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 Km value of 6.1 mg mL-1 and a Vmax 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.