title: Characterization of a κ-carrageenase from marine Cellulophaga lytica strain N5-2 and analysis of its degradation products.
abstract: A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration on SephadexG-200 and SephadexG-75. The purified enzyme was verified as a single protein on SDS-PAGE, and whose molecular weight was 40.8 kDa. The κ-carrageenase yielded a high activity of 1170 U/mg protein. For κ-carrageenase activity, the optimum temperature and pH were 35 °C and pH 7.0, respectively. The enzyme was stable at 40 °C for at least 2.5 h. The enzyme against κ-carrageenan gave a Km value of 1.647 mg/mL and a Vmax value of 8.7 μmol/min/mg when the reaction was carried out at 35 °C and pH 7.0. The degradation products of the k-carrageenase were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS) and 13C-NMR spectroscopy, and the results indicated that the enzyme was specific of the β-1,4 linkage and hydrolyzed κ-carrageenan into κ-neocarraoctaose-sulfate and κ-neocarrahexaose-sulfate first, and then broke κ-neocarraoctaose-sulfate into κ-neocarrabiose-sulfate and κ-neocarrahexaose-sulfate.
authors.forename: Ziang
authors.initials: Z
authors.lastname: Yao
authors.forename: Feifei
authors.initials: F
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Gao
authors.forename: Liming
authors.initials: L
authors.lastname: Jin
authors.affiliation: School of Life Science and Technology, Dalian University, Dalian 116622, China. haige-hu@163.com.
authors.forename: Haige
authors.initials: H
authors.lastname: Wu
mesh_terms: D002351:Carrageenan / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D041965:Flavobacteriaceae / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010802:Phylogeny; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
chemical_list: D002351:Carrageenan; D006026:Glycoside Hydrolases