Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2

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 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-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 620.08 U/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 2 h. More than 50% of its activity was maintained after incubation at pH 5.0-8.0 and 4 °C for 6 h. The Km and Vmax values for κ-carrageenan were 0.94 mg/ml and 13.42 mM/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.