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Home Archive 2019 № 6 METALLOPROTEASE FROM THE CULTURAL LIQUID OF Pleurotus osreatus Sakovich V. V., Stohniy Ye. M., Zhernossekov D. D., Rebriev A. V., Korolova D. S., Marunych R. Yu., Chernyshenko V. O.
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ISSN 2410-776X (Online)
ISSN 2410-7751 (Print)

"Biotechnologia Acta" V. 12, No 6, 2019
https://doi.org/10.15407/biotech12.06.035
Р. 35-45, Bibliography 27, English
Universal Decimal Classification: 577.151.6

METALLOPROTEASE FROM THE CULTURAL LIQUID OF Pleurotus osreatus

Sakovich V. V.1, Stohnii Ye. M.2, Zhernosekov D. D.1, Rebriev A. V.2, Korolova D. S.2, Marunych R. Yu.2, Chernyshenko V. O.2

1Polesskii State University, Pinsk, Republic of Belarus.
2Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine.

The aim of this work was to identify and to study physical and chemical properties of the enzyme preparation which was obtained from the cultural liquid of Pleurotus ostreatus.

The protease containing fraction was obtained from the cultural liquid by sodium chloride precipitation followed by dialysis and concentration procedures. Gelatinase and milk-clotting activity were defined by standard methods. The content of the protein component of the fraction was analysed by HPLC, Laemmli electrophoresis and MALDI-TOF analysis. Protease activity was proved by enzyme-electrophoresis. To identify the protease, mass-spectrometry was carried out using the MatrixScience database. To study the specificity of protease action, the series of chromogenic substrates were used: S2238, S236, S2251, S2765, Leu-pNa, Ala-pNa and S2302. The inhibitory analysis was carried out using EDTA, benzamidine, PMSF, PCMB.

The obtained fraction possessed maximal protease activity at 45 °C. Meanwhile maximal milk-clotting activity was observed at 35 °C. The highest milk-clotting activity was shown at pH 5.0 and less than 3.0. The highest protease activity was shown at pH 6.0. Using HPLC method, it was found the main protein component and some minor proteins. According to the electrophoresis results, the main protein component of the fraction had molecular mass 45 kDa. Enzyme electrophoresis demonstrated that protease activity of the fraction was present in the zone corresponding to 45 kDa. When identifying trypsinolysis products, no homology was found with other known proteinases. It was shown that the protease hydrolyzed peptide bonds which were formed by carboxyl group of amino acids with hydrophobic side chains. The enzyme was inhibited by EDTA (ІС50 = 2.5 mМ). The maximal enzyme activity towards gelatin and Leu-pNa was shown in the presence of 5 mM calcium chloride.

The new calcium-dependent metalloprotease with molecular weight 45 kDa was found in the cultural liquid of Pleurotus ostreatus. The enzyme had no homology with other known proteases and hydrolyzes peptide bonds formed by carboxyl groups of amino acids with hydrophobic side chains.

Key words: basidiomycetes, proteolytic enzymes, milk-clotting activity, physical and chemical properties.

© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2019

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Home Archive 2019 № 6 METALLOPROTEASE FROM THE CULTURAL LIQUID OF Pleurotus osreatus Sakovich V. V., Stohniy Ye. M., Zhernossekov D. D., Rebriev A. V., Korolova D. S., Marunych R. Yu., Chernyshenko V. O.

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