Production and Characterization of New Fibrinolytic Protease from Mucor subtillissimus UCP 1262 in Solid-State Fermentation

Thiago Pajeú Nascimento¹, Amanda Emmanuelle Sales², Camila Souza Porto², Romero Marcos Pedrosa Brandão², Galba Maria Campos Takaki³, Jose Antônio Couto Teixeira⁴, Tatiana Souza Porto^2,5, Ana Lúcia Figueiredo Porto^2*

¹Center for Biological Sciences, Federal University of Pernambuco, Recife, Brazil.
²Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, Recife, Brazil.
³Center for Research in Environmental Sciences, Catholic University of Pernambuco, Recife, Brazil.
⁴Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus of Gualtar, Braga, Portugal.
⁵Academic Unit of Garanhuns, Federal Rural University of Pernambuco, Garanhuns, Brazil.

DOI: 10.4236/aer.2015.33009                

Abstract

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.

Keywords

Mucor, Enzyme, Protease, Fibrinolytic, Wheat

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Mine, Y., Kwan, W.A.H. and Jiang, B. (2005) Fibrinolytic Enzymes in Asian Traditional Fermented Foods. Food Research International, 38, 243-250. http://dx.doi.org/10.1016/j.foodres.2004.04.008
[2]	Mukherjee, A.K., Rai, S.K., Thakur, R., Chattopadhyay, P. and Kar, S.K. (2012) Bafibrinase: A Non-Toxic, Non- Hemorrhagic, Direct-Acting Fibrinolytic Serine Protease from Bacillus sp. Strain AS-S20-I Exhibits in Vivo Anticoagulant Activity and Thrombolytic Potency. Biochimie, 94, 1300-1308. http://dx.doi.org/10.1016/j.biochi.2012.02.027
[3]	Simkhada, J.R., Mander, P., Cho, S.S. and Yoo, J.C. (2010) A Novel Fibrinolytic Protease from Streptomyces sp. CS684. Process Biochemistry, 45, 88-93. http://dx.doi.org/10.1016/j.procbio.2009.08.010
[4]	Montriwong, A., Kaewphuak, S., Rodtong, S., Roytrakul, S. and Yongsawatdigul, J. (2012) Novel Fibrinolytic Enzymes from Virgibacillus halodenitrificans SK1-3-7 Isolated from Fish Sauce Fermentation. Process Biochemistry, 47, 2379-2387. http://dx.doi.org/10.1016/j.procbio.2012.09.020
[5]	Banerjee, S., Prasanna, R. and Bagchi, S.N. (2013) Purification and Characterization of a Fibrino(geno)lytic Protease from Cultured Natural Isolate of a Cyanobacterium, Anabaena fertilissima. Journal of Applied Psychology, 25, 1111- 1122. http://dx.doi.org/10.1007/s10811-012-9946-6
[6]	Chang, C.T., Wang, P.M., Hung, Y.F. and Chung, Y.C. (2012) Purification and Biochemical Properties of a Fibrinolytic Enzyme from Bacillus subtilis-Fermented Red Bean. Food Chemistry, 133, 1611-1617. http://dx.doi.org/10.1016/j.foodchem.2012.02.061
[7]	Lu, C.L., Chen, S. and Chen, S.N. (2010) Purification and Characterization of a Novel Fibrinolytic Protease from Schizophyllum commune. Food Drug Analysis, 18, 69-76.
[8]	Medeiros Silva, G.M., Marques, D.A.V., Porto, T.S., Lima-Filho, J.L., Teixeira, J.A.C., Pessoa-Júnior, A. and Porto, A.L.F. (2013) Extraction of Fibrinolytic Proteases from Streptomyces sp. DPUA1576 Using PEG-Phosphate Aqueous Two-Phase Systems. Fluid Phase Equilibria, 339, 52-57. http://dx.doi.org/10.1016/j.fluid.2012.11.033
[9]	Shirasaka, N., Naitou, M., Okamura, K., Kusuda, M., Fukuta, Y. and Terashita, T. (2012) Purification and Characterization of a Fibrinolytic Protease from Aspergillus oryzae KSK-3. Mycoscience, 53, 354-364. http://dx.doi.org/10.1007/S10267-011-0179-3
[10]	Germano, S., Pandey, A., Osaku, C.A., Rocha, S.N. and Soccol, C.R. (2003) Characterization and Stability of Proteases from Penicillium sp. Produced by Solid-State Fermentation. Enzyme and Microbial Technology, 32, 246-251. http://dx.doi.org/10.1016/S0141-0229(02)00283-1
[11]	Yegin, S., Fernandez-Lahore, M., Salgado, A.J.L., Guvenc, U., Goksungur, Y. and Tari, C. (2011) Aspartic Proteinases from Mucor spp. in Cheese Manufacturing. Applied Microbiology and Biotechonology, 89, 949-960. http://dx.doi.org/10.1007/s00253-010-3020-6
[12]	Pandey, A., Soccol, C.R., Nigam, P., Brand, D., Mohanb, R. and Roussos, S. (2000) Biotechnological Potential of Coffee Pulp and Coffee Husk for Bioprocesses. Biochemical Engineering Journal, 6, 153-162. http://dx.doi.org/10.1016/S1369-703X(00)00084-X
[13]	Banerjee, R., Mukherjee, G. and Patra, K.C. (2005) Microbial Transformation of Tannin-Rich Substrate to Gallic Acid through Co-Culture Method. Bioresource Technology, 96, 949-953. http://dx.doi.org/10.1016/j.biortech.2004.08.004
[14]	Certik, M. and Adamechova, Z. (2009) Cereal-Based Bioproducts Containing Polyunsaturated Fatty Acids. Lipid Technology, 21, 11-12. http://dx.doi.org/10.1002/lite.200900058
[15]	Fernandez-Lahore, H.M., Fraile, E.R. and Cascone, O. (1998) Aspartic Proteinase Recovery from Solid-State Fermentation System. Journal Biotechnology, 62, 83-93. http://dx.doi.org/10.1016/S0168-1656(98)00048-0
[16]	Barrios-González, J. (2012) Solid-State Fermentation: Physiology of Solid Medium, Its Molecular Basis and Applications. Process Biochemistry, 47, 175-185. http://dx.doi.org/10.1016/j.procbio.2011.11.016
[17]	Ginther, C.L. (1979) Sporulation and the Production of Serine Protease and Cephamycin C by Streptomyces lactamdurans. Antimicrobial Agents and Chemotherapy, 15, 522-526. http://dx.doi.org/10.1128/AAC.15.4.522
[18]	Wang, S.L., Wu, Y.Y. and Liang, T.W. (2011) Purification and Biochemical Characterization of a Nattokinase by Conversion of Shrimp Shell with Bacillus subtilis TKU007. New Biotechnology, 28, 196-202. http://dx.doi.org/10.1016/j.nbt.2010.09.003
[19]	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. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[20]	Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I. and Lee, S. (1996) Purification and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. Strain CK 11-4 Screened from Chungkook-Jang. Applied Environmental Microbiology, 62, 2482-2488.
[21]	Statsoft Inc. Statistica (Data Analysis Software Systems) Version 8.0, 2008.
[22]	Oliveira, L.F., Nascimento, M.R.F., Borges, S.V., Ribeiro, P.C.N. and Ruback, V.R. (2002) Aproveitamento alterna- tivo da casca de maracujá-amarelo (Passiflora edulis F. Flavicarpa) para produção de doce em calda. Ciencia e Tecno- logia de Alimentos, 22, 259-262. http://dx.doi.org/10.1590/S0101-20612002000300011
[23]	Aguiar, T.M., Rodrigues, F.S., Santos, E.R. and Sabaa-Srur, A.U.O. (2010) Caracterização química e avaliação do valor nutritivo de sementes de acerola. Journal of the Brazilian Society for Food and Nutrition, 35, 91-102.
[24]	Henrique, W., Sampaio, A.A.M., Leme, P.R., Alleoni, G.F., Lanna, D.P.D. and Malheiros, E.B. (2003) Digestibilidade e Balanço de Nitrogênio em Ovinos Alimentados à Base de Dietas com Elevado Teor de Concentrado e Níveis Crescentes de Polpa Cítrica Peletizada. Revista Brasileira de Zootecnia, 32, 2007-2015. http://dx.doi.org/10.1590/S1516-35982003000800026
[25]	Ravikumar, G., Gomathi, D., Kalaiselvi, M. and Uma, C. (2012) A Protease from the Medicinal Mushroom Pleurotus sajor-caju; Production, Purification and Partial Characterization. Asian Pacific Journal Tropical Biomedicine, 2, S411- S417. http://dx.doi.org/10.1016/s2221-1691(12)60198-1
[26]	Silveira, C.M. and Furlong, E.B. (2007) Caracterização de compostos nitrogenados presentes em farelos fermentados em estado sólido. Ciencia e Tecnologia de Alimentos, 27, 805-811. http://dx.doi.org/10.1590/S0101-20612007000400021
[27]	Haltrich, D., Nidetzky, B., Kulbe, K.D., Steiner, W. and Zupan, S. (1996) Production of Fungal Xylanases. Bioresource Technology, 58, 137-161. http://dx.doi.org/10.1016/S0960-8524(96)00094-6
[28]	Alves, M.H., Campos-Takaki, G.M., Okada, K., Pessoa, I.H.F. and Milanez, A.I. (2005) Detection of Extracellular Protease in Mucor Species. Revista Ibero Americana de Micologia, 22, 114-117. http://dx.doi.org/10.1016/S1130-1406(05)70020-6
[29]	Alves, M.H., Campos-Takaki, G.M., Porto, A.L.F. and Milanez, A.I. (2002) Screening of Mucor spp. for the Production of Amylase, Lipase, Polygalacturonase and Protease. Brazilian Journal of Microbiology, 33, 325-330. http://dx.doi.org/10.1590/S1517-83822002000400009
[30]	Sathya, R., Pradeep, B.V., Angayarkann, J. and Palaniswamy, M. (2009) Production of Milk Clotting Protease by a Local Isolate of Mucor circinelloides under SSF Using Agro-Industrial Wastes. Biotechnology Bioprocess Engineering, 14, 788-794. http://dx.doi.org/10.1007/s12257-008-0304-0
[31]	Soares, F.E.F., Braga, F.R., Araújo, J.V., Geniêr, H.L.A., Gouveia, A.S. and Queiroz, J.H. (2013) Nematicidal Activity of Three Novel Extracellular Proteases of the Nematophagous Fungus Monacrosporium sinense. Parasitology Research, 112, 1557-1565. http://dx.doi.org/10.1007/s00436-013-3304-8
[32]	Tubesha, Z.A. and Al-Delaimy, K.S. (2003) Rennin-Like Milk Coagulant Enzyme Produced by Local Isolate of Mucor. International Journal of Dairy Technology, 56, 237-241. http://dx.doi.org/10.1046/j.1471-0307.2003.00113.x
[33]	Agrawal, D., Patidar, P., Banerjee, T. and Patil, S. (2004) Production of Alkaline Protease by Penicillium sp. under SSF Conditions and Its Application to Soy Protein Hydrolysis. Process Biochemistry, 39, 977-998. http://dx.doi.org/10.1016/S0032-9592(03)00212-7
[34]	Calik, P., Calik, G. and Ozdamar, T.H. (2001) Bioprocess Development for Serine Alkaline Protease Production: A Review. Reviews in Chemical Engineering, 17, 1-62. http://dx.doi.org/10.1515/REVCE.2001.17.S1.1
[35]	Ito, K., Kawase, T., Sammoto, H., Gomi, K., Kariyama, M. and Miyake, T. (2011) Uniform Culture in Solid-State Fermentation with Fungi and Its Efficient Enzyme Production. Journal of Bioscience and Bioengineering, 3, 300-305. http://dx.doi.org/10.1016/j.jbiosc.2010.11.008
[36]	Bhunia, B., Basak, B. and Dey, A. (2012) A Review on Production of Serine Alkaline Protease by Bacillus spp. Journal of Biochemical Technology, 3, 448-457.
[37]	Delabona, P.S., Pirota, R.D.P.B., Codima, C.A., Tremacoldi, C.R., Rodrigues, A.F. and Cristiane, S. (2013) Effect of Initial Moisture Content on Two Amazon Rainforest Aspergillus Strains Cultivated on Agro-Industrial Residues: Biomass-Degrading Enzymes Production and Characterization. Industrial Crops and Products, 42, 236-242. http://dx.doi.org/10.1016/j.indcrop.2012.05.035
[38]	Chutmanop, J., Chuichucherm, S., Chisti, Y. and Srinophakun, P. (2008) Protease Production by Aspergillus oryzae in Solid-State Fermentation Using Agroindustrial Substrates. Journal of Chemical Technology and Biotechnology, 83, 1012-1018. http://dx.doi.org/10.1002/jctb.1907
[39]	Thanapimmethaa, A., Luadsongkrama, A., Titapiwatanakunc, B. and Srinophakun, P. (2012) Value Added Waste of Jatropha curcas Residue: Optimization of Protease Production in Solid-State Fermentation by Taguchi DOE Methodology. Industrial Crops and Products, 37, 1-5. http://dx.doi.org/10.1016/j.indcrop.2011.11.003
[40]	Sugimato, S., Fugii, T., Moriymio, T., Johodo, T. and Nakamura, T. (2007) The Fibrinolytic Activity of a Novel Protease Derived from a Tempeh Producing Fungus, Fusarium sp. Bioscience, Biotechnology and Biochemistry, 71, 2184- 2189. http://dx.doi.org/10.1271/bbb.70153
[41]	Choi, D., Cha, W.S. and Park, N. (2011) Purification and Characterization of a Novel Fibrinolytic Enzyme from Fruiting Bodies of Korean Cordyceps militaris. Bioresource Techonology, 102, 3279-3285. http://dx.doi.org/10.1016/j.biortech.2010.10.002
[42]	Kim, J.S., Kim, J.E. and Choi, B.S. (2008) Purification and Characterization of Fibrinolytic Metalloprotease from Perenniporia fraxinea Mycelia. Mycological Research, 112, 990-998. http://dx.doi.org/10.1016/j.mycres.2008.01.029
[43]	Lee, S.Y., Kim, J.S. and Kim, J.E. (2005) Purification and Characterization of Fibrinolytic Enzyme from Cultured Mycelia of Armillaria mellea. Protein Expression and Purification, 43, 10-17. http://dx.doi.org/10.1016/j.pep.2005.05.004
[44]	Dunaevsky, Y.E., Matveeva, A.R., Beliakova, G.A., Domash, V.I. and Belozersky, M.A. (2007) Extracellular Alkaline Proteinase of Colletotrichum gloeosporioides. Biochemistry, 72, 345-350. http://dx.doi.org/10.1134/s0006297907030145
[45]	Kirk, P.M., Cannon, P.F., David, J.C. and Stalpers, J.A. (2001) Ainsworth & Bisby’s Dictionary of the Fungi. 9th Edition, CAB International, Oxon, 452.
[46]	Zanphorlin, L.M., Cabral, H., Arantes, E., Assis, D., Juliano, L., Juliano, M.A., Silva, R., Gomes, E. and Bonilla- Rodriguez, G.O. (2011) Purification and Characterization of a New Alkaline Serine Protease from the Thermophilic Fungus Myceliophthora sp. Process Biochemistry, 46, 2137-2143. http://dx.doi.org/10.1016/j.procbio.2011.08.014
[47]	Dienes, D., Borjesson, J., Hagglund, P., Tjerneld, F., Liden, G. and Reczey, K. (2007) Identification of a Trypsin-Like Serine Protease from Trichoderma reesei QM9414. Enzyme Microbiology and Technology, 40, 1087-1094. http://dx.doi.org/10.1016/j.enzmictec.2006.08.013
[48]	Pekkarinen, A., Mannonen, L., Jones, B.L. and Niku-Paavola, M.L. (2000) Production of Proteases by on Barley Grains and in Media Containing Cereal Fusarium Species Grown Proteins. Journal of Ceramic Science, 31, 253-261.http://dx.doi.org/10.1006/jcrs.2000.0305
[49]	Zanphorlin, L.M., Facchini, F.D.A., Vasconcelos, F., Bonugli-Santos, R.C., Rodrigues, A., Sette, L.D., Gomes, E. and Bonilla-Rodriguez, G.O. (2010) Production, Partial Characterization and Immobilization in Alginate Beads of an Alkaline Protease from a New Thermophilic Fungus Myceliophthora sp. The Journal of Microbiology, 48, 331-336. http://dx.doi.org/10.1007/s12275-010-9269-8
[50]	Heba, I., Abo-Elmagd, A. and Housseiny, M.M. (2012) Purification and Characterization of Carboxymethyl Cellulase and Protease by Ulocladium botrytis Preuss ATCC 18042 Using Water Hyacinth as a Substrate under Solid-State Fermentation. Annals of Microbiology, 62, 1547-1556. http://dx.doi.org/10.1007/s13213-011-0409-0
[51]	Roy, S.K., Dey, S.K., Raha, S.K. and Chakrabatry, S.L. (1990) Purification and Properties of an Extracellular Endoglucanase from Myceliophthora thermophila. Journal of General Microbiology, 136, 1967-1971. http://dx.doi.org/10.1099/00221287-136-10-1967