Characterization of Solubilized Forms of Bound ß-Amylase Released by Various Agents

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.