Submitted to: Journal of the American Society of Brewing Chemists
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/1997
Publication Date: N/A
Interpretive Summary: Malt is used to make beer. To make good beer, the malt needs to contain a specific, intermediate concentration of amino acids and small peptides. These compounds are formed in the malt by enzymes (proteinases) that break down proteins. In order to get the right amounts of amino acids and peptides, the proteinases must function correctly. In some of the barley varieties that are now grown, the proteinases break down too much protein, so that the resulting malts contain too much amino acid material. We are trying to figure out how to make the proteinases function more slowly, to lower the amount of amino acids they release. We showed earlier that there are small proteins in barley and malt that can stop some of the proteinases from working. If we could increase the amount of these inhibitors in barley, the altered barley should be produce a better malt. This paper reports how we purified, identified and characterized one of the barley inhibitors. Now that we know what the inhibitor is and how it works, we can use this knowledge to use either classical plant breeding or biotechnology methods to incorporate the genes for this inhibitor into future barleys. This should lead to improved barley lines that will form malt that is more perfectly suited for brewing.
Technical Abstract: Endoproteinases control the rate of hydrolysis of storage proteins during barley germination and are thus critically important to the malting process. We have shown that endoproteinases comprising all four proteinase classes are present in green malt, with the cysteine proteinases probably being most important for hydrolyzing storage proteins during malting. Compounds from both barley and malt inhibit some of these cysteine proteinases. This paper reports the purification and characterization of a 10-kDa barley protein, purified from both seed and beer extracts, that specifically inhibits green malt cysteine endoproteinases. Amino acid composition, MALDI mass spectrophotometric and sequence data indicate that the inhibitor is identical to LTP1-PAPI, a non-specific lipid-transfer protein. The protein did not inhibit the activities of either papain or subtilisin but did suppress the activities of many of the green malt cysteine endoproteinase activities that are separated on a 2-dimensional IEF x PAGE gel system. Some serine proteinases were also partially inhibited. The purified inhibitor totally inhibited the activity of a purified 31-kDa cysteine endoproteinase from green malt. In the absence of inhibitor, the 31-kDa enzyme rapidly hydrolyzed barley storage proteins. LTP1-PAPI may well play an important role in controlling protein hydrolysis during malting.