Lentinus villosus Klotzsch (1833) AM 169: a natural and renewable source of alkaline protease / Lentinus villosus Klotzsch (1833) AM 169: uma fonte natural e renovável de protease alcalina

Rodrigo da Silva Braga, Érica Caroline de Matos Brito, Raiane Áila Teixeira Souza, Maria Francisca Simas Teixeira, Salomão Rocha Martim


Mushrooms have stood out as sources of proteases with industrial applicability. The objective of this work was to evaluate the parameters that influence the synthesis of proteases by Lentinus villosus Klotzsch (1833) AM 169 and to characterize these enzymes. The matrix culture, prepared on potato dextrose agar supplemented with yeast extract, was maintained for 8 days at 25 ° C. Submerged fermentation was carried out in GYPG (glucose, yeast extract, peptone and gelatin). Azocasein solution (1%, w/v) was used as an enzyme substrate. The best conditions for protease production were: initial pH of the culture medium (6), fermentation time (12 days), age of the inoculum (6 days) and size of the inoculum (10%). The proteases were active at pH 8, at 60 ° C, stable at pH (5 to 9) and temperature (30 °C to 60 °C). These enzymes were classified as cysteine proteases, stimulated by Fe2+ and Mn2+. Lentinus villosus synthesizes proteases with potential for application in the detergent, textile, leather processing and bioremediation processes.


basidiomycetes, peptidases, submerged fermentation.

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AHMED, N. S. A., S. G. MOHAMMED, A. A. E HUSSEIN & M. A. E. SIDDIG, 2017. Optimization of conditions for protease production from Aspergillus niger under solid state fermentation. Global Advanced Research Journal of Microbiology 6(4): 21-29.

ALJAMMAS, H. A., H. A. FATHI & W. ALKHALAF, 2018. Study the influence of culture conditions on rennin production by Rhizomucor miehei using solid-state fermentations. Journal of Genetic Engineering and Biotechnology 16: 213-216. DOI: https://doi.org/10.1016/j.jgeb.2017.10.004

BANO, S., M. U. DAHOT & S. H. A. NAQVI, 2016. Optimization of culture conditions for the production of protease by Pleurotus eryngii. Pakistan Journal of Biotechnology 13(3): 193-198.

BENLUVANKAR, V., G. R. JEBAPRIYA & J. JOEL GNANADOSS, 2015. Protease production by Penicillium sp. LCJ228 under solid state fermentation using groundnut oilcake as substrate. International of Journal of Life Science & Pharma Research 5(1): 12-19.

BOND, J. S., 2019. Proteases: History, discovery, and roles in health and disease. Journal of Biological Chemistry 294(5): 1643-1651. DOI: https://doi.org/10.1074/jbc. TM118.004156

BOON, L., E. UGARTE-BERZAL, J. VANDOOREN & G. OPDENAKKER, 2020. Protease propeptide structures, mechanisms of activation, and functions. Critical Reviews in Biochemistry and Molecular Biology 55: 1-55. DOI: https://doi.org/10.1080/10409238.2020.1742090

BOUKHALFA-LEZZAR, H., H. LEGHLIMI, E. COPINET, F. DUCHIRON & A. MECHAKRA-MAZA, 2014. Utilization of tomato pomace as a substrate for neutral protease production by Aspergillus oryzae 2220 on solid-state fermentation. International Journal of Advanced Research 2(11): 338-346.

BRITO, E. C. M., R. S. BRAGA, M. F. S. TEIXEIRA & S. R. MARTIM, 2019. Produção e caracterização parcial de proteases aspárticas sintetizadas por Lentinus crinitus (L.) Fr. 1825 DPUA 1693 (Polyporaceae). Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais 14(3): 463-472.

CHITTOOR, J. THAZ, L. BALAJI & G. JAYARAMAN, 2016. Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme. Iranian Journal of Biotechnology 14(1): 23-32. DOI: https://doi:10.15171/ijb.1269

DAUDI, S., H. MUKTHAR, A. U. REHMAN & I. U. HAQ, 2015. Production of rennin-like acid protease by Mucor pusillus through submerged fermentation. Pakistan Journal of Botany 47(3): 1121-1127.

EL-BAKY, H. A., D. LINKE, M. NIMTZ & R. G. BERGER, 2011. PsoP1, a Milk-Clotting Aspartic Peptidase From the Basidiomycete Fungus Piptoporus Soloniensis. Journal of Agricultural and Food Chemistry 59(18): 10311-10316. DOI: https://doi.org/10.1021/jf2021495

GAIS, S., F. FAZOUANE & A. MECHAKRA, 2009. Production of Milk Clotting Protease by Rhizopus stolonifer through Optimization of Culture Conditions. International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering 3(6): 340-344.

GENIER, H. L. A., F. E. F. SOARES, J. H. QUEIROZ, A. S. GOUVEIA, J. V. ARAÚJO, F. R. BRAGA, I. R. PINHEIRO & M. C. M. KASUYA, 2015. Activity of the fungus Pleurotus ostreatus and of its proteases on Panagrellus sp. Larvae. African Journal of Biotechnology 14(17): 1496-1503. DOI: https://doi.org/10.5897/AJB2015.14447

GURUMALLESH, P., K. ALAGU, B. RAMAKRISHNAN & S. MUTHUSAMY, 2019. International Journal of Biological Macromolecules 128: 254-267. DOI: https://doi.org/10.1016/j.ijbiomac.2019.01.081

IBRAHIM, A. S. S., A. A. AL-SALAMAH, Y. B. ELBADAWI, M. A. EL-TAYEB & S. S. S. IBRAHIM, 2015. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electronic Journal of Biotechnology 18: 236-243. DOI: http://dx.doi.org/10.1016/j.ejbt.2015.04.001

INÁCIO, F. D., R. O. FERREIRA, C. A. V. ARAÚJO, T. BRUGNARI, R. CASTOLDI, R. M. PERALTA & C. G. M. SOUZA, 2015. Production of enzymes and biotransformation of orange waste by oyster mushroom, Pleurotus pulmonarius (Fr.) Quél. Advances in Microbiology 5: 1-8. DOI: http://dx.doi.org/10.4236/aim.2015.51001

IRFAN, M., A. RAUF, Q. SYED, M. NADEEM & S. BAIG, 2011. Exploitation of Different Agro-residues for Acid protease Production by Rhizopus sp. in Koji Fermentation. Food & Biotechnology Research Center (FBRC) Pakistan Council of Scientific & Industrial 5(1): 43-52. DOI: http://doi:10.5455/ijavms.20110215080443

KIRSCH, L. S., V. C. S. EBINUMA & M. F. S. TEIXEIRA, 2013. Mycelial biomass and biochemical properties of proteases produced by Lentinus citrinus DPUA 1535 (Higher Basidiomycetes) in Submerged Cultivation. International Journal of Medicinal Mushrooms 15(5): 505-515. DOI: http://dx.doi.org/10.1615/IntJMedMushr.v15.i5.80

KUMMARI, S. & S. PRASAD, 2015. Isolation and Screening of Protease Producing Bacteria from Soil. International Journal of Emerging Trends in Science and Technology 2 (5): 2516-2519.

LANKA, S., C. ANJALI & M. PYDIPALI, 2017. Enhanced production of alkaline protease by Aspergillus niger DEF 1 isolated from dairy form effluent and determination of its fibrinolytic ability. African Journal of Microbiology Research 11(11): 440-449, 2017. DOI: https://doi.org/10.5897/AJMR2016-8379

LEIGHTON, T. J., R. H. DOI, R. A. J. WARREN & R. A. KELLN, 1973. The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. Journal of Molecular Biology 76(1): 103-122. DOI: https://doi.org/10.1016/0022-2836(73)90083-1

LEONHARDT, R. H., U. KRINGS, R. BERGER & D. LINKE, 2016. Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius. Bioprocess and Biosystems Engineering 39(5): 845-853. DOI: https://doi.org/10.1007/s00449-016-1564-2.

MACHADO, A. R. G., S. R. MARTIM, M. M. ALECRIM & M. F. S. TEIXEIRA, 2017. Production and characterization of proteases from edible mushrooms cultivated on amazonic tubers. African Journal of Biotechnology 16(46): 2160-2166. DOI: https://doi.org/10.5897/AJB2017.16154

MAGALHÃES, A. A. S., T. A. SILVA, M. F. S. TEIXEIRA, R. F. CRUZ FILHO, S. D. SILVA, D. M. D. GOMES & J. O. PEREIRA, 2019. Produção e caracterização de enzimas proteolíticas de Lentinus crinitus (L.) Fr. 1825 DPUA 1693 do bioma amazônico (Polyporaceae). Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais 14(3): 453-461.

MAJUMDER, R., S. P. BANIK, L. RAMRAKHUIANI & S. KHOWALA, 2014. Bioremediation by alkaline protease (AkP) from edible mushroom Termitomyces clypeatus: optimization approach based on statistical design and characterization for diverse applications. Journal of Chemical Technology and Biotechnology 90(10): 1886-1896. DOI: https://doi.org/10.1002/jctb.4500

MALDONADO, R. R., J. F. M. BURKERT, E. AGUIAR-OLIVEIRA, L. DURRANT, M. A. MAZUTTI, F.MAUGERI FILHO & M. I. RODRIGUES, 2014. Elucidation of the effects of inoculum size and age on lipase production by Geotrichum candidum. Biotecnología Aplicada 31(3): 216-221.

MARTIM, S. R., L. S. C. SILVA, L. B. SOUZA, E. J. CARMO, M. M. ALECRIM, M. C. VASCONCELLOS, I. M. A. OLIVEIRA & M. F. S. TEIXEIRA, 2017a. Pleurotus albidus: A new source of milk-clotting proteases. African Journal of Microbiology Research 11(17): 660-667. DOI: https://doi.org/10.5897/AJMR2017.8520

MARTIM, S. R., L. S. C. SILVA, M. M. ALECRIM, B. C. SOUZA, I. M. A. OLIVEIRA & M. F. S. TEIXEIRA, 2017b. Proteases ácidas de cogumelo comestível da Amazônia para aplicabilidade industrial. Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais 12(3): 353-362.

MINITAB (2018). Minitab statistical software. LEAD Technologies, Inc. Version 18.0, 2017.

NIYONZIMA, F. N. & S. S. MORE, 2013. Screening and optimization of cultural parameters for an alkaline protease production by Aspergillus terreus GR. under submerged fermentation. International Journal of Pharma and Bio Sciences 4(1): 1016-1028.

OUESLATI, A. & MOUNIRHAOUALA, 2014. Operating conditions effects onenzyme activity: case enzyme protease. Journal of Engineering Research and Applications 4(9): 33-37.

RAVIKUMAR, G., D. GOMATHI, M. KALAISELVI & C. A. UMA, 2012. Protease from the medicinal mushroom Pleurotus sajor-caju; production, purification and partial characterization. Asian Pacific Journal of Tropical Biomedicine 2(1): 411-417. DOI: https://doi.org/10.1016/S2221-1691(12)60198-1

RAZZAQ, A., S. SHAMSI, A. ALI, Q. ALI, M. SAJJAD, A. MALIK, & M. ASHRAF, 2019. Microbial Proteases Applications. Frontiers in Bioengineering and Biotechnology 7(110): 1-20. DOI: https://doi: 10.3389/fbioe.2019.00110

SALES-CAMPOS, C., L. M. ARAUJO, M. T. A. MINHONI & M. C. N. ANDRADE, 2010. Análise Físico-química e Composição Nutricional da Matéria Prima e de Substratos pré e pós Cultivo de Pleurotus ostreatus. Interciencia 35(1): 70-76.

SHARMA, K. M., R. KUMAR, S. PANWAR & A. KUMAR, 2017. Microbial alkaline proteases: Optimization of production parameters and their properties. Journal of Genetic Engineering and Biotechnology 15(1): 115-126. DOI: https://doi.org/10.1016/j.jgeb.2017.02.001

SHIVASHANKAR, M. & B. PREMKUMARI, 2014. Enzyme kinetics of protease from hypsizygus ulmarius. International Journal of Pharma and Bio Sciences 5(2): 746-754.

SILVA, R. R., L. C. G. OLIVEIRA, M. A. JULIANO, L. JULIANO, A. H. C. OLIVEIRA, J. C. R. & H. CABRAL, 2017. Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium. Food Chemistry 225: 45-54. DOI: https://doi.org/10.1016/j.foodchem.2017.01.009

SOUZA, I. H. S., NOGUEIRA, J. P., DUTRA, R. P. & A. C. FREITAS, 2019. Stability study of pre-purified protease obtained from Aspergillus oryzae NRRL 1911 by solid state fermentation with canola cake as substrate at different pH and temperature. Brazilian Journal of Development 5(10): 18538-18552. DOI:10.34117/bjdv5n10-106

SRILAKSHMI, J., J. MADHAVI, S. LAVANYA & K. AMMANI, 2014. Commercial Potential of Fungal Protease: Past, Present and Future Prospects. Journal of Pharmaceutical, Chemical and Biological Sciences 2(4): 218-234.

SUN, J., Y. ZHAO, H. CHAI, H. WANG & T. B. NG, 2011. A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinose. Acta Biochimica Polonica 58(4): 567-572.

TAVANO, O. L., A. BERENGUER-MURCIA, F. SECUNDO & R. FERNANDEZ-LAFUENTE, 2018. Biotechnological Applications of Proteases in Food Technology. Comprehensive Reviews in Food Science and Food Safety 17: 412-436. DOI: https://doi.org/10.1111/1541-4337.12326

ZHANG, G., H. WANG, X. ZHANG & T. NG, 2010. Helvellisin, a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa. Journal of Bioscience and Bioengineering, 109(1): 20-24. DOI: https://doi.org/10.1016/j.jbiosc.2009.06.022

ZHANG, G., H. WANG, X. ZHANG & T. NG, 2011. A novel alkaline protease from wild edible mushroom Termitomyces albuminosus. Acta Biochimica Polonica 52(2): 269-263.

ZHOU, C., H. ZHOU, D. LI, H. ZHANG, H. WANG & F. LU, 2020. Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709. Microbial Cell Factories 19(45): 1-13. DOI: https://doi.org/10.1186/s12934-020-01307-2

DOI: https://doi.org/10.34117/bjdv6n11-127


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