Characterization of a κ-carrageenase from marine Cellulophaga lytica strain N5-2 and analysis of its degradation products

Int J Mol Sci. 2013 Dec 17;14(12):24592-602. doi: 10.3390/ijms141224592.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrageenan / analysis*
  • Carrageenan / isolation & purification
  • Carrageenan / metabolism
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Flavobacteriaceae / enzymology*
  • Glycoside Hydrolases / classification
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism*
  • Hydrogen-Ion Concentration
  • Magnetic Resonance Spectroscopy*
  • Molecular Weight
  • Phylogeny
  • Spectrometry, Mass, Electrospray Ionization*
  • Temperature

Substances

  • Carrageenan
  • Glycoside Hydrolases