Purification of kappa (k)-carrageenase from locally isolated Cellulosimicrobium cellulans

Abstract

Partial purification of the crude kappa (k)-carrageenase present in the culture filtrates of Cellulosimicrobium cellulans was carried out by fractional precipitation, using ammonium sulphate, acetone and ethanol individually. The highest recovered protein (37.08%) combined with enzyme activity was obtained with ammonium sulphate. The fraction precipitated by 90% ammonium sulphate was re-purified by anion exchange chromatography diethylaminoethyl (DEAE) cellulose, A-52 and 79 fractions were obtained. The loaded protein was separated into 4 peaks. The third protein peak was the major one which contained the most recovered enzyme activity (84.95%) from the eluted fractions. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. The k-carrageenase activity was fractionated into 2 peaks. The first peak was the major one containing 95.622% of the total recovered activity. The pooled fractions of the major protein component showed a specific k-carrageenase activity of 46.22 U/mg protein, yielding about 4.6 fold purification of the crude enzyme preparation. Some properties of purified k-carrageenase obtained from cellusimicrobium cellulans cultures were studied. The optimum reaction temperature of the purified k-carrageenase was 30°C and the maximum activity occurred at a reaction pH of 6.

Key words: Cellulosimicrobium cellulans, k-carrageenase, purification, sephadex G-100, diethylaminoethyl (DEAE) sephadex A-52.