Purification and characterization of carboxymethyl cellulase from Sinorhizobium fredii

Carboxymethyl cellulase (CMCase) is the key enzyme used by bacteria to decompose the plant root hairwall during symbiosis. In this study, CMCase activities were routinely assayed using carboxymethyl cellulose (CMC) as a substrate. Coupled with the catalytic reaction of BCA (2,2’-bicinchoninic acid) solution, the color developed at 570 nm was measured to determine the enzyme activity. Sinorhizobium fredii CCRC 15769 was utilized to produce CMCase for this study. The optimum growth conditions of the cultural medium were studied in order to obtain sufficient cells for further CMCase purification, and further, for large-scale fermentation production. BIII medium containing 0.3% myo-inositol proved to be a suitable carbon source for bacterial cell growth and specific activity of CMCase. A 22.4 g wet weight of cells can be harvested from 3.5 liters of fermented medium through continuous centrifugation. The cells were suspended in 50 mM potassium phosphate-citric acid (PCA) buffer at pH 5.2 and disintegrated through ultra-sonication to obtain crude extract. Then, certain precipitates were collected at 40-60% ammonium sulfate saturation via ammonium sulfate fractionation of the crude extract. The supernatant obtained following centrifugation was loaded onto a DEAE Sepharose anion-exchange column, and the active fractions were collected and dialyzed against 10 mM Tris-HCl buffered at pH 7.4. For further purification, the dialyzed fraction was loaded onto a Phenyl-Sepharose column. The active fractions were dialyzed and then assayed by SDS-PAGE activity stain to confirm that it contained CMCase activity at the fraction of 94 kDa. The characterizations of CMCase were demonstrated as follows: the optimal temperature and pH were 35°C and 7.0, respectively. The purification fold was 9.08, and the recovery yield was 26.4%, and the specific activity was 3.822 U mg.