Biochemical Characteristics of Polygalacturonase inSinorhizobium fredii CCRC 15769

• Main Authors: Yu-Jhun Lin, 林育諄
• Other Authors: Liang-Ping Lin
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/31599445510250847562

碩士 === 國立臺灣大學 === 農業化學研究所 === 91 === The polygalacturonase (PG) could be the key enzyme for the bacteria across the plant root hair-wall during symbiosis. In this study, a PG activity assay system has been applied in which the polygalacturonic acid was used as a substrate. Coupled with the catalytic reaction of BCA (2,2’-bicinchoninic acid) solution, the color developed was measured at 570 nm to determine the enzyme activity. The PG activity was greatest in BⅢ medium containing myo-inositol as carbon source. With pectin or flavonid (Naringenin, DHF, Genistein) adding as the inducer to induce PG activity, found the effect of improving PG activity by Genistein. The cell was suspended in PCA buffer and disintegrated by sonication to get crude enzyme extract. The optimal temperature and pH of crude enzyme were 35℃and 5.5. The crude extract was purified by means of ammonium sulfate fractionation, and the precipitate collected at 40∼60％ ammonium sulfate saturation. The supernatant obtained after centrifugation was loaded un a Phenyl Sepharose 6 hydrophobic column, the active fractions were collected and dialyzed against NaOAc buffer. The dialyzed fraction was applied on a DEAE Sepharose anion exchange column for further purification. The purification fold of PG was 52.49, the recovery was 0.04％, and the specific activity was 0.152 U/mg.