Isolation of a raw starch-binding fragment from barley alpha-amylase |
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Authors: | Wong D W Batt S B Tibbot B K Robertson G H |
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Affiliation: | (1) Western Regional Research Center, USDA-ARS, Albany, California, 94710;(2) Western Regional Research Center, USDA-ARS, Albany, California, 94710 |
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Abstract: | Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the 7-and 8-helices of the (/)8 domain. |
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Keywords: | /content/g1171gp1r666226w/xxlarge945.gif" alt=" agr" align=" BASELINE" BORDER=" 0" >-Amylase barley /content/g1171gp1r666226w/xxlarge945.gif" alt=" agr" align=" BASELINE" BORDER=" 0" >-amylase raw starch binding /content/g1171gp1r666226w/xxlarge946.gif" alt=" beta" align=" MIDDLE" BORDER=" 0" >-cyclodextrin |
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