Cloning and biochemical characterization of a novel κ-carrageenase from newly isolated marine bacterium Pedobacter hainanensis NJ-02

Enzymatic preparation of carrageenan oligosaccharides has drawn increasing attention due to its advantages of mild reaction conditions and excellent product-specificity. A novel gene (CgkA) encoding a new κ-carrageenase was cloned, heterogeneously expressed and characterized from a newly isolated marine bacterium Pedobacter hainanensis NJ-02. It consisted of 1539 bp and encoded 512 amino acid residues with a molecular weight of 57.12 kDa. Multiple alignment analysis indicated that CgkA belongs to glycoside hydrolase (GH) family 16 and was most homologous to κ-carrageenase of Zobellia sp. M-2 with identity of 50%. The recombinant enzyme showed maximal activity of 3659.72 U/mg at 40 °C and pH 7.0. Additionally, it could retain more than 80% of its maximal activity after being incubated at pH of 5.0-9.0 below 40 °C. K⁺ and Na⁺ with a wide range of concentration can activate the enzyme, while other divalent ions such as Cu²⁺, Zn²⁺ showed inhibitory effect on the enzyme. The ESI–MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the carrageenan into tetrasaccharides and hexasaccharides. The results suggest that it is an endo-type carrageenase and could be a valuable enzyme tool to produce carrageenan oligosaccharides with higher Dps.