Purification, Identification, and Partial Characterization of a Barley Protein that Inhibits Green Malt Endoproteinases

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 article 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, matrix-assisted laser desorption/ionization mass spectrometric and N-terminal amino acid sequence data indicated that the inhibitor is identical to barley lipid transfer protein 1 (probable amylase/proteinase inhibitor, PAPI), a nonspecific 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 two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis 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.