PURIFICATION, IDENTIFICATION, AND PARTIAL CHARACTERIZATION OF A BARLEY PROTEIN THAT INHIBITS GREEN MALT ENDOPROTEINASES

Endoproteinases control the rate of hydrolysis of storage proteins dur ing barley germination and are thus critically important to the maltin g process. We have shown that endoproteinases comprising all four prot einase classes are present in green malt, with the cysteine proteinase s probably being most important for hydrolyzing storage proteins durin g malting. Compounds from both barley and malt inhibit some of these c ysteine proteinases. This article reports the purification and charact erization of a 10-kDa barley protein, purified from both seed and beer extracts, that specifically inhibits green malt cysteine endoproteina ses. Amino acid composition, matrix-assisted laser desorption/ionizati on mass spectrometric and N-terminal amino acid sequence data indicate d that the inhibitor is identical to barley lipid transfer protein 1 ( probable amylase/proteinase inhibitor, PAPI), a nonspecific lipid-tran sfer protein. The protein did not inhibit the activities of either pap ain or subtilisin but did suppress the activities of many of the green malt cysteine endoproteinase activities that are separated on a two-d imensional isoelectric focusing and polyacrylamide gel electrophoresis system. Some serine proteinases were also partially inhibited. The pu rified inhibitor totally inhibited the activity of a purified 31-kDa c ysteine endoproteinase from green malt. In the absence of inhibitor, t he 31-kDa enzyme rapidly hydrolyzed barley storage proteins. LTP1-PAPI may well play an important role in controlling protein hydrolysis dur ing malting.