Akademik Veri Yönetim Sistemi
Vienna Young Scientists Symposium, Vienna, Avusturya, 1 - 02 Haziran 2017, ss.82-83
HYDROPHOBINS AS INACTIVE EXCIPIENTS IN THE PHARMACEUTICAL AND FOOD INDUSTRY Tatyana Yemelyanovaa , Vladimir Berezhinskiya , Agnes Przyluckaa , Feng Caia,b, Günseli Bayram Akcapinara,e, Hinrich Grothec , Erik Reimhultd ,Irina S. Druzhininaa a E166 - Institute of Chemical, Environmental and Biological Engineering b Nanjing Agricultural University, Nanjing, China c Institute of Materials Chemistry, TU Wien, Vienna, Austria d University of Natural Resources and Life Sciences, Vienna, Austria e Department of Statistics and Medical Informatics, Acibadem University, Istanbul, Turkey
INTRODUCTION
The pharmaceutical and food industries require not only bioactive ingredients but also inactive
excipients in formulation development. Pharmaceutical excipients are substances other than the
pharmacologically active drug, which are included in the manufacturing process or are contained in
a finished pharmaceutical product dosage form. They may be important for keeping the drug from
being released too early in the assimilation process or protect the product's stability so that it will be
at maximum effectiveness at time of use. Class II hydrophobins (HFBs) from Trichoderma spp. can
be potentially used as such inactive excipients. In this study, the class II HFB4 proteins from
different Trichoderma spp. were heterologously produced in Pichia pastoris. The extracellular
hydrophobins obtained from the fermentation process were purified and their antioxidant properties
tested.
EXPERIMENTS / FUNDAMENTAL OF THE PROBLEM / EXAMINATIONS
Filamentous fungi produce a diversity of hydrophobins, a family of low molecular weight
amphiphilic surface-active proteins containing four disulfide bridges and a large conserved and
exposed hydrophobic patch [Picture 1] [1]. Hydrophobins are traditionally split into class I and class
II, by their solubility and hydropathy plots of their amino acid sequences. Class I hydrophobins are
not soluble in water whereas the proteins from class II are easily
dissolved in the aqueous phase. The large potential of class II
hydrophobins in clinical applications has been described in
recent literature[1-8]. It was reported that class II HFBs (HFB4
and HFB7) of Trichoderma can enhance the rate of enzymatic
hydrolysis of aromatic-aliphatic polyesters such as PET [2].
Furthermore, class II hydrophobins have been successfully used
for generating stabilized foams in foam-rich products where
control of the air phase is especially important [3]. In contrast, the
formation of stabilized CO2 nanobubbles by class II
hydrophobins have been reported to induce gushing and are
considered to be a negative property of this protein in the
carbonated beverages industry [4].
Przylucka et al. (2017) have proposed the role a novel HFB7
from T. virens in the protection against oxidative stress[1]. The
antioxidant activity and ACE-inhibitory of class II HFB2 from
Trichoderma reesei was reported Khalesi et al. (2016) who
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demonstrated the reduction of free radicals of ABTS in the environment [5]. Currently, there are
only several antioxidants which are used in industrial applications, such as BHT (Butylated
Hydroxy Toluene), BHA (Butylated Hydroxy Anisol), sodium metabisulfite and ascorbic acid.
Some of these antioxidants have a negative impact on human health. As hydrophobins have been
shown to be immunologically inert [6-7], in this study, we test the antioxidant potential of a
collection of class II HFB4 proteins from Trichoderma and discuss the possibility of their use as
pharmaceutical excipients.
RESULTS AND DISCUSSION
Hydrophobins were originally detected because they enable fungi to grow at the interphase of solids
or water and air, which was brought about by their assembly into amphiphilic structures on the
outer fungal cell wall. In a previous study, an extended repertoire of class II HFBs was identified in
Trichoderma species. Among them, HFB4 from 160 different species of Trichoderma were studied
in detail. In some infrageneric groups of Trichoderma, these HFBs are under positive selection
pressure, and some of their residues are positively selected during their evolution. A set of
numerous HFB4 genes with different biochemical properties such as pI and hydrophobicity from
different Trichoderma species were expressed in P. pastoris [8]. Subsequently, the antioxidant
activity of different HFBs at certain concentrations was determined. The results of this study
allowed to detect a few particular HFB4 proteins that significantly reduce the presence of ABTS+
radicals in the solution in comparison with other HFB4s from Trichoderma species. The structural
analysis of these proteins will be presented and discussed. To test the interaction between HFB4
proteins and industrially, pharmaceutically, and biologically relevant enzymes were assessed using
quartz crystal microbalance with dissipation monitoring (QCM-D). For this purpose, quartz crystal
sensors, coated with a homogeneous film of either borosilicate or polyethylene terephthalate
presenting a hydrophilic and a hydrophobic surfaces, respectively, were used.
CONCLUSION
Class II HFB4 can be potentially used as an inactive excipient in the pharmaceutical or food
industry. Its unique amphiphilic properties are promising for the use as antioxidants and stabilizing
agents. The interaction between industrially important enzymes and HFBs is the major focus of
future investigations as well as their emulsifying properties.
REFERENCES
[1] Agnes Przylucka et al. “HFB7 – A novel orphan hydrophobin of the Harzianum and Virens clades of Trihoderma, is
involved in response to biotic and abiotic stresses”, Fungal Genetics and Biology, article in press, 2017
[2] Liliana Espino-Rammer et al. “Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic
Hydrolysis of Poly (Ethylene Terephthalate) when Expressed as Fusion Proteins”, Applied and Environmental
Microbiology, Pages 4230-4238, 2013
[3] ] Basheva E.S. et al. “Unique properties of bubbles and foam films stabilized by HFBII hydrophobin”, Langmuir 27,
Pages 2382–2392, 2011
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stability”, Langmuir 23, Pages 7995–8002, 2007
[5] Mohammadreza Khalesi et al. “Antioxidant activity and ACE-inhibitory of Class II hydrophobin from wild strain
Trichoderma reesei”, International Journal of Biological Macromolecules, Volume 91, Pages 174–179, 2016
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delivery”, BIOTRANS 2015, Page 554, 2015
[7] Mirkka Sarparanta et al. “Intravenous Delivery of Hydrophobin-Functionalized Porous Silicon Nanoparticles:
Stability, Plasma Protein Adsorption and Biodistribution”, Journal of Molecular Pharmaceutics, Pages 654–663, 2012
[8] Günseli Bayram Akcapinar et al. “Hydriphobins of Trichoderma as immobilizers of enzymes on polymeric surfaces
for biocatalyses”, BIOTRANS 2015, Page 525, 2015