Annual Progress Report on Malting Barley Research March, 2002

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Purification of a serine endopeptidase from green malt
An endopeptidase (SEP-1) was purified from green malt using a series of
chromatographic separations. Some of the biochemical properties of SEP-1 have been
characterized, using either gelatin or a synthetic peptide substrate (N-succinyl Ala-Ala-
Pro-Leu p-nitroanilide [AAPLpNA]). The enzyme had a molecular weight of 70 kD, as
estimated both on size-exclusion chromatography and SDS-PAGE. The hydrolysis of the
synthetic peptide was optimal at pH 6.5 and 50 °C, with a Km of 2.6 mM. Inhibition
studies indicated that the enzyme belonged to the class of serine endopeptidases. Amino
acid sequencing of the SEP-1 polypeptide showed that its structure was similar to those
of a group of enzymes that have been recently identified in plants, the cucumisin-like
peptidases. The cucumisins are a subfamily of the subtilases, which are enzymes that are
found in bacteria, yeasts and mammals. The roles of these enzymes in plants have not yet
been elucidated.
By studying the localization of SEP-1 in the various tissues of resting barley seeds and in
seeds that were malted for several days, we discovered that the enzymes was present in
small amounts in the scutellum-embryo tissue fractions that were excised from dry seeds.
This scutellar activity increased considerably during the course of malting, and the
enzyme started showing up in other seed tissues. In particular, in green malt it was
present in the highest amounts in the rootlets and shoots, in low amounts in aleurone and
it was completely absent from the SE. Its absence from the SE, where the storage protein
are deposited, implies that the enzyme probably cannot digest storage proteins. Our own
in vitro experiments, in which purified SEP-1 was incubated with storage protein
extracts, also indicated that the enzyme can not hydrolyze hordein proteins. However,
even if SEP-1 is not directly involved in the solubilization of storage proteins, it could
still indirectly affect this process by carrying out the limited hydrolysis of specific seed
proteins. Such hydrolyses could lead to: 1) the activation of other proteolytic enzymes
that, in turn, can directly hydrolyze hordeins, or 2) a limited processing of the storage
proteins that could render them more susceptible to proteolysis by the other malt
proteinases whose job it is to carry out the majority of the hydrolysis of the storage
proteins.
Publications
Jones, B. L. and Marinac, L. A. Purification and Partial Characterization of a Second
Cysteine Proteinase Inhibitor from Ungerminated Barley. J. Agric. Food Chem., 48 (2),
257 -264, 2000.
Fontanini, D., and Jones, B.L. A study of metallopeptidase isozymes from malted barley
(Hordeum vulgare cv. Morex). J. Agric. Food Chem., 49, 4903 - 4911, 2001
Jones, B.L., and Marinac, L. The effect of mashing on malt endoproteolytic activities.
J. Agric. Food Chem., 50, 858-864, 2002.
Jones, B. L. Interactions of malt and barley (Hordeum vulgare L.) Endoproteinases with
their endogenous inhibitors. J. Agric. Food Chem., 49, 5975-5981, 2001.