Characterization and purification of hydrolytic enzymes in Sinorhizobium fredii CCRC15769

Abstract

Rhizobial symbiosis provides the nitrogen for the leguminous plant through fixation of the gaseous nitrogen component of air. For the bacteria in the plant root-hair wall, carboxymethylcellulase (CM-cellulase, EC 3.2.1.4) may be the key enzyme in this symbiotic process, with polygalacturonase (pectinase, EC 3.2.1.15) another critical enzyme involved early in the mechanism of nitrogen supply. The precise cytosolic location, function and expression of CM-cellulase are still uncertain, however. To detect the relevant enzyme activity in Sinorhizobium fredii CCRC15769, various assay methods were used including double-layer plate assay and quantitation of reducing sugar products. After sonication of the cell pellet, ammonium sulphate precipitation, gel filtration, and ion-exchange chromatography are the preferred methods for derivation of the purified protein, with CM-cellulase characterized as follows: purification ratio, 33.35; recovery, 10.8%; and specific activity, 0.053 U mg⁻¹. The endoglucanase in the purified samples was resolved using native and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis; it was then assayed with an ultrathin CM-cellulose overlay stained with Congo Red. Two CM-cellulase isozymes were determined by native activity stain assay, with gel-filtration revealing molecular weights of approximately 196 and 30 kD; the SDS-PAGE activity gel resolved four enzyme subunits of 94, 67, 37, and 30 kD. It is suggested that the CM-cellulase in S.fredii CCRC15769 is a two-isozyme form, one a trimer of 196 kD (94, 67 and 37 kD), and the other a 30 kD monomer.