African Journal of Biotechnology Vol. 4 (8), pp. 838-843, August 2005          
ISSN 1684–5315 © 2005 Academic Journals

Full Length Research Paper

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

M. K. Bakare¹, I. O. Adewale^2*, A. O. Ajayi³, A. I.Okoh³, O. O. Shonukan³

¹Department of General Studies, Osun State College of  Technology, Esa-Oke, Osun State, Nigeria.

²Department of Biochemistry, Obafemi Awolowo  University, Ile-Ife, Nigeria.

³Department of Microbiology, Obafemi Awolowo  University, Ile-Ife, Nigeria.

*Corresponding Author’s E- mail: olusanjo2002@yahoo.co.uk,  iadewale@oauife.edu.ng.

Accepted 17 June, 2005

Pseudomonas fluorescens, was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources.  Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants.  The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source.  A total of fifty mutants were isolated.  All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt₄) and catabolite repression resistant mutant24 (CRRmt₂₄), respectively.  It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity.  However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study.  It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration.  The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression.

Key words: Pseudomonas fluorescens, ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio.