| Summary: | 博士 === 國立臺灣大學 === 農業化學研究所 === 91 === Rhizobia symbiosis provides the nitrogen for the legume plant through fixation of the gaseous nitrogen component of air. For the bacteria in the plant root-hair wall, carboxymethyl cellulase (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.
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 sulfate precipitation, ion-exchange chromatography and electroelution are the preferred methods for derivation of the purified protein, with CM-cellulase characterized as follows: purification fold, 36.30; recovery, 9.8%; and specific activity, 0.053 U mg-1. The endoglucanase in the purified samples was resolved using native and sodium dodecyl sulfate polyacrylamide-gel electrophoresis; it was then assayed with an ultrathin CM-cellulose overlay stained with Congo Red. Two CM-cellulase isozymes were determined from 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.
Rhizobial cells of 3 strains of fast growing Sinorhizobium fredii CCRC15769, USDA205, and slow growing Bradyrhizobium japonicum USDA110 were imaged with atomic force microscopy（AFM）in air condition, and AFM results show that the two strains have considerable similarity in the surface morphology, and it offers the two and three dimension structure and details the morphology of bacteria as short rod shaped, 1.5-2.2 μm long, 0.5-0.7 μm wide and 0.4 μm high. Imaging of the cell fixed on the mica over 3 hours was applied for direct observation of the surface degradation. The detail observation of Sinorhizobium fredii CCRC15769 on cell surface revealed the protein-channel-liked structure of 40 nm in diameter. The observation of purified CM-cellulase isozyme 1（IS1）from Sinorhizobium fredii CCRC15769 resolved the size of about in diameter, in air and liquid mode.
Electron microscope immunogold-labeling experiments for CM-cellulase labeling were performed using ultrathin sections of Sinorhizobium fredii CCRC15769. Osmium tetroxide was used for pretreatment fixation, without detriment to the cell antigenicity of the CM-cellulase in the S. fredii CCRC15769. Specific immunogold labeling was not detected in Bradyrhizobium japonicum USDA110, however, it was observed in S. fredii USDA205, seemingly demonstrating highly contrasting intra- and interspecies cellulase similarity (high and low, respectively). Preliminary results indicate that antigenic determinants of membrane-bound carboxymethyl cellulase are exposed to the periplasmic space surrounding the cytoplasmic membrane, with the 10-nm immunogold particles hardly ever observed in the cytoplasm or other intracellular spaces. Flavonoid genistein induction in the early log phase of Sinorhizobium fredii CCRC15769 caused the aggregiation of CM-cellulase towards two sites of cell, revealing the critical role of the enzyme in the early symbiosis.
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