Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2
Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2
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海洋细菌溶纤维噬菌体 N5-2 菌株中新型 κ-卡拉胶酶的克隆、鉴定和表征
DOI：
10.1016/j.ijbiomac.2017.07.071
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发表时间：
2017-12-01
影响因子：
8.2
通讯作者：
Yao, Ziang
中科院分区：
文献类型：
--
作者：
Cui, Hongli;Peng, Yuxin;Yao, Ziang
从海洋细菌Cellulophaga lytica菌株N5-2中克隆并异源表达了一个新的κ-卡拉胶酶基因（Cly-kappa-car）。该基因全长1488 bp，开放阅读框架，编码495个氨基酸残基。推导的蛋白质的计算分子量为55.24 kDa，估计等电点为9.90。多重比对分析显示Cly-kappa-CAR与来自Zobellia sp. M-2（46%）、Zobellia galactanivorans（42%）和Rhodopirellula islandica（38%）的κ-卡拉胶酶具有相同性。重组Cly-kappa-CAR（R-Cly-kappa-CAR）在35 ℃、pH 7.0、0.7% κ-角叉菜胶和0.6%NaCl存在下具有620.08 U/mg的最大比活性。在低于35 ℃热处理2小时后，它保留了>75%的初始活性。在pH5.0 -8.0、4 ℃条件下培养6 h，酶活力仍保持在50%以上。κ-角叉菜胶的K-m和V-max值分别为0.94 mg/ml和13.42 mM/min/mg。R-Cly-kappa-CAR水解产物的薄层色谱分析显示，该酶将κ-卡拉胶水解成新-κ-卡拉胶八糖和新-κ-卡拉胶六糖。R-Cly-kappa-CAR是一种新型的κ-卡拉胶酶，可用于生产具有多种生物活性的高聚合度κ-卡拉胶寡糖。(C)2017由Elsevier B. V.出版
A novel kappa-carrageenase gene (Cly-kappa-car) was cloned and heterologously expressed from marine bacterium Cellulophaga lytica strain N5-2. The gene comprised an open reading frame of 1488 bp encoding 495 amino acid residues. The deduced protein had a calculated molecular weight of 55.24 kDa with an estimated isoelectric point of 9.90. Multiple alignment analysis revealed that Cly-kappa-CAR shared identity with kappa-carrageenases from Zobellia sp. M-2 (46%), Zobellia galactanivorans (42%) and Rhodopirellula islandica (38%). Recombinant Cly-kappa-CAR (R-Cly-kappa-CAR) had maximum specific activity of 620.08 U/mg at 35 degrees C, pH 7.0, 0.7% kappa-carrageenan and in the presence of 0.6% NaCl. It retained >75% of its initial activity after heat treatment below 35 degrees C for 2 h. More than 50% of its activity was maintained after incubation at pH 5.0-8.0 and 4 degrees C for 6 h. The K-m and V-max values for kappa-carrageenan were 0.94 mg/ml and 13.42 mM/min/mg, respectively. Thin layer chromatographic analysis of the R-Cly-kappa-CAR hydrolysis products revealed that the enzyme hydrolyzed kappa-carrageenan into neo-kappa-carraoctaose and neo-kappa-carrahexaose. R-Cly-kappa-CAR is a novel kappa-carrageenase enzyme and could be a valuable tool to produce high degree of polymerization kappa-carrageenan oligosaccharides with various biological activities. (C) 2017 Published by Elsevier B.V.