中文摘要：

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1194bp，编码一个由398个氨基酸残基细成的蛋白酶，该蛋白酶的分子量为48ku，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肛卡拉胶；该酶的最适温度和pH分别是55℃和pH9．0，重组酶在40℃保温1h可保持95．0％的活性，在45℃保温1h残余酶活与对照比较仍保留60．0％，在pH8．0-10．0的缓冲液中处理30min，仍能保持80％以上的酶活力，低浓度的Na^＋和K^＋以及1mmol／L的Sn^2＋、Fe^3＋、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＋和心以及1mmol／L的Cd^2＋、Ba^2＋、Mg^＋、Fe^2＋、Ca^2＋对重组酶活性有不同程度的抑制作用。

英文摘要：

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 x-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-carmgeenase belonged to the GH16 glycohydrolase family. The enzymatic properties of the recombinant κ-carrageenase were studied, and this enzyme specifically degraded κ-carmgeenan. The results also indicated that the optimal temperature and pH for the enzyme were 55 ℃ and pH 9.0, respectively. The recombinant κ-carmgeenase 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 Sn^2＋, Fe^3＋, ethylene diamine tetraacetic acid （EDTA）, dithiothreitol （DTT）, Tween-20, Tween-80, Triton X-100, fl-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 Cd^2＋, Ba^2＋, Mg^2＋, Fe^2＋, and Ca^2＋.