| Summary: | 碩士 === 國立臺灣大學 === 農業化學研究所 === 87 === A novel insoluble alkaline invertase was isolated from rice (Oryza sativa) suspension cells. The alkaline invertase (ITa) could be released by 1.0 M NaCl and purified further by ammonium sulfate fractionation, Con A-Sepharose affinity chromatography (non-retained), gel-filtration (Sepharose CL-6B) and DEAE-Sepharose chromatography. Another bound-form invertase (ITb) can be released by 5% EDTA from cell fragments after treated 1.0 M NaCl and purified further through the same steps. The molecular masses of ITa and ITb estimated by Superose 6 (FPLC) were 250 kD, and subunit of molecular mass determined by SDS-PAGE was 60 kD. It is apparently a homotetrameric enzyme. The characteristics of these two alkaline invertases are similar. The pH optimum of ITa was 7.6. The optimum temperature was 40℃ - 50℃. Isoelectric focusing analysis indicated that the pI value of ITa was 4.7. ITa could hydrolyze sucrose and raffinose, but not maltose. The Km values of ITa and ITb for sucrose as substrate were 15.28 mM and 14.02 mM, respectively. This insoluble alkaline invertase did not have affinity toward Con A. Glucose and fructose inhibited the enzyme activity. The additional protein such as BSA could activate ITa. The enzyme activity was inhibited by Tris and metal ions (Cu2+, Ag+). Some thiol reagents (HgCl2, PCMB) inhibited ITa, while DTT could activate its activity. It suggested that sulf hydryl-group(s) either in the active site or nearly active site of ITa, which did participate the sucrose hydrolysis.
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