Studies On Chemical And Biological Properties Of κ-Neocarratetraose And κ-Neocarrahexaose By Enzymatic Depolymerization

69 Strains with carrageenan degrading activity were isolated from marine environment after repeating enrichment and selection. After treated by ultraviolet and EMS, a mutant M2-4-4 was produced from Cytophaga MCA-2, with a higher enzyme activity of 66U/ml. Bacterial growth and enzyme production condition was designed as follows: NaCl 15‰, carrageenan 2‰, NaNO3 2‰, yeast extract l‰, stirrer speed 150r/min, initial pH 7.5 and temperature 32℃ in shaking incubator for 36 h. From the cell free supernatant of cultures grown on crude k-carrageenan, the carrageenase was purified by ammonium sulfate fractionation, ion-exchange chromatography on Q-Sepharose FF and gel filtration on Sephacryl S-100HR. The purified carrageenase was detected as a single protein upon SDS-PAGE, with a molecular weight at 30kDa. Optimal activity was found at pH6-8 and a temperature range from 32 to 38℃. However, the enzyme is totally inactivated by heat treatment at 55℃ for 30min or 60℃ for 10min. It is stable over the pH range 5-8. Al3+, Hg2+ and Pb2+ can intensively inhibit the enzyme activity. After fractionated by Q-Sepharose FF, the composition and structure of main enzyme depolymerized end-products was examined by MALDI-TOF-MS, 13C-NMR and IR spectroscopy. Carrageenase from Cytophaga M2-4-4 acts as an endo-enzyme, specifically hydrolyzing the β-glycosidic linkage between 3,6-anhydro-D-galactose and D-galactose. The main depolymerized end-products were determined as k-neocarratetraose sulfate and k-neocarrahexaose sulfate and with 3-linkedβ-D-galactopyranose 4-sulphate as the reducing end.The anti-tumor activities of enzymatic depolymerized products with different molecular weight and sulfur content were determined by using Sarcoma 180 tumor in mouse. A carrageenan oligosaccharide with a molecular weight of 1726, sulfur content 15.1% administered orally at a dose of 100 mg/kg mouse markedly inhibit tumor formation. The activities on superoxide dismutase and catalase are enhanced considerably, which suggests that Neo-carrageenan oligosaccharide is effective in promoting the antioxidation ability and eliminating danger from free radicals. It also shows obvious antioxidative activities in vitro, including inhibition of LPO, removing of OH, H2O2 and O2-. The preparations show special effects on immunological regulation, especially the phagocytosis ratio and phagocytosis index of macrophage, and lysozyme activity of serum, which might be great beneficial for the anti-tumor activity.