Submitted to: Proceedings International Barley Genetics Symposium
Publication Type: Proceedings
Publication Acceptance Date: 9/1/2000
Publication Date: N/A
Interpretive Summary: Malt is used to make beer. For this, the malt needs to contain a specific, intermediate level of amino acids and small peptides. These compounds are formed in the malt by enzymes (proteinases) that degrade proteins. In addition, barley and malt contain small proteins, called inhibitors, that can stop some proteinase enzymes from operating. We study these enzymes and inhibitors to determine how they control the degradation of barley proteins during malting and brewing and how this affects brewing quality. To study the enzymes in detail we need to purify them but, because they instantly join together with the inhibitors to form complexes when dissolved, this is not now possible. In this study, we measured the sizes of extracted proteins to determine whether the inhibitors were bound to the enzymes (giving a large complex) or were not bound (small size proteins). We tried to disrupt the inhibitor- enzyme complex using gentle methods that would not destroy the ability of the proteinases to function. We were not able to do this; dissociation of the inhibitor-enzyme complex always inactivated the enzymes. Other methods for dissociating these proteins need to be found. A method was devised for purifying the inhibitors by first separating the enzyme-inhibitor complex from other small proteins and then disrupting the complex and purifying the small inhibitor molecules. Using this method we prepared samples that contained good amounts of partially purified inhibitors. This information will help barley researchers to more readily purify the inhibitors for study and it shows that, while the method works well for characterizing inhibitor-enzyme interactions, other methods must be found for purifying the enzymes.
Technical Abstract: When brewing, the barley storage proteins must be partially solubilized during the malting and mashing processes to ensure that acceptable worts are formed during brewing. This solubilization is controlled by endoproteinases that form during malting and by their endogenous barley and malt protease inhibitors. We are studying these endoproteinases and inhibitors to define how the protein hydrolysis system operates, so that the protein degradation process can be regulated more precisely to produce improved worts. This work reports how we have used size separation chromatography to study the interactions between barley malt endoproteinases and their endogenous inhibitors and to carry out 'affinity' purifications of some of the inhibitors. The study showed that as soon as malt was dissolved, the proteinases and inhibitors formed a tight complex. Tests using heating, detergents, denaturing agents and the reducing agent cysteine indicated that the complex could not be dissociated without destroying the proteinase activities. It had been hoped that this dissociation would occur under gentle conditions so that the released proteinases could be studied further and characterized. Because the inhibitors bound strongly to the proteases that they inhibited, it seemed likely that this inhibitor-enzyme complex could be purified and then decomposed with heating to release active inhibitor molecules. Purifying the complex will remove many of the noninhibiting small proteins that occur in malt, making it relatively easy to finish purifying the inhibitors. This method worked well; ample inhibitors were obtained but they were not completely pure. We are now purifying the inhibitors from these fractions.