将来源于Pseudoalteromonas carrageenovora ASY5菌株的κ-卡拉胶酶基因克隆到载体pET-28a上，并转化到大肠杆菌BL21(DE3)中表达。克隆得到的κ-卡拉胶酶基因序列全长1194 bp，编码一个由398个氨基酸残基组成的蛋白酶，该蛋白酶的分子量为48 ku，结构域分析表明该κ-卡拉酶符合GH16家族的特点。对重组κ-卡拉胶酶的酶学性质进行考察：重组κ-卡拉胶酶只能够专一性的降解κ-卡拉胶；该酶的最适温度和pH分别是55 ℃和pH 9.0，重组酶在40 ℃保温1 h可保持95.0%的活性，在45 ℃保温1 h残余酶活与对照比较仍保留60.0%，在pH 8.0~10.0的缓冲液中处理30 min，仍能保持80%以上的酶活力，低浓度的Na+和K+以及1 mmol/L的Sn2+、Fe3+、EDTA、DTT、Tween-20、Tween-80和Triton X-100、β-mercaptoethanol、SDS、Urea对重组酶活具有显著的促进作用；而高浓度的Na+和K+以及1 mmol/L的Cd2+、Ba2+、Mg2+、Fe2+、Ca2+对重组酶活性有不同程度的抑制作用。

A κ-carrageenase gene from the bacterium Pseudoalteromonas carrageenovora ASY5 was cloned into a pET-28a expression vector and expressed in Escherichia coli BL21 (DE3). The length of the obtained κ-carrageenase gene sequence after cloning was 1194 base pairs (bp), and this gene encoded a protease (molecular weight: 48 ku) composed of 398 amino acid (AA) residues. Based on domain analysis, this κ-carrageenase belonged to the GH16 glycohydrolase family. The enzymatic properties of the recombinant κ-carrageenase were studied, and this enzyme specifically degraded κ-carrageenan. The results also indicated that the optimal temperature and pH for the enzyme were 55 ℃ and pH 9.0, respectively. The recombinant κ-carrageenase retained more than 95.0% and 60.0% of the maximum enzymatic activity after 1 h of incubation at 40 ℃ and below 45 ℃, respectively, and retained more than 80.0% of the maximum enzymatic activity after 30 min of incubation at a pH range of 8.0~10.0. The activity of the recombinant enzyme was significantly promoted at low concentrations of Na+ and K+, and 1 mmol/L Sn2+, Fe3+, ethylene diamine tetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20, Tween-80, Triton X-100, β-mercaptoethanol, sodium dodecyl sulfate (SDS), and urea. The enzymatic activity was inhibited to various degrees by high concentrations of Na+ and K+ and 1 mmol/L Cd2+, Ba2+, Mg2+, Fe2+, and Ca2+.

国家自然科学基金项目（31401632）；福建省重大专项专题项目（2015NZ0001-1）；厦门市科技计划项目（3502Z20120005）；厦门南方海洋研究中心项目（13GZP004NF10）