Characterization of Solubilized Forms of Bound ß-Amylase Released by Various Agents
2000; Wiley; Volume: 106; Issue: 2 Linguagem: Inglês
10.1002/j.2050-0416.2000.tb00042.x
ISSN2050-0416
Autores Tópico(s)Pineapple and bromelain studies
ResumoJournal of the Institute of BrewingVolume 106, Issue 2 p. 71-82 Free Access Characterization of Solubilized Forms of Bound ß-Amylase Released by Various Agents E. T. Buttimer, E. T. Buttimer DuPont Cereals Innovation Centre, Block B, The Mill Site, 40 Station Road, Cambridge CB1 2UJSearch for more papers by this authorD. E. Briggs, D. E. Briggs Birmingham Malting and Brewing Group, School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TTSearch for more papers by this author E. T. Buttimer, E. T. Buttimer DuPont Cereals Innovation Centre, Block B, The Mill Site, 40 Station Road, Cambridge CB1 2UJSearch for more papers by this authorD. E. Briggs, D. E. Briggs Birmingham Malting and Brewing Group, School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TTSearch for more papers by this author First published: 16 May 2012 https://doi.org/10.1002/j.2050-0416.2000.tb00042.xCitations: 15 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract In a previous study the patterns of release of bound β-amylase caused by various agents were investigated. In the present study the released β-amylase isoforms were purified and characterized. The agents used to release bound β-amylase at different temperatures were papain, thiols, starchy endosperm extract and several substances with amphipathic characteristics. The isoforms released were compared to those present in soluble β-amylase from 8-day malt. They were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), native polyacrylamide gel electrophoresis (PAGE), western blotting and attempts were made to use matrix assisted lazer desorption ionization (MALDI) mass spectrometry. β-Amylase released by papain was similar in molecular weight (57 kDa by SDS-PAGE) to soluble β-amylase from 8-day malt (56 kDa). The isoforms released by thiol reagents were larger (57–58 and 60–62 kDa) and were similar to the soluble β-amylase extracted from barley (56–57 and 60–61 kDa). Agents with amphipathic characteristics (bovine serum albumin and the detergents CHAPS and Triton X-100) released higher molecular weight material (62 kDa). Extracts from starchy endosperm containing proteolytic activity released a β-amylase isoform, intermediate in size (58 kDa) between that released by papain and those released by thiol-containing and amphipathic agents. Heat-treated starchy endosperm extract (lacking protease activity) caused limited (15%) release of β-amylase isoforms (58 and 61 kDa). These resembled the isoforms released by thiol and hydrophobic agents. The β-amylase isoforms exhibited heterogeneity when analyzed by IEF, native PAGE and MALDI. The results confirm that a complex combination of mechanisms is involved in the release of bound β-amylase during the germination of barley. 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