ISSN 2410-776X (Online)
ISSN 2410-7751 (Print)
"Biotechnologia Acta" V. 12, No 6, 2019
Р. 35-45, Bibliography 27, English
Universal Decimal Classification: 577.151.6
https://doi.org/10.15407/biotech12.06.035
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
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.
References
1. Emmons D. B. Estimating cheese yield losses from proteolysis during cheese making. Journal of Dairy Science, 1990, 73 (8), 2016?2021. https://doi.org/10.3168/jds.S0022-0302(90)78880-7
2. Preetha S., Boopathy R. Influence of culture conditions on the production of milk-clotting enzyme from Rhizomucor. World Journal of Microbiology & Biotechnology, 1994, 10, 527?530. https://doi.org/10.1007/BF00367659
3. Emmanuel V. Pontual Belany, E. Carvalho Ranilson, S. Ranilson, C. Bezerra, Luana, B. B. Coelho, Thiago H. Napole?o, Patr?cia M. G. Paiva. Caseinolytic and milk-clotting activities from Moringaoleifera flowers. Food Chemistry. 2012, 135 (6), 1848?1854. https://doi.org/10.1016/j.foodchem.2012.06.087
4. Lebedeva G. V., Proskuryakov M. T. Purification and characterization of milkclotting enzymes from oyster mushroom (Pleurotus ostreatus (Fr.) Kumm). Applied Biochemistry and Microbiology. 2009, 45 (6), 623?625. https://doi.org/10.1134/S0003683809060088
5. D’jakonova G. V. The study of some physicochemical properties of milk-clotting enzymes of oyster mushroom. 03.01.04 VAK RF, Kazan state University, Rostov-on-don. 2010, P 44. (In Russian).
6. Dahmardeh M., Dahmardeh M., Hossienabadi R., Safarpoor H., Dahmardeh M. Comparative study on cultivation and yield performance of Pleurotus ostreatus (oyster mushroom) grown on different substrates (wheat straw and barley straw) and supplemented at various levels of spawn. J. Food Agri. Environ. 2010, 8 (3/4), part 2, 996?998. https://www.researchgate.net/publication/224886012
7. Sakovich V. V., Zhernosekov D. D. Selection of optimal nutrient media and conditions of submerged cultivation for the cultivation of oyster mushroom (Pleurotus ostreatus). Сurrent issues of science, Nezhin. 2018, P. 88. (In Russian).
8. Sakovich V. V., Grusha A. M., Zhernosekov D. D. Guidelines for obtaining a drug with milkclotting activity from Pleurotus ostreatus. Veterinary journal of Belarus. 2018, P. 63?67. (In Russian).
9. Sakovich V. V., Zhuk O. N. The effect of mycelium extract and oyster mushroom culture liquid (Pleurotus ostreatus) on the formation of a cheese bunch. Scientific potential of youth: XI intern. scient. and practical conf. Pinsk. 2017, P. 340?342. (In Russian).
10. Rudakova N. L. Secreted metalloproteinase Bacillus intermedius: obtaining a homogeneous preparation of the enzyme and the study of physico-chemical properties. Scientific notes of Kazan state University. 2010, 40 (2), 145?154. (In Russian).
11. Leighton T. J., Doi R. H., Warren R. A. J., Kelln R. A. The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. J. Mol. Bio. 1973, 76 (1, 5), 103–122. https://doi.org/10.1016/0022-2836(73)90083-1
12. Pyatnitsky N. P., Proskuryakov M. T. Determination of the activity of chymotrypsin at a speed of milk has been curdled and strainedacetate mixtures. 17th century. Conf. physiologists of the South of Russia. Т. 2., Stavropol. 1969, Р. 80. (In Russian).
13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227 (5259), 680–685. https://doi.org/10.1038/227680a0
14. Chapman J. R. . Mass Spectrometry of Pro teins and Peptides. Humana Press. 2000, 538. https://doi.org/10.1385/1592590454
15. Gilliland G. L., Oliva M. T., Dill J. Functional implications of the threedimensional structure of bovine chymosin. Advances in Experimental Medicine and Biology. 1991, 306, 273–371. https://doi.org/10.1007/978-1-4684-6012-4_3
16. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976, 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3
17. Rao M. B., Tanksale A. M., Ghatge M. S., Despande V. V. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 1998, 62, 597–635. PMCID: PMC98927 https://doi.org/10.1128/MMBR.62.3.597-635.1998
18. Wang N., Wang Y., Li G., Guo W., Liu D. Expression and characterization of camel chymosinin Pichiapastoris. Protein Expr. Purif. 2015, 111, 75–81. https://doi.org/10.1016/j.pep.2015.03.012
19. Kim J. H., Kim Y. S. Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. Biosci. Biotechnol. Biochem. 2001, 65 (2), 56–62. https://doi.org/10.1271/bbb.65.356
20. Neveen M., Mohamed N., Sayed M., Sandy M., Esraa E., Mahmoud M. Mahmoud, Alaa M. Mahmoud, Ali M. Ebrahim, Rania R. Abdel-kader, Arwa S. Mohamad, Wesam S. Purification and characterization of Milk-Clotting Enzyme from the edible mushroom Pleurotus albidus. Research Journal of Pharmaceutical, Biological and Chemical Sciencesю 2018, 9 (5). ttps://doi.org/10.2174/092986610791498966
21. Poza M, Sieiro C, Carreira L, Barros-Velzquez J, Villa T. G. Production and characterization of the milk-clotting protease of Myxococcusxanthus strain 422. Ind. Microbiol. Biotechnol. 2003, 30 (12), 691. https://doi.org/10.1007/s10295-003-0100-y
22. McMahon D. J., Brown R. J., Richardson G. H., Ernstrom C. A. Effects of calcium, phosphate and bulk culture media on milk coagulation properties. J. Dairy Sci. 1984, 67, 930. https://doi.org/10.3168/jds.S0022-0302(84)81391-00
23. He X., Ren F., Guo H., Zhang W., Song X., Gan B. Purification and properties of a milkclotting enzyme produced by Bacillus amyloliquefaciens D4. Korean Journal of Chemical Engineering, 2011, 28 (1), 203–208. https://doi.org/10.1007/s11814-010-0347-8
24. Kumar Sapkota1, Se-eun Park, Bong-Suk choi, Seung Kim, Hyun-Hwa lee, Chun-Sung Kim, Hong-Sung Chun. Purification, characterization, and cloning of fibrinolyticmetalloprotease from Pleurotus ostreatus mycelia. Microbiol. biotechnol. 2007, 17 (8), 1271–1283.
25. Gershkovich A. A., Kibirev V. K. Chromogenic and fluorogenic peptide substrates of proteolytic enzymes. Bioorganic chemistry. 1988, 14 (11), 1461–1488. (In Russian).
26. Raposo S., Domingos A. Purification and characterization of milk-clotting aspartic proteinases from Centaureacalcitrapa cell suspension cultures. Process Biochemistry. 2008, 43, 139–144. https://doi.org/10.1016/j.procbio.2007.11.003