Abstract: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+.
