Abstract

Cellulases 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, CRRmt₄and 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

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