ISSN 0003-6838, Applied Biochemistry and Microbiology, 2018, Vol. 54, No. 2, pp. 206–210. © Pleiades Publishing, Inc., 2018.
Original Russian Text © E.S. Zvonareva, A.A. Osmolovskiy, V.G. Kreier, N.A. Baranova, I.B. Kotova, N.S. Egorov, 2018, published in Prikladnaya Biokhimiya i Mikrobiologiya,
2018, Vol. 54, No. 2, pp. 195–200.
Production of Proteinase with Plasmin-Like and Prekallikrein
Activating Activity by the Micromycete Aspergillus terreus
E. S. Zvonareva
, A. A. Osmolovskiy
*, V. G. Kreier
, N. A. Baranova
, I. B. Kotova
, and N. S. Egorov
Department of Biology, Lomonosov Moscow State University, Moscow, 119234 Russia
International Center of Biotechnology, Lomonosov Moscow State University, Moscow, 119234 Russia
Received September 18, 2017
Abstract⎯The effect of nitrogen sources in the fermentation medium and the cultivation conditions on the
production of proteinases with plasmin-like and prekallikrein activation activity by micromycete Aspergillus
terreus 2 was investigated. The highest secretion of proteinases was achieved when the micromycete was cul-
tivated on a medium containing both amine- and mineral nitrogen sources at an initial pH of 5.5 and at 28°С.
It was established that the extracellular micromycete proteinases are equally capable of hydrolyzing fibrin and
Keywords: Aspergillus proteinase, plasmin-like activity, fibrinolytic enzymes, prekallikrein activators
Proteolytic enzymes that act upon proteins of the
human hemostasis system are produced by multiple
representatives of the fungi of Aspergillus genus [1–3].
Among them proteinases possessing fibrinolytic (plas-
min-like) activity and the ability to activate proen-
zymes of the plasma hemostasis have been identified.
The activating activity of the isolated proteinases from
all known producers is accompanied by another pro-
teolytic activity specific to various substrates [2, 4].
Furthermore, the targets activated by the extracellular
proteinases of different micromycetes could be differ-
ent. For example, it was shown that the proteinase
from Aspergillus ochraceus activated protein C and fac-
tor Х in blood plasma, while the proteinase from
A. terreus activated prekallikrein .
Prekallikrein and its active form kallikrein com-
prise a part of the kallikrein–kinin system, which plays
a role in the blood clotting reaction, inflammation
processes, and blood pressure control. Although cases
of prekallikrein deficiency in blood are rare and non-
symptomatic, its concentration in plasma is often
determined with diagnostic kits [6–8]. Cheaper and
more specific proteinases of fungal origin that activate
prekallikrein can be used for diagnostic determination
of this proenzyme.
It is known that the maximal production of pro-
teinases by micromycetes depends on cultivation con-
ditions, specifically on the medium composition and
nitrogen content [9–13]. It was demonstrated that,
with mixed-type nitrogen sources for cultivation of
A. ochraceus, it was possible to select a nutrient
medium that facilitated the production of proteinases
by this strain that were active towards plasma hemo-
stasis proteins and exhibited the maximal ability for
proenzyme activation and minimal hydrolytic activity
and vice versa . Thus, the selection of the medium
composition and cultivation conditions for micromy-
cete A. terreus seems to be a promising approach for the
targeted production of proteinase activating preka-
The objective of this investigation was to study the
conditions of the production of proteinases with plas-
min-like and kallikrein-activating activity by the
micromycete A. terreus 2.
Producer and cultivation conditions. The micromy-
cete A. terreus 2 strain, which was previously investi-
gated as a producer of proteinases exhibiting plasmin-
like and prekallikrein activating activity, was used as
the object of the study [2, 5].
Micromycete was cultivated in 750-mL flasks con-
taining 100 mL of medium on an orbital shaker
(200 rpm) at 28°C. To prepare seed material, spores
were washed from the surface of culture propagated in
tubes on a slanted wort agar medium for 7 days at 25°C
into a nutrient medium containing wort, glucose, and
peptone . After 2 days of cultivation, part of the
biomass was transferred to fermentation medium no. 1
with the following composition (%): glucose—3.0,