Isolation and characterization of endopeptidases from barley (Hordeum vulgare L. cv. Morex) green malt

Cysteine peptidases are among the most abundant of the germinating barley (green malt) proteolytic activities and are considered the main players in the degradation of storage proteins. Peptidases belonging to other proteolytic enzyme classes are present in germinating barley, but their roles in germination are yet to be defined. This study was conducted to investigate some of the green malt serine- and metallopeptidases, whose roles in protein degradation were unknown.; A group of enzymes were isolated from Hordeum vulgare L. cv. Morex green malt. They hydrolyzed gelatin optimally at pH 8.0, and the digestion was inhibited by the metal chelating agents 1,10-phenanthroline and EDTA, indicating that they were all metallopeptidases. The enzymes all had very similar biochemical characteristics, suggesting that they were probably isoforms. The metallopeptidases were absent (or inactive) in resting seeds, but their activities increased rapidly after seed imbibition. They were abundant in the aleurone tissue but were undetectable in the starchy endosperm. In vitro, they readily degraded the barley hordein storage proteins, especially the D component. This study shows that although the isolated metallopeptidases are probably not directly involved in degrading hordeins during barley germination, they can digest them in the mashing step of the brewing process.; A green malt endopeptidase was purified until it comprised a single band of 70 kD on SDS-PAGE. It digested the substrate N-succinyl Ala-Ala-Pro-Leu p-nitroanilide optimally at pH 6.5 and 50°C and was stable over a broad pH range. Both inhibition and amino acid sequence studies indicated that the enzyme belonged to the serine endopeptidase class. The enzyme was most similar to the plant subtilisin-like endopeptidases (EC 3.4.21) that resemble cucumisin (EC 3.4.21.25). Some enzyme activity occurred in the embryonic axis/scutellum tissue of resting seeds. After imbibition, it was present in all of the seed tissues except for the starchy endosperm, and its levels increased through the sixth day of germination. On the basis of these results, the enzyme may carry out a limited proteolysis of specific seed proteins or it may be involved in recognizing and/or reacting to environmental stimuli.