Study On κ-carrageenase Of Marine Bacterium Pseudoalteromonas Sp.QY203

Carrageenans are sulfated galactans consisting of D-galactose residues linked byalternating α-1,3and β-1,4linkages which are a commercially important group in thecell walls of red seaweeds. The three most industrially exploited carrageenans, namely,κ-, ι-and λ-carrageenan, are distinguished by the presence of one, two or threeester-sulfate groups per repeating disaccharide unit, respectively. Carrageenans arewidely used for industrial applications owing to their unique physicochemicalproperties. The κ-Carrageen-derived sulfated oligosaccharides have been reported toshow potential anti-viral, anti-tumor, anti-inflammation, antioxidant andimmunoloregulation activities.The κ-Carrageenases （EC3.2.1.83） can cleave the internal β-1,4linkages ofκ-carrageenans to yield oligogalactans of the neocarrabiose or neoagarobiose series. Itis a useful tool for the structural analysis of the cell walls and protoplast isolationfrom red alga.Carrageenans provide a unique polysaccharide-polysaccharidase systemto investigate the influence of ester-sulfate groups on the structure/functionrelationships of the hydrolases that degrade sulfated polysaccharides.In this study, a new κ-carrageenase CgkP from marine bacterium Pseudoalteromonassp. QY203was purified and characterized. Identification of strain QY203wasaccomplished using the16S rDNA region of the rDNA sequence. The phylogenetictree proves that the strain QY203belongs to the genus Pseudoalteromonas.The extracellular κ-carrageenase CgkP was purified to homogeneity and gave a singleband on SDS-PAGE with a molecular mass of34.0kDa. CgkP was most active at45°C and pH7.2. The addition of NaCl enhanced its activity markedly. Anothermonovalent metal ion K+also enhanced its activity. However, all of tested divalentand trivalent metal ions, such as Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Al3+, Fe3+, showed a significantly inhibitory effect except for Mg²⁺. CgkP was stable over a broad pH range（6.0-9.0） and remained70%of original activity after incubation at40°C for48h.CgkP remained80%of the activity after incubation for1h at45°C.One of the encoding gene of κ-carrageenase was cloned using degenerate PCR andinverse PCR. The amino acid sequence of CgkX deduced from the full-length genewas aligned using NCBI Basic Local Alignment Search Tool （BLAST）. The sequenceof cgkX exhibited95%homology with cgkA from Pseudoalteromonascarrageenovora ATCC43555. From sequence and protein domain analyses, CgkPbelongs to family16of the glycoside hydrolases.It exhibited endo-κ-carrageenase activity to depolymerize the κ-carrageenanmolecules into mainly disaccharide and tetrasaccharide. When κ-carrageenan wascompletely degraded with CgkP, the reaction mixture was analyzed using TLC. Theresults indicated that the end products were disaccharide and tetrasaccharide. Theenzyme hydrolysates were applied to a Biogel-P6column and most of them wereeluted as two distinct peaks. These two main end products were further identified asκ-carrageenan derived neocarratetraose and neocarrahexraose by electrosprayionization mass spectrometry.In this study, an endo-κ-carrageenase CgkP was purified from Pseudoalteromonas sp. QY203. Theprocedures for the production and purification of κ-carrageenase CgkP are simple and reliable.CgkP showed higher specific activity and thermostability compared with other κ-carrageenasereported previously. Therefore, CgkP is of interest of preparation of κ-carrageenan derivedoligosaccharides and protoplast of the red algae.