580 Part V: Fruits, Vegetables, and Cereals
activities have also been found in whole meal
extracts from ungerminated rye seeds by Brijs et al.
(1999). During germination, proteolytic activity in-
creases during the first 3 days to then remain con-
stant (Brijs et al. 2002). Milling fractions of germi-
nated rye (Brijs et al. 2002) have much higher
proteolytic activities than those of ungerminated rye
(Brijs et al. 1999). The enzymes are mainly present
in the bran and shorts fractions. Ungerminated rye
mainly contains two classes of proteases (serine and
aspartic proteases) (Brijs et al. 1999), whereas all
four protease classes are present during germination
(Brijs et al. 2002). Germinated rye contains mainly
aspartic and cysteine protease activities; serine and
metalloproteases are less abundant.
Aspartic proteases have been purified from bran
of ungerminated rye (Brijs 2001). Two heterodimer-
ic aspartic proteases were found: a larger 48 k
enzyme, consisting of 32 k and 16 k subunits which
are probably linked by disulphide bridges and a
smaller one of 40 k, consisting of two noncovalently
bound 29 k and 11 k subunits. Both proteins have
isoelectric points close to pI 4.6. Rye aspartic pro-
teases show optimal activity around pH 3.0 and
45°C and can be completely inhibited by pepstatin A
(Brijs 2001). Amino acid sequence alignment
showed that the rye aspartic proteases resemble
those from wheat (Bleukx et al. 1998) and barley
(Sarkkinen et al. 1992). Cysteine proteases were iso-
lated from the starchy endosperm of 3-day germi-
nated rye (Brijs 2001). SDS-PAGE revealed three
protein bands with apparent molecular weights of
67 k, 43 k, and 30 k, although it was not established
that all three protein bands are cysteine proteases.
The rye cysteine proteases are optimally active
around pH 4.0 and at 45°C and can be totally inhib-
ited by L-transepoxysuccinyl-L leucylamido-(4-
guanidino)butane (or E-64).
Protease inhibitors can reduce the activity of pro-
teases and have also been found in rye. Boisen
(1983) and Lyons et al. (1987) isolated a trypsin
inhibitor from rye endosperm and rye seed, respec-
tively. The inhibitor had a molecular weight of about
12 to 14 k, contained four disulphide bridges, and
was resistant to trypsin and elastase but was com-
pletely inactivated by chymotrypsin. The amino acid
composition was very similar to that of the barley
and wheat trypsin inhibitors.
Mosolov and Shul’gin (1986) purified a subtilisin
inhibitor from rye that belongs to the Kunitz-type
inhibitor family. The inhibitor consisted of two
forms with pI values of 6.8 and 7.1 and had a molec-
ular weight of about 20 k. It inhibited subtilisin (at a
molar ration of 1:1) and fungal proteinases from the
genus Aspergillus,but was inactive against trypsin,
chymotrypsin, and pancreatic elastase. The amino
acid composition of the rye subtilisin inhibitor was
similar to that from its wheat and barley counter-
parts.
Hejgaard (2001) purified and characterized six
serine protease inhibitors, also called “serpins,” from
rye grain. The serpins each have different regulatory
functions based on different reactive center loop
sequences, allowing for specific associations with
the substrate-binding subsites of the proteases. The
reactive center loops of the rye serpins were similar
to those of wheat serpins. Five of the six serpins
contained one or two glutamine residues at the reac-
tive center bond, which were differently positioned
than in the wheat reactive center loops. The rye ser-
pins have a molecular weight of 43 k and inhibit
proteases with chymotrypsin-like specificity, but not
pancreatic elastase or proteases with trypsin-like
specificity.Protein Physicochemical PropertiesThe properties of rye proteins, for example, their
foam-forming and emulsifying capacities, have hith-
erto not been studied well. Ludwig and Ludwig
(1989) isolated a protein mixture from rye flour that
exhibited low foam stability but excellent emulsify-
ing properties at pH 3.5. Meuser et al. (2001) reported
on a rye water-soluble protein with foam-forming
capacity. It had a molecular weight of 12.3 k and an
isoelectric point of 5.97. The optimum pH value for
the water-soluble rye protein to form stable foam was
pH 6.0. The protein did not appear to be located in
specific morphological parts of the rye kernel.
Wannerberger et al. (1997) found that secalins are
more surface-active than gliadins. The lower molecu-
lar weight secalins spread more rapidly at the air-
water interface, resulting in a higher surface pressure,
than did the higher molecular weight gliadins. The
surface pressure developed by both secalin and
gliadin decreased with decreasing pH. Different rye
milling fractions showed differences in surface be-
havior in relation to their protein content. The milling
fraction with the highest protein content spread
fastest and reached the highest surface pressure value.