Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens
AbstractCellulases from the wild-type (WT) and two improved mutants (catabolite repression resistant mutant 4 and 24, abbreviated CRRmt4 and CRRmt24, respectively) of Pseudomonas fluorescens were purified to apparent homogeneity by ammonium sulphate precipitation, ion exchange chromatography on DEAE Sephadex A-50 and gel filtration on Sephadex G-100. Purification fold of about 5 was obtained for the WT and CRRmt24 while purification fold of about 7 was achieved for CRRmt4 by ammonium sulphate precipitation. Ion exchange chromatography gave purification fold of about 24, 22 and 25 for WT, CRRmt4 and CRRmt24, respectively. Gel filtration chromatography step yielded a homogeneous preparation with a specific activity of 6.8, 5.9 and 6.9 units/mg protein for the WT, CRRmt4and CRRmt24, respectively. The purified cellulase gave a single protein band on polyacrylamide gel electrophoresis. The molecular weights of the three cellulases were estimated to be 36, 26 and 36 kDa for the wild-type,
CRRmt4 and CRRmt24, respectively. Km values of 3.6, 3.1, and 5.3 mg/ml were obtained for the wild-type, CRRmt4 and CRRmt24, respectively. The optimum pH value for the purified cellulases was 6.5 – 7.0 and
the enzymes were optimally active at temperature of 35°C. The activities of the purified cellulases were stimulated by low concentrations (10-30 mM) of Na+ and Mg++ while EDTA was found to inhibit enzyme activity at all concentrations.