DEVELOPMENT OF ENTEROBACTER CLOACAE IP8 MUTANT STRAIN PRODUCING THERMOSTABLE CELLULASE

Abstract

Mutagenesis of Enterobacter cloacae IP8 for enhanced cellulase production was carried out using ultraviolet (UV) irradiation and ethyl methanesulphonate (EMS) treatment. The mutant strain exhibited cellulolytic activity which was 2.18-fold higher than that of the wildtype strain. The optimal conditions for cellulase production were an incubation period of 28 h, a temperature of 45 oC, and pH 7.0, using CMC and peptone as carbon and nitrogen sources, respectively. The cellulases from both strains were purified by using ammonium sulfate precipitation, CM Sephadex C-50, and Biogel P-100 column chromatography. The specific activity of the purified cellulase from the mutant strain was 29.47 U/mg while that of wildtype cellulase was 21.5 U/mg. Biochemical characterization of the purified enzyme revealed the optimum pH and temperature of 8.0 and 65 oC, respectively, for the cellulase from the mutant strain, and 7.0 and 60 oC for the wild-type cellulase. The mutant cellulase was thermally stable up to 70 oC retaining 86.5% of its original activity after 180 h. Metal ions Na+ and Ca2+ remarkably enhanced the activity of the cellulase from both strains while Al3+ and the chelating agent, EDTA, strongly inhibited the activity. Mutagenesis of E. cloacae IP8 using combined UV and EMS treatment led to the development of mutant strain with enhanced capacity for the production of cellulase exhibiting novel properties such as thermostability, alkalinity, low Km, and high Vmax values. Therefore, the enzyme from the mutant strain of E. cloacae IP8 has the potential for broad industrial applications.