摘要
We isolated a bacterial strain (HC4) that is able to degrade κ-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa.With 16S rRNA gene sequencing,we identified the strain as Tamlana sp.,and then purified an extracellular κ-carrageenase from a culture of Tamlana sp.HC4 by ammonium sulfate precipitation,Sephadex G-200 gel filtration chromatography,and DE-cellulose 52 anion-exchange chromatography.The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa.The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30°C,respectively.The enzyme is stable over the range of pH 7.2-8.6 below 45°C.The enzyme activity is strongly inhibited by Zn 2+ and Cu 2+ at 1 mmol/L.The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant (K m) at 7.63 mg/ml.Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13 C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level of κ-neocarrabiose sulfate.
We isolated a bacterial strain (HC4) that is able to degrade k-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular K-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30~C, respectively. The enzyme is stable over the range ofpH 7.2-8.6 below 45℃. The enzyme activity is strongly inhibited by Zn2+ and Cu2+ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant (Kin) at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level ofκ-neocarrabiose sulfate.
基金
Supported by the Open Project of Key Laboratory of Mariculture and Biotechnology, Ministry of Agriculture, Dalian Ocean University (No.K2006-12)