Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2003
Publication Date: 8/20/2003
Citation: JONES, B.L., FONTANINI, D. TRYPSIN/ALPHA-AMYLASE INHIBITORS INACTIVATE ENDOGENOUS BARLEY/MALT SERINE ENDOPROTEINASES. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003. Interpretive Summary: In order to produce malt that will make good beer, a portion of the proteins of the barley that is malted must be degraded into amino acids and peptides. Enzymes that form during malting (germination) carry out this process, but the barley and malt both contain proteins (called inhibitors) that can stop these enzymes from acting. In order to develope malting barleys whose seeds will germinate well and form malts that are optimally adapted for beer production, we need to understand exactly how the inhibitors and enzymes interact. This paper describes the isolation and characterization of proteins (named `CM' proteins) that inhibit the activity of one set of protein-degrading enzymes (the `serine' class enzymes) that occur in germinating barley seeds. The inhibitory proteins were identified by their amino acid sequences and their molecular sizes. Not all of the CM proteins acted as inhibitors and not all of the serine proteinases were inhibited. It had not previously been shown that any barley or malt proteins could inhibit the activities of barley or malt serine proteinases. This research shows that the activities of the serine class protein-degrading enzymes are controlled during seed germination. It allows scientists to study in detail the effects of the inhibitors on germination. This knowledge will make it easier for plant breeders to produce new barleys that have improved germination characteristics and malting quality.
Technical Abstract: Barley malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low-molecular-weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. We now report that some proteins that were concentrated using an `affinity' method inhibited the activity of a malt serine endoproteinase. The inhibited enzyme was serine endoproteinase 1 (SEP-1) and the inhibition was quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI mass spectrometry showed that only the alpha-amylase-trypsin inhibitors' or chloroform:methanol (CM-) proteins, most of which had truncated N- and C- termini, comprised the inhibitory fractions. When a traditional CM-protein fraction was prepared, some of its components inhibited the activity of SEP-1 and some did not. This is the first purification and identification of barley/malt proteins that can inhibit an endogenous serine proteinase. It shows that the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.