Keywords
cellulases, ion exchange chromatography, factorial design, optimization, enzyme purification celulasas, cromatografía de intercambio iónico, diseño factorial, optimización, purificación de enzimas
How to Cite
Morales Borrell, D., Baryolo González, L., Mora González, N., Ramos Sánchez, L. B., & Pérez Sánchez, A. (2022). Salt precipitation of a crude extract with cellulolytic activity produced by Aspergillus niger UC33. Revista Colombiana De Investigaciones Agroindustriales, 9(2), 69–85. https://doi.org/10.23850/24220582.4872
Abstract
The purification of cellulase enzymes is a critical step for their production because it is often very expensive. The objective of this study was to evaluate the saline precipitation of cellulases produced by Aspergillus niger UC33 in solid-state fermentation, to increase the yield and purity of the enzyme at the lowest possible cost. The Design-Expert 11.1.2 program was used for the experimental design and the optimization of the experimental conditions. Ammonium sulfate between 60 and 90 % saturation was used for precipitation, among 1 and 3 h of precipitation time were employed and the temperatures applied were 4 and 25 °C. The optimal conditions found to maximize yield and purity were 68.97% saturation with ammonium sulfate, precipitation time of 1.28 h and temperature of 25 °C. Under these conditions, the yield, purity and cost values were: 74.81%, 1.26 and USD $ 0.2609, respectively. The molecular weight of the enzyme was 38.47 kDa and the cellulolytic activity was 31.854 IU.
References
Abdullah, R., Akhtar, A., Nisar, K., Kaleem, A., Iqtedar1, M., Iftikhar, T., Saleem, F., y Aslam, F. (2021). Process optimization for enhanced production of cellulases from locally isolated fungal strain by submerged fermentation. Bioscience Journal, 37, e37021. https://doi.org/10.14393/BJ-v37n0a2021-53815
Afzal, M., Qureshi, M. Z., Khan, S., Khan, M. I., Ikram, H., Ashraf, A., Iqbal, A., y Quresh, N. A. (2019). Production, purification and optimization of cellulase by Bacillus licheniformis HI-08 isolated from the hindgut of wood-feeding termite. International Journal of Agriculture & Biology, 21(1), 125-134. https://doi.org/10.17957/IJAB/15.0872
Ahmed, K., Munawar, S., y Naz, S. (2015). Purification and characterization of cellulase from Aspergillus niger (Van Tieghem, 1867). Science International (Lahore), 27(5), 4341-4344. http://www.sci-int.com/pdf/636341779348991064.pdf
Amritkar, N., Kamat, M., y Lali, A. (2004). Expanded bed affinity purification of bacterial alpha-amylase and cellulase on composite substrate analogue–cellulose matrices. Process Biochemistry, 39(5), 565–570. https://doi.org/10.1016/S0032-9592(03)00123-7
Bakare, M. K., Adewale, I. O., Ajayi, A., y Shonukan, O. O. (2005). Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. African Journal of Biotechnology, 4 (9), 898-904. https://www.ajol.info/index.php/ajb/article/download/71111/60085
Behera, S. S., y Ray, R. C. (2016). Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies. International Journal of Biological Macromolecules, 86, 656-669. http://dx.doi.org/10.1016/j.ijbiomac.2015.10.090
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 (1-2): 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
Cerda, A., Gea, T., Vargas-García, M. C., y Sánchez, A. (2017). Towards a competitive solid state fermentation: Cellulases production from coffee husk by sequential batch operation and role of microbial diversity. Science of the Total Environment, 589, 56-65. http://dx.doi.org/10.1016/j.scitotenv.2017.02.184
Chaplin, M. F. (1994). Monosaccharides. In M. F. En Chaplin, M. F. y Kennedy, J. F. (Eds.), Carbohydrate Analysis: A Practical Approach, 24-25. Oxford University Press.
Das, A., Paul, T., Halder, S. K., Jana, A., Ghosh, K., Maity, C., Das, P. K. M., Pati, B., y Mondal, K. C. (2013). Low cost single-step purification of endoglucanase from Aspergillus fumigatus ABK-9. Indian Journal of Experimental Biology, 51 (11), 954-959. http://nopr.niscair.res.in/bitstream/123456789/23461/1/IJEB%2051(11)%20954-959.pdf
Devi, M. C., y Kumar, M. S. (2012). Production, optimization and partial purification of cellulase by Aspergillus niger fermented with paper and timber sawmill industrial wastes. Journal of Microbiology and Biotechnology Research, 2 (1): 120-128. https://www.semanticscholar.org/paper/Production%2C-Optimization-and-Partial-purification-Devi-Kumar/59dc009e5be5abd213284c2529f92d086aa2063b
Doi, R. H., y Kosugi, A. (2004). Cellulosomes: Plant-cell-wall degrading enzyme complexes. Nature Reviews: Microbiology, 2, 541-551. https://doi.org/10.1038/nrmicro925
Ejaz, U., Sohail, M., y Ghanemi, A. (2021). Cellulases: From bioactivity to a variety of industrial applications. Biomimetics, 6 (3). https://doi.org/10.3390/biomimetics6030044
EnCor Biotechnology Inc. (2022). Ammonium sulfate calculator. http://www.encorbio.com/protocols/AM-SO4.htm
Faheina, G. d. S., Amorim, M. V. da F. S., Souza, C. G. d., Sousa, D. M. d., Sousa, K. A. d., y Pinto, G. A. S. (2015). Strategies to increase cellulase production with submerged fermentation using fungi isolated from the Brazilian biome. Acta Scientiarum. Biological Science, 37 (1), 15-22. https://doi.org/10.4025/actascibiolsci.v37i1.23483
Farinas, C. S., Scarpelini, L. M., Miranda, E. A., y Neto, V. B. (2011). Evaluation of operational parameters on the precipitation of endoglucanasa and xylanase produced by solid state fermentation of Aspergillus niger. Brazilian Journal of Chemical Engineering, 28 (1), 17-26. https://www.scielo.br/j/bjce/a/7bmDY3gM5syf6mW4zDmVskS/?format=pdf&lang=en
Hamdan, N. T., y Jasim, H. M. (2018). Purification and characterization of cellulase enzyme from Trichoderma longibrachiatum isolated in Iraqi soil. IOSR Journal of Biotechnology and Biochemistry, 4 (1), 32-41. https://doi.org/10.9790/264X-04013241
Han, W., y He, M. (2010). The application of exogenous cellulase to improve soil fertility and plant growth due to acceleration of straw decomposition. Bioresource Technology, 101 (10), 3724–3731. https://doi.org/10.1016/j.biortech.2009.12.104
Hasunuma, T., Okazaki, F., Okai, N., Hara, K. Y., Ishii, J., y Kondo, A. (2013). A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresource Technology, 135, 513-522. http://doi.org/10.1016/j.biortech.2012.10.047
Heukeshoven, J., y Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6 (3), 103-112. https://doi.org/10.1002/elps.1150060302
Hölker, U., Höfer, M., y Lenz, J. (2004). Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl Microbiol Biotechnol, 64, 175-186. https://doi.org/10.1007/s00253-003-1504-3
Ijaz, A., Anwar, Z., Irshad, M., Iqbal, Z., Arshad, M., Javed, M., Zulfiqar, M., Rehman, A., y Ahmad, A. (2014). Purification and kinetic characterization of statistically optimized cellulase produced from Aspergillus niger. Romanian Biotechnological Letters, 19 (6), 9835-9845. https://e-repository.org/rbl/vol.19/iss.6/4.pdf
ITW Reagents Division. (2022). Ammonium Sulfate for molecular biology. PanReac AppliChem. https://www.itwreagents.com/rest-of-world/en/product/ammonium-sulfate-for-molecular-biology/A3485
Iqbal, H. M. N., Ahmed, I., Zia, M. A., y Irfan, M. (2011). Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility. Advances in Bioscience and Biotechnology, 2 (3), 149-156. https://doi.org/10.4236/abb.2011.23024
Islam, F., y Roy, N. (2018). Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. BMC Research Notes, 11, 1-6. https://doi.org/10.1186/s13104-018-3558-4
Juturu, V., y Wu, J. C. (2014). Microbial cellulases: Engineering, production and applications. Renewable and Sustainable Energy Reviews, 33, 188-203. http://doi.org/10.1016/j.rser.2014.01.077
Kumar, B., Bhardwaj, N., Alam, A., Agrawal, K., Prasad, H., y Verma, P. (2018). Production, purification and characterization of an acid/alkali and thermo tolerant cellulase from Schizophyllum commune NAIMCCF03379 and its application in hydrolysis of lignocellulosic wastes. AMB Express, 8 (173), 1-16. https://doi.org/10.1186/s13568-018-0696-y
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature, 227, 680-685. https://doi.org/10.1038/227680a0
Lee, K. C., Tong, W. Y., Ibrahim, D., Arai, T., Murata, Y., Mori, Y., y Kosugi, A. (2016). Evaluation of enzymatic deinking of non-impact ink laser-printed paper using crude enzyme from Penicillium rolfsii c3-2(1) IBRL. Appl Biochem Biotechnol, 181 (1), 451-463. https://doi.org/10.1007/s12010-016-2223-4
Leon-Revelo, G., Cujilema-Quitio, M. C., Baryolo, L., Rosero, E., Córdova, J., y Ramos-Sánchez, L. B. (2017). Efecto del pH en la producción de celulasas de Aspergillus niger en fermentación sólida. Revista Centro Azúcar, 44 (2), 27-38. http://centroazucar.uclv.edu.cu/index.php/centro_azucar/article/view/98/91
Liu, Y., Guo, H., Gu, J., y Qin, W. (2019). Optimize purification of a cellulase from Bacillus velezensis A4 by aqueous two-phase system (ATPS) using response surface methodology. Process Biochemistry, 87, 196-203. https://doi.org/10.1016/j.procbio.2019.08.017
Lodha, A., Pawar, S., y Rathod, V. (2020). Optimised cellulase production from fungal co-culture of Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 under solid state fermentation. Journal of Environmental Chemical Engineering, 8 (5), 103958. https://doi.org/10.1016/j.jece.2020.103958
Malik, W. A., y Javed, S. (2021). Biochemical characterization of cellulase from Bacillus subtilis strain and its effect on digestibility and structural modifications of lignocellulose rich biomass. Frontiers in Bioengineering and Biotechnology, 9, 1-15. https://doi.org/10.3389/fbioe.2021.800265
Manan, M. A., y Webb, C. (2017). Design aspects of solid state fermentation as applied to microbial bioprocessing. Journal of Applied Biotechnology & Bioengineering, 4 (1), 511-532. https://doi.org/10.15406/jabb.2017.04.00094
Marín, R. M. (2007). Caracterización y expresión recombinante de una celulasa de origen antártico. (Tesis de pregrado, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile). Repositorio Institucional. Santiago de Chile. https://repositorio.uchile.cl/tesis/uchile/2007/marin_ra/sources/marin_ra.pdf
Mrudula, S., y Murugammal, R. (2011). Production of cellulase by Aspergillus niger under submerged and solid state fermentation using coir waste as a substrate. Brazilian Journal of Microbiology, 42 (3), 1119-1127. https://doi.org/10.1590/S1517-83822011000300033
Nehad, E. A., Yoness, M. F., y Reem, A. A. (2019). Optimization and purification of cellulase produced by Penicillium decumbens and its application. Egyptian Pharmaceutical Journal, 18(4): 391-402. https://doi.org/10.4103/epj.epj_31_19
Oliveira, J. S. D. , de Araujo, P. C. E., Alves, E. de A., Ribeiro, G. de M., y dos Santos, E. S. (2020). Recovery and purification of cellulolytic enzymes from Aspergillus fumigatus CCT 7873 using an aqueous two-phase micellar system. Annals of Microbiology, 70 (23): 1-12. https://doi.org/10.1186/s13213-020-01573-w
Pathak, P., Bhardwaj, N. K., y Singh, A. K. (2014). Production of crude cellulase and xylanase from Trichoderma harzianum PPDDN10 NFCCI-2925 and its application in photocopier waste paper recycling. Applied Biochemistry and Biotechnology, 172 (8), 3776-3797. https://doi.org/10.1007/s12010-014-0758-9
Rabinovich, M. L., Melnick, M. S., y Bolobova, A. V. (2002). The structure and mechanism of action of cellulolytic enzymes. Biochemistry (Moscow), 67 (8), 850-871. https://doi.org/10.1023/A:1019958419032
Raju, P. S., y Bawa, A. S. (2006). Food additives in fruit processing. In Hui, Y. H. (Ed.), Handbook of Fruits and Fruit Processing, 145-170. Blackwell Publishing. https://doi.org/10.1002/9780470277737.ch9
Sajith, S., Sreedevi, S., Priji, P., Unni, K. N., y Benjamin, S. (2014). Production and partial purification of cellulase from a novel fungus, Aspergillus flavus BS1. Annals of Microbiology, 64, 763-771. https://doi.org/10.1007/s13213-013-0711-0
Sathya, T. A., y Khan, M. (2014). Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry. Journal of Food Science, 79 (11), R2149 - R2156. https://doi.org/10.1111/1750-3841.12677
Singhania, R. R., Sukumaran, R. K., Patel, A. K., Larroche, C., y Pandey, A. (2010). Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme and Microbial Technology, 46 (7), 541-549. https://doi.org/10.1016/j.enzmictec.2010.03.010
Siqueira, J. G. W., Rodrigues, C., Vandenberghe, L. P. d. S., Woiciechowski, A. L., y Soccol, C. R. (2020). Current advances in on-site cellulase production and application on lignocellulosic biomass conversion to biofuels: A review. Biomass and Bioenergy, 132, 105419. https://doi.org/10.1016/j.biombioe.2019.105419
Sukumaran, R. K., Singhania, R. R., y Pandey, A. (2005). Microbial cellulases - Production, application and challenges. Journal of Scientific & Industrial Research, 64 (11), 832-844. https://www.researchgate.net/profile/Reeta-Singhania/publication/228635285_ Microbial_cellulases-Production_applications_and_challenges/links/09e41507fb356886 1a000000/Microbial-cellulases-Production-applications-and-challenges.pdf
Sulyman, A. O., Igunnu, A., y Malomo, S. O. (2020). Isolation, purification and characterization of cellulase produced by Aspergillus niger cultured on Arachis hypogaea shells. Heliyon, 6 (12), e05668. https://doi.org/10.1016/j.heliyon.2020.e05668
Wu, J. C., Ng, K. R., Chong, J., Yang, K. J., Ping, X. L., Nam, C. T., y Nugroho, A. J. (2010). Recovery of cellulases by adsorption/desorption using cation exchange resins. Korean Journal of Chemical Engineering, 27 (2), 46-473. https://doi.org/10.1007/s11814-010-0096-8
Yang, S.-T., El-Enshasy, H., y Thongchul, N. (2013). Bioprocessing technologies in biorefinery for sustainable production of fuels, chemicals, and polymers. John Wiley & Sons, Inc. New Jersey, USA. https://es.b-ok.lat/book/2159136/f07836
Zahra, T., Irfan, M., Nadeem, M., Ghazanfar, M., Ahmad, Q., Ali, S., Siddique, F., Yasmeen, Z., y Franco, M. (2020). Cellulase production by Trichoderma viride in submerged fermentation using response surface methodology. Punjab University Journal of Zoology, 35(2), 223-228. https://dx.doi.org/10.17582/journal.pujz/2020.35.2.223.228
Zapata, Y. M., Galviz-Quezada, A., y Osorio, V. M. (2018). Cellulases production on paper and sawdust using native Trichoderma asperellum. Universitas Scientiarum, 23(3), 419-436. https://doi.org/10.11144/Javeriana.SC23-3.cpop
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Downloads
Download data is not yet available.
