis products revealed that the enzyme hydrolyzed κ-carrageenan into neo-κ-carraoctaose and neo-κ-carrahexaose. R-Cly-κ-CAR is a novel κ-carrageenase enzyme and could be a valuable tool to produce high degree of polymerization κ-carrageenan oligosaccharides with various biological activities.
从海洋细菌 Cellulophaga lytica 菌株 N5-2 中克隆并异源表达了一种新型 κ-卡拉胶酶基因（Cly-κ-car）。该基因包含一个 1488bp 的开放阅读框，编码 495 个氨基酸残基。推导的蛋白质的计算分子量为 55.24kDa，估计等电点为 9.90。多重比对分析表明，Cly-κ-CAR 与 Zobellia sp. M-2（46%）、Zobellia galactanivorans（42%）和 Rhodopirellula islandica（38%）的 κ-卡拉胶酶具有同源性。重组 Cly-κ-CAR（R-Cly-κ-CAR）在 35°C、pH7.0、0.7%κ-卡拉胶和 0.6%NaCl 存在下的最大比活性为 620.08U/mg。在 35°C 以下热处理 2 小时后，其保留了初始活性的>75%。在 pH5.0-8.0 和 4°C 孵育 6 小时后，其活性保持在 50%以上。κ-卡拉胶的 K 和 V 值分别为 0.94mg/ml 和 13.42mM/min/mg。R-Cly-κ-CAR 水解产物的薄层色谱分析表明，该酶将 κ-卡拉胶水解成 neo-κ-carraoctaose 和 neo-κ-carrahexaose。R-Cly-κ-CAR 是一种新型的 κ-卡拉胶酶，可作为生产具有各种生物活性的高聚合度 κ-卡拉胶寡糖的有价值工具。
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