PRODUCTION AND CHARACTERIZATION OF CELLULOLYTIC ENZYMES BY ASPERGILLUS NIGER AND RHIZOPUS SP. BY SOLID STATE FERMENTATION OF PRICKLY PEAR
DOI:
https://doi.org/10.1590/1983-21252016v29n126rcKeywords:
Filamentous fungi. Response surface methodology. Bioprocesses. Semi-arid region. Enzymatic characterization.Abstract
Prickly palm cactus husk was used as a solid-state fermentation support substrate for the production of cellulolytic enzymes using Aspergillus niger and Rhizopus sp. A Box-Behnken design was used to evaluate the effects of water activity, fermentation time and temperature on endoglucanase and total cellulase production. Response Surface Methodology showed that optimum conditions for endoglucanase production were achieved at after 70.35 h of fermentation at 29.56°C and a water activity of 0.875 for Aspergillus niger and after 68.12 h at 30.41°C for Rhizopus sp. Optimum conditions for total cellulase production were achieved after 74.27 h of fermentation at 31.22°C for Aspergillus niger and after 72.48 h and 27.86°C for Rhizopus sp. Water activity had a significant effect on Aspergillus niger endoglucanase production only. In industrial applications, enzymatic characterization is important for optimizing variables such as temperature and pH. In this study we showed that endoglucanase and total cellulase had a high level of thermostability and pH stability in all the enzymatic extracts. Enzymatic deactivation kinetic experiments indicated that the enzymes remained active after the freezing of the crude extract. Based on the results, bioconversion of cactus is an excellent alternative for the production of thermostable enzymes.Downloads
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References
AGUIAR, C. M.; SERGIO, L. L. Produção de celulases por Aspergillus niger e cinética da desativação celulásica. Acta Scientiarum Technology, Maringá, v. 33, n. 4, p. 385-391, 2011.
AHMAD, Z.; BUTT, M. S.; RIAZ, M. Partial Purification and Charactrization of Xylanase produced from Aspergillus niger using Wheat Bran. Pakistan Journal of Agriculture Science, Faisalabad, v. 50, n. 3, p. 433-437, 2013.
AMIN, F. et al. Utilization of Wheat Bran for Enhanced Production of Exopoligalacturonase by Penicillium notatum using Response Surface Methodology. Pakistan Journal of Agricultural Sciences, Faisalabad, v. 50, n. 3, p. 469-477, 2013.
AOAC - Association of Official Analytical Chemistry, 1995. Official methods of analysis. 16th ed. AOAC International, Arlington.
ARAÚJO, L. F. et al. Protein Enrichment of Cactus Pear (Opuntia ficus – indica Mill) using Saccharomyces cerevisiae in Solid-State Fermentation. Brazilian Archives of Biology and Technology, Curitiba, v. 48, special, p. 161-168, 2005.
BAGGA, P. S.; SANDHU, D. K.; SHARMA, S. Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans. Journal of Applied Bacterioloogy, Malden, v. 68, n. 1, p. 61-68, 1990.
BELMESSIKH, A. et al. Statistical optimization of cullture médium for neutral protease production by Aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato pomace. Journal of the Taiwan Institute of Chemical Engineers, Taiwan, v. 44, n. 3, p. 377-385, 2013.
CAMASSOLA, M. et al. Characterization of the cellulase complex of Penicillium echinulatum. Biocatalysis and Biotransformation, Madrid, v. 22, n. 5-6, p. 391-396, 2004.
CHIACCHIO, F. P. B.; MESQUITA, A. S.; SANTOS, J. R. Palma forrageira: uma oportunidade econômica ainda desperdiçada para o semiárido baiano. Revista Bahia Agrícola, Salvador, v. 7, n. 3, p. 39-49, 2006.
COLLA, L. M.; PRENTICE-HERNANDE, C. Congelamento e descongelamento – sua influência sobre os alimentos. Vetor - Revista de Ciências Exatas e Engenharias, Rio Grande, v. 13, n. 1, p. 53-66, 2003.
COUTO, S. R.; SANROMÁN, M. A. Application of solid-state fermentation to ligninolytic enzyme production. Biochemical Engineering Journal, Illinois, v. 22, n. 3, p. 211–219, 2005.
DUBEUX, J. C. B. et al. Productivity of Opuntia fícus-indica (L.) Miller under different N and P fertilization and plant population in north-east Brazil. Journal of Arid Environments, Chubut, v. 67, n. 3, p. 357-372, 2006.
FERREIRA, C. A. et al. Use of multivariate techniques in genetic divergence evaluation among cactus forage (Opuntia ficus-indica Mill.). Revista Brasileira de Zootecnia, Viçosa, v. 32, n. 6, p. 1560-1568, 2003.
FOROOTANFAR, H. et al. Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile. Bioresource Technology, Trivandrum, v. 102, n. 2, p. 1808-1814, 2011.
GALEMBECK, F.; BARBOSA, C. A. S.; SOUSA, R. A. Aproveitamento Sustentável de Biomassa e de Recursos Naturais na Inovação Química. Química Nova, São Paulo, v. 32, n. 3, p. 571-581, 2009.
GERVAIS, P.; MOLIN, P. The role of water in solid-state fermentation. Biochemical Engineering Journal, Illinois, v. 13, n. 2-3, p. 85-101, 2003.
GHORAI, S. et al. Fungal Biotechnology in Food and Feed Processing. Food Research International, Toronto, v. 42, n. 5-6, p. 577-587, 2009.
GHOSE, T. K. Measurement of cellulase activities. Pure & Applied Chemistry, Durhan, v. 59, n. 2, p. 257-268, 1987.
GOMES, E. et al. Enzimas termoestáveis: fontes, produção e aplicação industrial. Química Nova, São Paulo, v. 30, n. 1, p. 136-145, 2007.
GOMES, I. et al. Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Applied Microbiology and Biotechnology, New York, v. 53, n. 4, p. 461-468, 2000.
JANG, H. D.; CHEN, K. S. Production and characterization of thermostable cellulases from Streptomyces transformant T3-1. World Journal of Microbiology and Biotechnology, Valencia, v. 19, n. 2, p. 263–268, 2003.
JORGENSEN, H.; OLSSON, L. Production of cellulases by Penicillium brasilianum IBT 20888 Effect of substrate on hydrolytic performance. Enzyme and Microbial Technology, Georgia, v. 38, n. 3/4, p. 381–390, 2006.
MANAVALAN, T. et al. Characterization of optimized production, purification and application of laccase from Ganoderma lucidum. Biochemical Enginnering Journal, Illinois, v. 70, n. 1, p. 106–114, 2013.
MILLER, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, Washington, v. 31, n. 2, p. 426-428, 1959.
NOBEL, P. S. et al. Temperature limitations for cultivation of edible cacti in California. Madroño, California, v. 49, n. 4, p. 228-236, 2002.
NOBEL, P. S.; ZUTTA, B. R. Tempeture tolerances for stams and roots of two cultivated cacti, Nopalea cochenilliferra and Opuntia robusta: Acclimation, light, and droyght. Journal of Arid Environments, Chubut, v. 72, n. 5, p. 633–642, 2008.
OLIVEIRA, F.T. et al. Palma forrageira: adaptação e importância para os ecossistemas áridos e semiáridos. Revista Verde de Agroecologia e Desenvolvimento Sustentável, Mossoró, v. 5, n. 4, p. 27–37, 2010.
OMEMU, A.M. et al. Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil. African Journal of Biotechnology, Abraka, v. 4, n. 1, p. 19–25, 2005.
OSMA, J. F.; TOCA-HERRERA, J. L.; RODRIGUEZ-COUTO, S. Cost analysis in laccase production. Journal of Environmental Management, California, v. 92, n. 11, p. 2907-2912, 2011.
PALMA-FERNANDEZ, E. R.; GOMES, E.; SILVA, R. Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus. Folia Microbiologica, Praga, v. 47, n. 6, p. 685-690, 2002.
PANDEY, A. Solid-state fermentation. Biochemical Engineering Journal, Illinois, v. 13, n. 2/3, p. 81-84, 2003.
PELIZER, L. H.; PONTIERI, M. H.; MORAES, I. O. Utilização de resíduos agroindustriais em processos biotecnológicos como perspectiva de redução do impacto ambiental. Journal Technology Management & Innovation, Santiago, v. 2, n. 1, p. 118-127, 2007.
RABELO, S. C. et al. Production of bioethanol, methane, and heat from sugarcane bagasse in a biorefinery concept. Bioresource Technology, Trivandrum, v. 102, n. 17, p. 7887-7895, 2011.
SANCHEZ, C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnology Advances, Rehovot, v. 27, n. 21, p. 85-94, 2009.
SANTANA, R. S. M. et al. Produção de amiloglucosidase utilizando como substrato a palma forrageira. Revista Caatinga, Mossoró, v. 25, n. 1, p. 188-193, 2012.
SANTOS, T. C. et al. Effect of solid state fermentation on nutritional content and evaluation of degradability in cactus pear. Revista Caatinga, Mossoró, v. 28, n. 3, p. 248- 254, 2015.
SANTOS, T. C. et al. Application of response surface methodology for producing cellulolytic enzymes by solid-state fermentation from the puple mombin (Spondias purpurea L.) Residue. Food Science and Biotechnology, Seoul, v. 22, n. 1, p. 1-7, 2013.
SANTOS, T. C. et al. Optimisation of solid state fermentation of potato peel for the production of cellulolytic enzymes. Food Chemistry, Reading, v. 133, n. 4, p. 1299-1304, 2012.
SANTOS, T. C. et al. Optimization of productions of cellulolytic enzymes by Aspergillus niger using residue of mango a substrate. Ciência Rural, Santa Maria, v. 41, n. 12, p. 2210-2216, 2011.
SILVA, J. D. et al. Análise de alimentos: Métodos químicos e biológicos. 3. ed. Viçosa, MG: Editora UFV, 2002. 235 p.
SINGHANIA, R. R. et al. Recent Advances in Solid-state Fermentation. Biochemical Engineering Journal, Illinois, v. 44, n. 1, p. 13-18, 2009.
SNIFFEN, C. J. et al. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal Science, Champaign, v. 70, n. 7, p. 3562-3577, 1992.
SOCCOL, C. R. et al. Bioethanol from lignocelluloses: Status and perspectives in Brazil. Bioresource Technology, Trivandrum, v. 101, n. 13, p. 4820-4825, 2010.
VAN SOEST, P. J.; ROBERTSON, J. B.; LEWIS, B. A. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. Journal of Dairy Science. Champaign, v. 74, n. 10, p. 3583-3597, 1991.
VASCONCELOS, A. G. V. et al. Micropropagation of pricly-pear cv. Miúda (Nopalea cochenillifera - Salm Dyck). Revista Brasileira de Ciências Agrárias, Recife, v. 2, n. 1, p. 28-31, 2007.
ZHANG, Y. H. P.; HIMMEL, M. E.; MIELENZ, J. R. Outlook for cellulose improvement: Screening and selection strategies. Biotechnology Advanced, Rehovot, v. 24, n. 5, p. 452-481, 2006.
AHMAD, Z.; BUTT, M. S.; RIAZ, M. Partial Purification and Charactrization of Xylanase produced from Aspergillus niger using Wheat Bran. Pakistan Journal of Agriculture Science, Faisalabad, v. 50, n. 3, p. 433-437, 2013.
AMIN, F. et al. Utilization of Wheat Bran for Enhanced Production of Exopoligalacturonase by Penicillium notatum using Response Surface Methodology. Pakistan Journal of Agricultural Sciences, Faisalabad, v. 50, n. 3, p. 469-477, 2013.
AOAC - Association of Official Analytical Chemistry, 1995. Official methods of analysis. 16th ed. AOAC International, Arlington.
ARAÚJO, L. F. et al. Protein Enrichment of Cactus Pear (Opuntia ficus – indica Mill) using Saccharomyces cerevisiae in Solid-State Fermentation. Brazilian Archives of Biology and Technology, Curitiba, v. 48, special, p. 161-168, 2005.
BAGGA, P. S.; SANDHU, D. K.; SHARMA, S. Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans. Journal of Applied Bacterioloogy, Malden, v. 68, n. 1, p. 61-68, 1990.
BELMESSIKH, A. et al. Statistical optimization of cullture médium for neutral protease production by Aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato pomace. Journal of the Taiwan Institute of Chemical Engineers, Taiwan, v. 44, n. 3, p. 377-385, 2013.
CAMASSOLA, M. et al. Characterization of the cellulase complex of Penicillium echinulatum. Biocatalysis and Biotransformation, Madrid, v. 22, n. 5-6, p. 391-396, 2004.
CHIACCHIO, F. P. B.; MESQUITA, A. S.; SANTOS, J. R. Palma forrageira: uma oportunidade econômica ainda desperdiçada para o semiárido baiano. Revista Bahia Agrícola, Salvador, v. 7, n. 3, p. 39-49, 2006.
COLLA, L. M.; PRENTICE-HERNANDE, C. Congelamento e descongelamento – sua influência sobre os alimentos. Vetor - Revista de Ciências Exatas e Engenharias, Rio Grande, v. 13, n. 1, p. 53-66, 2003.
COUTO, S. R.; SANROMÁN, M. A. Application of solid-state fermentation to ligninolytic enzyme production. Biochemical Engineering Journal, Illinois, v. 22, n. 3, p. 211–219, 2005.
DUBEUX, J. C. B. et al. Productivity of Opuntia fícus-indica (L.) Miller under different N and P fertilization and plant population in north-east Brazil. Journal of Arid Environments, Chubut, v. 67, n. 3, p. 357-372, 2006.
FERREIRA, C. A. et al. Use of multivariate techniques in genetic divergence evaluation among cactus forage (Opuntia ficus-indica Mill.). Revista Brasileira de Zootecnia, Viçosa, v. 32, n. 6, p. 1560-1568, 2003.
FOROOTANFAR, H. et al. Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile. Bioresource Technology, Trivandrum, v. 102, n. 2, p. 1808-1814, 2011.
GALEMBECK, F.; BARBOSA, C. A. S.; SOUSA, R. A. Aproveitamento Sustentável de Biomassa e de Recursos Naturais na Inovação Química. Química Nova, São Paulo, v. 32, n. 3, p. 571-581, 2009.
GERVAIS, P.; MOLIN, P. The role of water in solid-state fermentation. Biochemical Engineering Journal, Illinois, v. 13, n. 2-3, p. 85-101, 2003.
GHORAI, S. et al. Fungal Biotechnology in Food and Feed Processing. Food Research International, Toronto, v. 42, n. 5-6, p. 577-587, 2009.
GHOSE, T. K. Measurement of cellulase activities. Pure & Applied Chemistry, Durhan, v. 59, n. 2, p. 257-268, 1987.
GOMES, E. et al. Enzimas termoestáveis: fontes, produção e aplicação industrial. Química Nova, São Paulo, v. 30, n. 1, p. 136-145, 2007.
GOMES, I. et al. Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Applied Microbiology and Biotechnology, New York, v. 53, n. 4, p. 461-468, 2000.
JANG, H. D.; CHEN, K. S. Production and characterization of thermostable cellulases from Streptomyces transformant T3-1. World Journal of Microbiology and Biotechnology, Valencia, v. 19, n. 2, p. 263–268, 2003.
JORGENSEN, H.; OLSSON, L. Production of cellulases by Penicillium brasilianum IBT 20888 Effect of substrate on hydrolytic performance. Enzyme and Microbial Technology, Georgia, v. 38, n. 3/4, p. 381–390, 2006.
MANAVALAN, T. et al. Characterization of optimized production, purification and application of laccase from Ganoderma lucidum. Biochemical Enginnering Journal, Illinois, v. 70, n. 1, p. 106–114, 2013.
MILLER, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, Washington, v. 31, n. 2, p. 426-428, 1959.
NOBEL, P. S. et al. Temperature limitations for cultivation of edible cacti in California. Madroño, California, v. 49, n. 4, p. 228-236, 2002.
NOBEL, P. S.; ZUTTA, B. R. Tempeture tolerances for stams and roots of two cultivated cacti, Nopalea cochenilliferra and Opuntia robusta: Acclimation, light, and droyght. Journal of Arid Environments, Chubut, v. 72, n. 5, p. 633–642, 2008.
OLIVEIRA, F.T. et al. Palma forrageira: adaptação e importância para os ecossistemas áridos e semiáridos. Revista Verde de Agroecologia e Desenvolvimento Sustentável, Mossoró, v. 5, n. 4, p. 27–37, 2010.
OMEMU, A.M. et al. Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil. African Journal of Biotechnology, Abraka, v. 4, n. 1, p. 19–25, 2005.
OSMA, J. F.; TOCA-HERRERA, J. L.; RODRIGUEZ-COUTO, S. Cost analysis in laccase production. Journal of Environmental Management, California, v. 92, n. 11, p. 2907-2912, 2011.
PALMA-FERNANDEZ, E. R.; GOMES, E.; SILVA, R. Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus. Folia Microbiologica, Praga, v. 47, n. 6, p. 685-690, 2002.
PANDEY, A. Solid-state fermentation. Biochemical Engineering Journal, Illinois, v. 13, n. 2/3, p. 81-84, 2003.
PELIZER, L. H.; PONTIERI, M. H.; MORAES, I. O. Utilização de resíduos agroindustriais em processos biotecnológicos como perspectiva de redução do impacto ambiental. Journal Technology Management & Innovation, Santiago, v. 2, n. 1, p. 118-127, 2007.
RABELO, S. C. et al. Production of bioethanol, methane, and heat from sugarcane bagasse in a biorefinery concept. Bioresource Technology, Trivandrum, v. 102, n. 17, p. 7887-7895, 2011.
SANCHEZ, C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnology Advances, Rehovot, v. 27, n. 21, p. 85-94, 2009.
SANTANA, R. S. M. et al. Produção de amiloglucosidase utilizando como substrato a palma forrageira. Revista Caatinga, Mossoró, v. 25, n. 1, p. 188-193, 2012.
SANTOS, T. C. et al. Effect of solid state fermentation on nutritional content and evaluation of degradability in cactus pear. Revista Caatinga, Mossoró, v. 28, n. 3, p. 248- 254, 2015.
SANTOS, T. C. et al. Application of response surface methodology for producing cellulolytic enzymes by solid-state fermentation from the puple mombin (Spondias purpurea L.) Residue. Food Science and Biotechnology, Seoul, v. 22, n. 1, p. 1-7, 2013.
SANTOS, T. C. et al. Optimisation of solid state fermentation of potato peel for the production of cellulolytic enzymes. Food Chemistry, Reading, v. 133, n. 4, p. 1299-1304, 2012.
SANTOS, T. C. et al. Optimization of productions of cellulolytic enzymes by Aspergillus niger using residue of mango a substrate. Ciência Rural, Santa Maria, v. 41, n. 12, p. 2210-2216, 2011.
SILVA, J. D. et al. Análise de alimentos: Métodos químicos e biológicos. 3. ed. Viçosa, MG: Editora UFV, 2002. 235 p.
SINGHANIA, R. R. et al. Recent Advances in Solid-state Fermentation. Biochemical Engineering Journal, Illinois, v. 44, n. 1, p. 13-18, 2009.
SNIFFEN, C. J. et al. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal Science, Champaign, v. 70, n. 7, p. 3562-3577, 1992.
SOCCOL, C. R. et al. Bioethanol from lignocelluloses: Status and perspectives in Brazil. Bioresource Technology, Trivandrum, v. 101, n. 13, p. 4820-4825, 2010.
VAN SOEST, P. J.; ROBERTSON, J. B.; LEWIS, B. A. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. Journal of Dairy Science. Champaign, v. 74, n. 10, p. 3583-3597, 1991.
VASCONCELOS, A. G. V. et al. Micropropagation of pricly-pear cv. Miúda (Nopalea cochenillifera - Salm Dyck). Revista Brasileira de Ciências Agrárias, Recife, v. 2, n. 1, p. 28-31, 2007.
ZHANG, Y. H. P.; HIMMEL, M. E.; MIELENZ, J. R. Outlook for cellulose improvement: Screening and selection strategies. Biotechnology Advanced, Rehovot, v. 24, n. 5, p. 452-481, 2006.
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12-03-2016
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