Page 28 - Book of Abstracts

5 International Scientific Online Conference DOI: https://doi.org/10.15414/2021.9788055224015

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DERHAMNOSILATION OF FLAVONOIDS BY α-L-RHAMNOSIDASE PENICILLIUM TARDUM
Nataliia Borzova, Olena Gudzenko

Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine,
Kyiv, Ukraine; E-mail: nvb.imv@gmail.com

Plants are the major source of flavonoids in the human diet. These natural substances with
variable phenolic structures have various biological properties: anti-inflammatory, antioxidant,
antimutagenic, antiproliferative, and antiatherogenic, and therefore are applicated in the
treatment of neurodegenerative and cardiovascular diseases. Removing the carbohydrate
component of flavonoids (rhamnose, glucose, rutinose, hesperidose) often increases their
activity and efficacy, both as medicines and as dietary supplements. For enzymatic hydrolysis
of glycosides, α-L-rhamnosidase, an enzyme that cleaves terminal L-rhamnose residues from a
wide range of glycoconjugates, can be used.
This work aimed to study the substrate specificity of the purified α-L-rhamnosidase from
Penicillium tardum, and to evaluate the potential of the enzyme for use in the pharmaceutical
and food industries. Gel filtration on Toyopearl HW-60 and ion-exchange chromatography on
DEAE Toyopearl 650m were used for purification of the enzyme from micromycetes cultural
liquid. Naringin, rutin, neohesperidin, hesperidin and narirutin (0.5-1 mM), and synthetic p-
nitrophenyl (p-NP) substrates p-NP-α-L-rhamnose, p-NP-β-D-glucose, p-NP-β-D-galactose, and
p-NP-N-acetyl-β-D-glucosamine (1 mM) were used to assay the α-L-rhamnosidase substrate
specificity. The changes in the concentration of substrates were measuring by HPLC. Hydrolysis
efficiency (%) is the residual concentration of substrates after enzymatic hydrolysis. The
hydrolysis efficiency (%) was estimated from the residual substrate concentration after
enzymatic hydrolysis. The maximum rate (Vmax) and Michaelis constant (Km) were calculated
according to Dixon as well as to Lineweaver-Burk at 37 °C at the pH 5.0.
The high affinity of α-L-rhamnosidase P. tardum toward natural and synthetic
rhamnoglycosides has been shown. Thus α-L-rhamnosidase effectively hydrolyzed naringin,
neohesperidin, hesperidin, rutin, narirutin, and p-NP-α-L-rhamnose. α-L-Rhamnosidase P.
tardum exhibits a higher affinity for the synthetic substrate p-NP-α-L-rhamnose compared to
the natural flavonoid naringin (Km 0.7 and 1.34 mM, respectively). The hydrolysis efficiency of
substrates containing α-1,2-linked rhamnose was maximal under experimental conditions (98
% for naringin and 97 % for neohesperidin). The rate of hydrolysis of narirutin and hesperidin
was slightly lower (65 and 70 %, respectively), and the least efficient α-L-rhamnosidase P.
tardum was hydrolyzed rutin (10 %). However, it should be remembered that the aglycone part
of flavonoids is also important in determining the selectivity of the enzyme to natural
substrates.
Thus, the high hydrolyzing ability of α-L-rhamnosidase P. tardum to plant flavonoids was
shown, and it's open up broad prospects for using this enzyme in the medicine and food
industry.
Keywords: Penicillium tardum, α-L-rhamnosidase, flavonoids, derhamnosilation.

5 International Scientific Conference Agrobiodiversity for Improving the Nutrition, Health, Quality of Life and |27
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Spiritual Human Development
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