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Purification and characterization of κ-carrageenase from marine bacterium mutant strain pseudoalteromonas sp. aj5-13 and its degraded products

MA, Y-X., DONG, S-L., JIANG, X-L., LI, J., MOU, H-J.
Journal of food biochemistry 2010 v.34 no.3 pp. 661-678
macroalgae, ribosomal RNA, enzymes, polyacrylamide gel electrophoresis, chromatography, Apostichopus japonicus, gels, filtration, immunology, structure-activity relationships, Pseudoalteromonas, polysaccharides, Rhodophyta, cell walls, intestines, molecular weight, metabolism, amino acid sequences, antioxidant activity, ammonium sulfate, proteins, fractionation, biotechnology, pH, nucleotide sequences, bacteria
A κ-carrageenan-degrading bacterial strain AJ5 isolated from the intestine of Apostichopus japonicus was identified as Pseudoalteromonas sp. based on the phenotypic characters and 16S rRNA gene sequencing. The mutant Pseudoalteromonas sp. AJ5-13 with κ-carrageenase activity of 61 U/mg protein was obtained from Pseudoalteromonas sp. AJ5 using mutagenesis technique. An extracellular κ-carrageenase was purified from Pseudoalteromonas sp. AJ5-13 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-cellulose 52). The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel κ-carrageenase. The pI and Km of the enzyme were 8.5 and 9.8 ± 0.2 mg/mL, respectively. The enzyme exhibited maximal activity at pH 8.0 and 55C. It hydrolyzed the β-1, 4-glycosidic linkages of κ-carrageenan yielding κ-neocarrabiose, -tetraose, -hexaose, -octaose and -decaose sulfates as the main end-products. κ-Carrageenases degrade κ-carrageenan by hydrolyzing the β-1,4 linkages to a series of oligosaccharides. Thus, it is expected that like other κ-carrageenases, the κ-carrageenase isolated from Pseudoalteromonas sp. AJ5-13 would also be useful in seaweed biotechnology, pharmacy and immunology. κ-Carrageenases can be applied to study the composition and structure of carrageenans from different red alga, and to study the bacterial κ-carrageenan metabolism. They also provide the opportunity to investigate the structure-function relationship of the hydrolases that degrade self-associating sulfated polysaccharides. Examples of the practical applications of κ-carrageenases include their use in degrading the cell walls of seaweeds to obtain protoplasts, and in hydrolyzing κ-carrageenan to produce oligosaccharides. κ-Carrageenan-oligosaccharides have various potential biological properties, such as antiviral, antitumor, antioxidant activities, cytoprotection, immunomodulation, etc.