Cloning, expression, and biochemical characterization of κ-carrageenase from marine bacterium Cellulophaga sp. P-1

A novel cold-adapted κ-carrageenase (Car2891) was identified from the marine bacterium Cellulophaga sp. P-1 isolated from Chondrus. The enzyme was recombinantly expressed in E. coli BL21(DE3) and biochemically characterized. Car2891 exhibited optimal activity at 30 °C and pH 8.0, maintaining high stability and catalytic performance from 0 °C to 30 °C under neutral-weakly alkaline conditions. Its activity was enhanced by Na⁺, Mg²⁺, and Mn²⁺ but strongly inhibited by Hg²⁺, Cu²⁺, Ni²⁺, and EDTA. Kinetic analysis revealed a K_m value of 6.1 mg mL^-1 and a V_max value of 31.65 mg mL^-1 min^-1 for κ-carrageenan. Hydrolysis pattern analysis revealed an endo-acting cleavage mechanism, initially yielding hexa- and tetrasaccharides, with disaccharides and tetrasaccharides as the final major products. These properties demonstrate Car2891's potential as an efficient and controllable biocatalyst for the environmentally friendly processing of κ-carrageenan and the scalable production of functional oligosaccharides.