| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 94 === Abstract
To understand the physiological function of VrPE-1, a putative asparaginyl endopeptidase, in the cotyledons of germinated mung beans (Vigana radiate), a polyclonal antibody against VrPE-1 and an enzymatic activity of asparaginyl peptidase with Z-Ala-Ala-Asn-MCA as a substrate were utilized for the analysis of the expression and the purification of VrPE-1 from germinated mung beans. By Western blot analysis, VrPE-1 occurs in cotyledons, hypocotyls and roots, but not in leaves, of germinated mung beans on 5 DAI (days-after-imbibition), and its expression in cotyledons reaches a maximum on 5 DAI.
Ammonium sulfate fraction (0-45% saturation) of the crude protein extract from 5-DAI cotyledons was chromatographed on a Q-Sepharose column. The fractions exhibiting asparaginyl peptidase activity were pooled and further chromatographed on a butyl-Sepharose 4 column. The chromatogram of butyl-Sepharose 4 showed a single peak of asparaginyl peptidase activity.
The enzymatically active fractions were collected and pooled for further biochemical characterization. The resulting pooled fraction showed several bands on SDS-polyacrylamide gel and only one band with a molecular mass of 18 kDa on Western blot. The pooled fraction had storing pH optimum around 5.0 for the asparaginyl peptidase activity with Z-Ala-Ala-Asn-MCA as a substrate. The fraction showed about 50% enzymatic inhibition with 10 μM PMSF, 1 �嵱 leupeptin or 10 μM NEM, and small effect with 1 �嵱 E-64 inhibitor.
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