Proteolytic Activity of Fungus Pleurotus Ostreatus Mycelial Culture in Deep-Liquid Cultivation
Keywords:
proteases, mycelial culture, extracellular proteases, protein substrates, pH-dependence of proteolysis, class-specific protease inhibitorsAbstract
It was stated that proteinases of Pleurotus mycelium homogenate cleaved gelatin at pH 2.8–11.0 (proteolysis maximums at 5.0–8.5 and 10.6), casein at pH 2.8–9.0 (maximums at 2.8, 4.0–5.0, and 6.2–8.2), and hemoglobin at pH 2.8–11.0 (maximums at 4.6–5.6 and 7.0–8.2). At 10–3 M concentration of group-specific inhibitors, gelatinolytic activity was moderately (38–61%) suppressed by diisopropylfluorophosphate and metal-binding reagents. The activity of ‘alkaline’ gelatinases was completely suppressed by diisopropylfluorophosphate, p-chloromercuribenzoate, and o-phenanthroline. Caseinolytic activity at pH 2.8 and 5.2 was completely inhibited by pepstatin and at pH 5.2 – by EDTA. At pH 7.6 the caseinolytic activity was completely suppressed by diisopropylfluorophosphate and p-chloromercuribenzoate. Gelatinolytic activity of extracellular proteinases appeared at pH 3–11 (proteolysis maximum at 5.8–10.6), their caseinolytic activity – only at pH 7.6–8.2, and hemoglobinolytic activity – in the range of pH 7.6–10.0 (maximum at 8.6–9.4). However, judging by the protein concentration, the activity of extracellular proteinases exceeds that of intracellular enzymes in some cases by an order of magnitude. According to the results of the inhibitory analysis, gelatinolytic and caseinolytic activities of the culture fluid are provided mainly by serine proteinases. However, gelatinolytic activity of intracellular proteinases at pH 9.2 and extracellular proteinases at pH 7.6 was weakly sensitive to the class-specific inhibitors used in this study. This suggests the existence of some other type of proteinases.
References
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19. Никандров, В.Н. Регуляторные белки: функциональные свойства молекул и механизмы их биологического действия/ В.Н. Никандров, Н.С. Пыжова // Известия НАН Беларуси. Серия мед.-биол наук. – 2003. – № 3. – С. 75-89.
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References
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7. Wiebe MG. Mycoprotein from Fusarium venenatum: a well-established product for human consumption : Applied Microbiology and Biotechnology, 2002, no. 58 (4), pp. 421–427.
8. Babitskaya V.G., Shchebra V.V., Oleshko V.S., Osadchaya O.V. Pleurotus ostreatus – produtsent kompleksa biologicheski aktivnykh veshchestv [Pleurotus ostreatus - producer of a complex of biologically active substances]. Prikladnaya biokhimiya i mikrobiologiya. [Applied Biochemistry and Microbiology], 1996, – T. 32, no. 2, pp. 203–210. (In Russian)
9. Deepalakshmi K., Mirunalini S. Pleurotus ostreatus: an oyster mushroom with nutritional and medicinal properties: Journal of Biochemical Technology, 2014, Vol. 5, no.2, pp. 718–726.
10. Dohmae N., Hayashi K., Miki K., Tsumuraya Y., Hashimoto Y. Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies: Biosci. Biotech. Biochem, 1995, Vol. 59, no 11, pp. 2074–2080.
11. Palmieri G., Bianco C., Cennamo G., Giardina P., Marino G., Monti M., Sannia G. Purification, characterization and functional role of novel extracellular protease from Pleurotus ostreatus: Appl. Environt. Microb, 2001, Vol. 67, no 6., pp. 2754–2759.
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17. Nikandrov V.N., Pyizhova N.S. Metodyi issledovaniya proteoliza. Glava 5. [Methods for the study of proteolysis. Chapter 5]. Sovremennyie problemyi biohimii. Metodyi issledovaniy [Modern problems of biochemistry. Research methods]. Minsk: Visheyshaya shkola, 2013, рр. 132-157. (In Russian)
18. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding: Anal. Biochem, 1976, Vol. 72, pp. 248–254.
19. Nikandrov V.N., Pyzhova N.S. Regulyatornyye belki: funktsional'nyye svoystva molekul i mekhanizmy ikh biologicheskogo deystviya [Regulatory proteins: functional properties of molecules and mechanisms of their biological action]: Izvestiya NAN Belarusi. Seriya med.-biol nauk [Proceedings of the NAS of Belarus. Biomedical Sciences Series], 2003, no. 3, pp. 75-89.
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2. Рождественская, Л.Н. Анализ вызовов и современных тенденций развития технологий на рынке белков / Л.Н. Рождественская, Е.С. Бычкова, А.Л. Бычков // Пищевая промышленность. –2018. – № 5. – С. 42–47.
3. Single cell protein – state-of-the-art, industrial landscape and patents 2001–2016 / A. Ritala [et al.] // Frontiers in Microbiol. – 2017. – Vol. B. art. 2009. – P. 1–18.
4. Стахеев, И.В. Биотехнология малотоннажного производства микробного протеина / И.В. Стахеев, Э.И. Коломиец, Н.А. Здор. – Минск : «Навука i тэхнiка». – 1991. – 264 с.
5. Jӓrvinen, R. Phisiological effect of Pekilo single cell protein in pigs / R. Jӓrvinen, R. Savonen, A. Ahlstrӧm // Agricultural and Food Science. – 1980. – Vol. 52. – P. 14–23.
6. Use of a series of chemostat cultures to isolate 'improved' variants of the Quorn mycoprotein fungus, Fusarium graminearum A3/5 / M.G. Wiebe [et al.] // Microbiology. – 1994. – 140 ( Pt 11). – Р. 3015–3021.
7. Wiebe, M.G. Mycoprotein from Fusarium venenatum: a well-established product for human consumption / M.G. Wiebe // Applied Microbiology and Biotechnology. – 2002. – № 58 (4). – P.421–427.
8. Бабицкая, В.Г. Pleurotus ostreatus – продуцент комплекса биологически активных веществ / В.Г. Бабицкая [и др.] // Прикладная биохимия и микробиология.– 1996. – Т. 32. – № 2. – С. 203–210.
9. Deepalakshmi, K. Pleurotus ostreatus: an oyster mushroom with nutritional and medicinal properties / K. Deepalakshmi, S. Mirunalini // Journal of Biochemical Technology. – 2014. – Vol. 5. – No.2. –P. 718–726.
10. Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies / N. Dohmae [et al.] // Biosci. Biotech. Biochem. – 1995. – Vol. 59, No 11. – P. 2074–2080.
11. Purification, characterization and functional role of novel extracellular protease from Pleurotus ostreatus / G. Palmieri [et al.] // Appl. Environt. Microb. – 2001. – Vol. 67, No 6. – P. 2754–2759.
12. Shaba, A.M. Screening of Plеurotus ostreatus and Gleophylum sepiarium strains for extracellular protease enzyme production / A.M. Shaba, J. Baba // Biopas. – 2012. – Vol. 5, No 1. – P. 187–190.
13. Влияние хлорида марганца (II) на протеолитическую активность гриба вешенка обыкновенная (Pleurotus ostreatus) при глубинном культивировании / О.Н. Жук [и др.] // Веснік Палескага дзяржаўнага універсітэта. Серыя прыродазнаўчых навук : научно-практический журнал. - 2017. - № 2. - С. 62-68.
14. Особенности роста и развития культуры гриба вешенка обыкновенная (Pleurotus ostreatus) в присутствии ионов марганца (II) / О. Н. Жук [и др.] // Веснік Палескага дзяржаўнага універсітэта. Серыя прыродазнаўчых навук : научно-практический журнал. - 2017. - № 2. - С. 43-50.
15. Кульгавеня, А.Д. О рн-зависимости протеиназ гриба вешенка обыкновенная (pleurotus ostreatus) при глубинном культивировании / А.Д. Кульгавеня // Материалы XIII международной молодежной науч.-практ. конференции «Научный потенциал молодежи – будущему Беларуси». – Пинск : ПолесГУ. – 2019. – Ч. 3. – С. 60–62.
16. Чугай, А.С. Апробация питательных сред на основе корнеплодов для глубинного культивирования вешенки обыкновенной / А.С. Чугай, Е.С. Гришан, Н.С. Коломацкая // Материалы X международной молодежной науч.-практ. конференции «Научный потенциал молодежи – будущему Беларуси». – Пинск : ПолесГУ. – 2016. – Ч. I. – С. 520–522.
17. Никандров, В.Н. Методы исследования протеолиза. Глава 5 / В.Н. Никандров, Н.С. Пыжова // Современные проблемы биохимии. Методы исследований. – Минск : Выш. шк. – 2013. – С. 132–157.
18. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding / M. M. Bradford // Anal. Biochem. – 1976. – Vol. 72. – P. 248–254.
19. Никандров, В.Н. Регуляторные белки: функциональные свойства молекул и механизмы их биологического действия/ В.Н. Никандров, Н.С. Пыжова // Известия НАН Беларуси. Серия мед.-биол наук. – 2003. – № 3. – С. 75-89.
20. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database / N.D. Rawlings [et al.] // Nucleic Acids Research. – 2018. – Vol. 46. – P. 624–632.
References
1. Kudinov P.I., Shchekoldina T.V., Sliz'kaya A.S. Sovremennoye sostoyaniye i struktura mirovykh resursov rastitel'nogo belka [Сurrent status and structure of vegetable protein world resources] Izvestiya vuzov. Pishchevaya tekhnologiya [University News. Food technology], 2012, no. 5–6, pp. 7–10. (In Russian)
2. Rozhdestvenskaya L.N., Bychkova Ye.S., Bychkov A.L. Analiz vyzovov i sovremennykh tendentsiy razvitiya tekhnologiy na rynke belkov [Analysis of challenges and current trends in development technology in the protein market] Pishchevaya promyshlennost' [Food Industry], 2018,no. 5,pp. 42–47. (In Russian)
3. Ritala A., Hӓkkinen S.T., Toivari M.,. Wiebe M.G Single cell protein – state-of-the-art, industrial landscape and patents 2001–2016: Frontiers in Microbiol, 2017, Vol. B. art. 2009, pp. 1–18.
4. Stakheyev I.V., Kolomiyets E.I., Zdor N.A. Biotekhnologiya malotonnazhnogo proizvodstva mikrobnogo proteina [Biotechnology of small tonnage production of microbial protein]: Minsk: «Navuka i tekhnika» [Science and Technology], 1991, 264 p. (In Russian)
5. Jӓrvinen R., Savonen R., Ahlstrӧm A. Phisiological effect of Pekilo single cell protein in pigs: Agricultural and Food Science, 1980, Vol. 52, pp. 14–23.
6. Wiebe MG., Robson GD., Oliver SG., Trinci AP. Use of a series of chemostat cultures to isolate 'improved' variants of the Quorn mycoprotein fungus, Fusarium graminearum A3/5: Microbiology, 1994, 140 (Pt 11), pp. 3015–3021.
7. Wiebe MG. Mycoprotein from Fusarium venenatum: a well-established product for human consumption : Applied Microbiology and Biotechnology, 2002, no. 58 (4), pp. 421–427.
8. Babitskaya V.G., Shchebra V.V., Oleshko V.S., Osadchaya O.V. Pleurotus ostreatus – produtsent kompleksa biologicheski aktivnykh veshchestv [Pleurotus ostreatus - producer of a complex of biologically active substances]. Prikladnaya biokhimiya i mikrobiologiya. [Applied Biochemistry and Microbiology], 1996, – T. 32, no. 2, pp. 203–210. (In Russian)
9. Deepalakshmi K., Mirunalini S. Pleurotus ostreatus: an oyster mushroom with nutritional and medicinal properties: Journal of Biochemical Technology, 2014, Vol. 5, no.2, pp. 718–726.
10. Dohmae N., Hayashi K., Miki K., Tsumuraya Y., Hashimoto Y. Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies: Biosci. Biotech. Biochem, 1995, Vol. 59, no 11, pp. 2074–2080.
11. Palmieri G., Bianco C., Cennamo G., Giardina P., Marino G., Monti M., Sannia G. Purification, characterization and functional role of novel extracellular protease from Pleurotus ostreatus: Appl. Environt. Microb, 2001, Vol. 67, no 6., pp. 2754–2759.
12. Shaba A.M., Baba J. Screening of Plеurotus ostreatus and Gleophylum sepiarium strains for extracellular protease enzyme production: Biopas, 2012, Vol. 5, no 1, pp. 187–190.
13. Zhuk O.N., Il'yuchik I.A., Kul'gavenya A.D., Nikandrov V.N. Vliyaniye khlorida margantsa (II) na proteoliticheskuyu aktivnost' griba veshenka obyknovennaya (Pleurotus ostreatus) pri glubinnom kul'tivirovanii [Effect of Manganese (II) Chloride on the Proteolytic Activity of Pleurotus Ostreatus Mushroom at Periodic Culture]. Vestnik Polesskogo gosudarstvennogo universiteta. Serija prirodovedcheskih nauk [Bulletin of Polessky State University. Series in Natural Sciences], 2017, no. 2, pp. 62–68. (In Russian)
14. Zhuk O.N., Bokova O.V., Sakovich V.V., Nikandrov V.N. Osobennosti rosta i razvitiya kul'tury griba veshenka obyknovennaya (Pleurotus ostreatus) v prisutstvii ionov margantsa (II) [Distinguishing Feature of Growth and Development of Pleurotus Ostreatus Mushroom in the Presence of Margants Ions (II)]. Vestnik Polesskogo gosudarstvennogo universiteta. Serija prirodovedcheskih nauk [Bulletin of Polessky State University. Series in Natural Sciences], 2017, no. 2, pp. 43–50. (In Russian)
15. Kul'gavenya A.D. O rn-zavisimosti proteinaz griba veshenka obyknovennaya (pleurotus ostreatus) pri glubinnom kul'tivirovanii [About pH dependence of the proteases of the oyster mushroom fungus (pleurotus ostreatus) during deep cultivation]. Materialy XIII mezhdunarodnoy molodezhnoy nauch.-prakt. konferentsii «Nauchnyy potentsial molodezhi – budushchemu Belarusi», Pinsk, 2019, CH. 3, pp. 60–62. (In Russian)
16. Chugay A.S., Grishan Ye.S., Kolomatskaya N.S. Aprobatsiya pitatel'nykh sred na osnove korneplodov dlya glubinnogo kul'tivirovaniya veshenki obyknovennoy / [Testing of nutrient media based on root crops for deep cultivation of oyster mushroom]. Materialy X mezhdunarodnoy molodezhnoy nauch.-prakt. konferentsii «Nauchnyy potentsial molodezhi – budushchemu Belarusi», Pinsk, 2016. – CH. I. – pp. 520–522. (In Russian)
17. Nikandrov V.N., Pyizhova N.S. Metodyi issledovaniya proteoliza. Glava 5. [Methods for the study of proteolysis. Chapter 5]. Sovremennyie problemyi biohimii. Metodyi issledovaniy [Modern problems of biochemistry. Research methods]. Minsk: Visheyshaya shkola, 2013, рр. 132-157. (In Russian)
18. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding: Anal. Biochem, 1976, Vol. 72, pp. 248–254.
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