ISSN 0003-6838, Applied Biochemistry and Microbiology, 2018, Vol. 54, No. 2, pp. 215–219. © Pleiades Publishing, Inc., 2018.
Original Russian Text © V.A. Kuzovlev, Zh.D. Beskempirova, D.A. Shansharova, O.V. Fursov, A.A. Khakimzhanov, 2018, published in Prikladnaya Biokhimiya i Mikrobiologiya,
2018, Vol. 54, No. 2, pp. 206–211.
Properties and Specific Functional Features of Wheat Grain
V. A. Kuzovlev
, Zh. D. Beskempirova
, D. A. Shansharova
, O. V. Fursov
, and A. A. Khakimzhanov
Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, 050012 Kazakhstan
Almaty University of Technology, Almaty, 050012 Kazakhstan
Received September 25, 2017
Abstract⎯A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from
wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point
values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values.
This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point
of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity
requires the presence of Ca
cations. The inhibition of excess α-amylase in wheat grain with a low falling
number by the purified protein is studied.
Keywords: Triticum aestivum L., bifunctional inhibitor, α-amylase, isozymes, subtilisin, falling number
Cereal grains contain manifold amylase inhibitors
of a protein nature. Most of them are active towards
exogenous α-amylases (1,4-glucan-4-glucohydro-
lases, EC 184.108.40.206) of bacteria, insects, and mammals.
There are a few protein inhibitors known to act on
their own (endogenous) α-amylases, but they are
rather poorly studied [1–4]. The group of endogenous
α-amylase inhibitors is regarded as a component of
the plant defense system (immunity). The physiologi-
cal role of endogenous α-amylase inhibitors most
likely consists of regulation of the endogenous enzyme
activity during grain ripening and germination, though
there is still no direct evidence for this assumption.
Many representatives of both groups are ascribed to
the PR protein family, which is involved in pathogen-
esis [5, 6].
The best-known protein inhibitor of grain α-amy-
lases is the bifunctional α-amylase/subtilisin inhibitor
(BFI), which was first discovered in barley grain and
designated BASI (barley amylase/subtilisin inhibitor)
[7, 8]. This inhibitor is able to suppress the activities of
barley endogenous α-amylase and Bacillus sp. serine
proteinase. BASI-like inhibitors were later discovered
in the grains of other cereal species, such as wheat, rye,
rice, and triticale [9–12]. So far, BASI has been the
center of attention because of its important role in reg-
ulation of the α-amylase content in malting barley,
which influences malt quality. This is an issue of con-
siderable applied interest. As was also demonstrated,
BASI is involved in plant protection against microbial
and fungal pathogens, as well as abiotic stresses. The
protein structure, regulation of its activity, its func-
tioning in grain, and genetic polymorphism have been
studied in detail [13–16]. A BFI is also present in
wheat grain and is referred to as WASI (wheat amy-
lase/subtilisin inhibitor). The data on this protein are
limited. However, this protein inhibitor, along with
protective functions, can play a certain role in the con-
trol of flour quality and its bread-backing characteris-
tics by inhibiting undesirable excess α-amylase activity
in the ripening grain and during grain storage.
The α-amylase of germinating wheat grain displays
a high polymorphism. This enzyme is represented by
two major groups, namely, α-AMY1 with basic pI val-
ues (6.3–7.5) and α-AMY2 with acid pI values (4.9–
6.0). The isozymes of these groups are encoded by dif-
ferent gene subfamilies and somewhat differ in their
structure, properties, regulation, and functions in the
kernel [17, 18]. α-AMY1 (“germination” α-amylase)
plays an important role in starch hydrolysis, namely, in
the initial attack of starch grains. This enzyme activity
drastically increases as a result of grain damage caused
by preharvest sprouting (PHS) or the presence of spe-
cific α-amylase form, late maturity α-amylase (LMA).
This considerably decreases the quality of flour and
bread [19, 20]. Thus, the study of BFIs as natural regu-
lators of grain α-amylase is an important problem.
The goal of this work was to isolate a highly purified
BFI of α-amylase/subtilisin from wheat grain and
examine some of its biochemical properties and spe-
cific functional features.