caa a22 4500 475744462 CHVBK 20180406123509.0 cr unu---uuuuu 170329e20000701xx s 000 0 eng 10.1023/A:1026435430097 doi (NATIONALLICENCE)springer-10.1023/A:1026435430097 Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase [Elektronische Daten] [Dominic Wong, Sarah Batt, Brian Tibbot, George Robertson] Barley α-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 β-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 β-sheets in domain C and the α7-and α8-helices of the (α/β)8 domain. Plenum Publishing Corporation, 2000 α-Amylase nationallicence barley α-amylase nationallicence raw starch binding nationallicence β-cyclodextrin nationallicence Wong Dominic Western Regional Research Center, USDA-ARS, 94710, Albany, California aut Batt Sarah Western Regional Research Center, USDA-ARS, 94710, Albany, California aut Tibbot Brian Western Regional Research Center, USDA-ARS, 94710, Albany, California aut Robertson George Western Regional Research Center, USDA-ARS, 94710, Albany, California aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 373-377 0277-8033 19:5<373 2000 19 10930 https://doi.org/10.1023/A:1026435430097 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026435430097 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wong Dominic Western Regional Research Center, USDA-ARS, 94710, Albany, California aut NATIONALLICENCE 700 1- Batt Sarah Western Regional Research Center, USDA-ARS, 94710, Albany, California aut NATIONALLICENCE 700 1- Tibbot Brian Western Regional Research Center, USDA-ARS, 94710, Albany, California aut NATIONALLICENCE 700 1- Robertson George Western Regional Research Center, USDA-ARS, 94710, Albany, California aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 373-377 0277-8033 19:5<373 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer