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Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products
School of Life Science and Technology, Dalian University, Dalian 116622, China
School of Life Science, Dalian Nationalities University, Dalian 116600, China
* Author to whom correspondence should be addressed.
Received: 16 September 2013; in revised form: 4 December 2013 / Accepted: 6 December 2013 / Published: 17 December 2013
Abstract: A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 188.8.131.52) 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.
Keywords: κ-carrageenase; Cellulophaga lytica strain N5-2; characterization; degradation product
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Yao, Z.; Wang, F.; Gao, Z.; Jin, L.; Wu, H. Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products. Int. J. Mol. Sci. 2013, 14, 24592-24602.
Yao Z, Wang F, Gao Z, Jin L, Wu H. Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products. International Journal of Molecular Sciences. 2013; 14(12):24592-24602.
Yao, Ziang; Wang, Feifei; Gao, Zheng; Jin, Liming; Wu, Haige. 2013. "Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products." Int. J. Mol. Sci. 14, no. 12: 24592-24602.