CHARACTERIZATION OF AN ASPARTIC PROTEASE PRODUCED BY Amylomyces rouxii
Keywords:
Amylomyces rouxii, aspartic protease, inhibitor protease
Abstract
The zigomycete Amylomyces rouxii was isolated, in our laboratory, from effluents of paper industries, it was showed that this fungus produces extracellular tyrosinase. In this work, we show that this fungus produces also an extracellular protease. The extracellular protease was partially purified using two-step purification, by (NH4)2SO4 fractionation and DEAE-sepharose anion exchange chromatography with 4.25-fold factor purification. The optimal pH for protease activity, using hemoglobin as substrate, was 3.5. Results from protein purification revealed that this protease is an aspartic protease. Protease activity was inhibited 73% by pepstatin A, a specific inhibitor of acid proteases. Results allow conclude that A. rouxii produce an extracellular aspartic protease that has similar characteristics to aspartic proteases produced by fungi as Mucor and Rhizopus.
References
Anson, M.L. (1938). The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology 22,79-89.
Asakura, T., Watanabe, H., Ab, K. and Arai, S. (1997). Oryzasin as an aspartic proteinase occurring in rice seeds: purification, characterization, and application to milk clotting. Journal of Agricultural and Food Chemistry 45, 1070-1075.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
Chen, C-C., Cho, Y.C., Lai, C-C. and Hsu, W-H. (2009). Purification and characterization of a new rhizopuspepsin from Rhizopus oryzae NBRC 4749. Journal of Agricultural and Food Chemistry 57, 6742-6747.
Etoh, Y., Shoun, H., Ogino, T., Fujiwara, S., Arima, K. and Beppu, T. (1982). Proton magnetic resonance spectroscopy of an essential histidyl residue in a milk-clotting acid protease, Mucor rennin. Journal of Biochemistry 91, 2039-2046.
Fernández-Lahore, H.M., Auday, R.M., Fraile, E.R., Biscoglio de Jiménez, B.M., Pripignani, L., Machalinski, C. and Cascone, O. (1999). Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures. Journal of Pepetide Research 53, 599-605.
Fox, P.F, Law, J, McSweeney P.L.H. and Wallace, J. (1993). Biochemistry of cheese ripening. In: Fox PF, editor. Cheese chemistry, Physics and Microbiology, vol. II. London: Chapman & Hall. P. 389-438.
Girelli, A.M., Mattei, E. and Messina, A. (2006). Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography. Analytical Chemistry Acta 580, 271-277.
Gray, G.L., Hayenga, K., Cullen, D., Wilson, L.J. and Norton, S. (1986). Primary structure of Mucor miehei aspartic protease: evidence for a zymogen intermediate. Gene 48, 41-53.
Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M. and Lomascolo, A. (2006). Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. Journal of Applied Microbiology 100, 219-232.
Kim, G.Y., Shim, J., Kang, M.S. and Moon, S.H. (2008). Optimized coverage of gold nanoparticles at tyrosinase electrode for measurement of a pesticide in various water samples. Journal of Hazardous Material 165, 141-147.
Kito, H., Abe, A., Sujaya, I.N., Oda, Y., Asano, K. and Sone, T. (2009). Molecular characterization of the relationship among Amylomyces rouxii, Rhizopus oryzae, and Rhizopus delemar. Bioscience Biotechnology and Biochemistry 73, 861-864.
Kudryavtseva, O.A., Dunaevsky, Ya.E., Kamzolkina, O.V. and Belozersky, M.A. (2008). Fungal proteolytic enzymes: features of the extracellular proteases of xylotrophic basidiomycetes. Micobiology 77, 725-737.
Kumar, S., Sharma, N.S., Saharan, M.R. and Singh, R. (2005). Extracellular acid protease from Rhizopus oryzae: purification and characterization. Process Biochemistry 40, 701-1705.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
Liu, Y. and Yang, Q. (2007). Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum. FEMS Microbiology Letters 277, 173-181.
Montiel, A.M., Fernández ,F.J., Marcial, J., Soriano, J., Barrios-González, J. and Tomasini, A. (2004). A fungal phenoloxidase (tyrosinase) involved in pentachlorophenol degradation. Biotechnology Letters 26, 1353-1357.
Nielsen, T.H., Deiting, U. and Sitt, M. (1997). A -amylase in potato is induced by storage at low temperature. Plant Physiology 113, 503-510
Palmieri, G., Bianco, C., Cennamo, G., Giardina, P., Merino ,G., Monti, M. and Sannia, G. (2001). Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus. Applied and Environmental Microbiology 67, 2754-2759.
Preetha, S. and Boopathy, R. (1997). Purification and characterization of a milk clotting protease from Rhizomucor miehei. World Journal of Microbiology and Biotechnology 13, 573-578.
Raposo, S. and Domingos, A. (2008). Purification and characterization milkclotting aspartic proteinases from Centaurea calcitrapa cell suspension cultures. Process Biochemistry 43, 139-144.
Selinheimo, E., Saloheimo, M., Ahola, E., Westerholm-Parvinen, A., Kalkkinen, N., Buchert, J. and Cruz, K. (2006). Production and characterization of a secreted, Cterminally processed tyrosinase from the filamentous fungus Trichoderma reesei. The FEBS Journal 273, 4322-4325.
Seo, S.Y., Sharma, V.K. and Sharma, N. (2003). Mushroom tyrosinase: Recent prospect. Journal of Agricultural and Food Chemistry 51, 2837-2853.
Suárez, B., Sanz, L., Chamorro, I., Rey, M., González, F.J., Llobell, A. and Monte, E. (2005). Proteomic analysis of secreted proteins from Trichoderma harzianum identification of a fungal cell wall-induced aspartic protease. Fungal Genetics Biology 42, 924-934
Tomasini, A., Villareal-Arellanos, H.R. and Barrios-González, J. (1996). Resistencia de una cepa de Rhizopus sp. al crecer en medios conteniendo pentaclorofenol. Avances en Ingeniería Química 6, 36-40.
Tomasini, A., Flores, V., Cortés, D. and Barrios-González, J. (2001). An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol. World Journal of Microbiology and Biotechnology 17, 201-205.
Tonouchi, N., Shoun, H., Uozumi, T. and Beppu, T. (1986). Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus. Nucleic Acids Research 14, 7557-7569.
Vioque, M., Gomez, R., Sanchez, E., Mata, C., Tejeda, L., Fernandez-Salguero, J. (2000). Chemical and microbiological characteristics of ewe’s milk cheese manufactured with extracts from flowers of Cynara cardunculus and Cynara humilis as coagulants. Journal of Agriculture and Food Chemistry 48, 451- 456.
Vishwanatha, K.S., Appu-Rao, A.G. and Singh, S.A. (2009). Characterization of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chemistry 114, 402-407.
Asakura, T., Watanabe, H., Ab, K. and Arai, S. (1997). Oryzasin as an aspartic proteinase occurring in rice seeds: purification, characterization, and application to milk clotting. Journal of Agricultural and Food Chemistry 45, 1070-1075.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
Chen, C-C., Cho, Y.C., Lai, C-C. and Hsu, W-H. (2009). Purification and characterization of a new rhizopuspepsin from Rhizopus oryzae NBRC 4749. Journal of Agricultural and Food Chemistry 57, 6742-6747.
Etoh, Y., Shoun, H., Ogino, T., Fujiwara, S., Arima, K. and Beppu, T. (1982). Proton magnetic resonance spectroscopy of an essential histidyl residue in a milk-clotting acid protease, Mucor rennin. Journal of Biochemistry 91, 2039-2046.
Fernández-Lahore, H.M., Auday, R.M., Fraile, E.R., Biscoglio de Jiménez, B.M., Pripignani, L., Machalinski, C. and Cascone, O. (1999). Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures. Journal of Pepetide Research 53, 599-605.
Fox, P.F, Law, J, McSweeney P.L.H. and Wallace, J. (1993). Biochemistry of cheese ripening. In: Fox PF, editor. Cheese chemistry, Physics and Microbiology, vol. II. London: Chapman & Hall. P. 389-438.
Girelli, A.M., Mattei, E. and Messina, A. (2006). Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography. Analytical Chemistry Acta 580, 271-277.
Gray, G.L., Hayenga, K., Cullen, D., Wilson, L.J. and Norton, S. (1986). Primary structure of Mucor miehei aspartic protease: evidence for a zymogen intermediate. Gene 48, 41-53.
Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M. and Lomascolo, A. (2006). Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. Journal of Applied Microbiology 100, 219-232.
Kim, G.Y., Shim, J., Kang, M.S. and Moon, S.H. (2008). Optimized coverage of gold nanoparticles at tyrosinase electrode for measurement of a pesticide in various water samples. Journal of Hazardous Material 165, 141-147.
Kito, H., Abe, A., Sujaya, I.N., Oda, Y., Asano, K. and Sone, T. (2009). Molecular characterization of the relationship among Amylomyces rouxii, Rhizopus oryzae, and Rhizopus delemar. Bioscience Biotechnology and Biochemistry 73, 861-864.
Kudryavtseva, O.A., Dunaevsky, Ya.E., Kamzolkina, O.V. and Belozersky, M.A. (2008). Fungal proteolytic enzymes: features of the extracellular proteases of xylotrophic basidiomycetes. Micobiology 77, 725-737.
Kumar, S., Sharma, N.S., Saharan, M.R. and Singh, R. (2005). Extracellular acid protease from Rhizopus oryzae: purification and characterization. Process Biochemistry 40, 701-1705.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
Liu, Y. and Yang, Q. (2007). Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum. FEMS Microbiology Letters 277, 173-181.
Montiel, A.M., Fernández ,F.J., Marcial, J., Soriano, J., Barrios-González, J. and Tomasini, A. (2004). A fungal phenoloxidase (tyrosinase) involved in pentachlorophenol degradation. Biotechnology Letters 26, 1353-1357.
Nielsen, T.H., Deiting, U. and Sitt, M. (1997). A -amylase in potato is induced by storage at low temperature. Plant Physiology 113, 503-510
Palmieri, G., Bianco, C., Cennamo, G., Giardina, P., Merino ,G., Monti, M. and Sannia, G. (2001). Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus. Applied and Environmental Microbiology 67, 2754-2759.
Preetha, S. and Boopathy, R. (1997). Purification and characterization of a milk clotting protease from Rhizomucor miehei. World Journal of Microbiology and Biotechnology 13, 573-578.
Raposo, S. and Domingos, A. (2008). Purification and characterization milkclotting aspartic proteinases from Centaurea calcitrapa cell suspension cultures. Process Biochemistry 43, 139-144.
Selinheimo, E., Saloheimo, M., Ahola, E., Westerholm-Parvinen, A., Kalkkinen, N., Buchert, J. and Cruz, K. (2006). Production and characterization of a secreted, Cterminally processed tyrosinase from the filamentous fungus Trichoderma reesei. The FEBS Journal 273, 4322-4325.
Seo, S.Y., Sharma, V.K. and Sharma, N. (2003). Mushroom tyrosinase: Recent prospect. Journal of Agricultural and Food Chemistry 51, 2837-2853.
Suárez, B., Sanz, L., Chamorro, I., Rey, M., González, F.J., Llobell, A. and Monte, E. (2005). Proteomic analysis of secreted proteins from Trichoderma harzianum identification of a fungal cell wall-induced aspartic protease. Fungal Genetics Biology 42, 924-934
Tomasini, A., Villareal-Arellanos, H.R. and Barrios-González, J. (1996). Resistencia de una cepa de Rhizopus sp. al crecer en medios conteniendo pentaclorofenol. Avances en Ingeniería Química 6, 36-40.
Tomasini, A., Flores, V., Cortés, D. and Barrios-González, J. (2001). An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol. World Journal of Microbiology and Biotechnology 17, 201-205.
Tonouchi, N., Shoun, H., Uozumi, T. and Beppu, T. (1986). Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus. Nucleic Acids Research 14, 7557-7569.
Vioque, M., Gomez, R., Sanchez, E., Mata, C., Tejeda, L., Fernandez-Salguero, J. (2000). Chemical and microbiological characteristics of ewe’s milk cheese manufactured with extracts from flowers of Cynara cardunculus and Cynara humilis as coagulants. Journal of Agriculture and Food Chemistry 48, 451- 456.
Vishwanatha, K.S., Appu-Rao, A.G. and Singh, S.A. (2009). Characterization of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chemistry 114, 402-407.
Published
2020-04-21
How to Cite
Marcial, J., Pérez de los Santos, A., Fernández, F., Díaz-Godínez, G., Montiel-González, A., & Tomasini, A. (2020). CHARACTERIZATION OF AN ASPARTIC PROTEASE PRODUCED BY Amylomyces rouxii. Revista Mexicana De Ingeniería Química, 10(1), 9-16. Retrieved from http://www.rmiq.org/ojs311/index.php/rmiq/article/view/1603
Section
Biotechnology
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