Isolation and characterization of polygalacturonase producing thermophilic Aspergillus niger isolated from decayed tomato fruits
DOI:
https://doi.org/10.18006/2024.12(3).379.389Keywords:
Aspergillus niger, Column chromatography, Polygalacturonase, Solid-state fermentationAbstract
This study aimed to isolate a fungal strain capable of producing acidophilic and thermostable polygalacturonase. In this study, the fungal isolate was isolated from decaying tomatoes. Based on the colony characteristics, microscopic and morphological observations, the isolated fungal pathogen has been identified as Aspergillus niger. The isolated fungus was used in solid-state fermentation to produce an acidic polygalacturonase enzyme. The enzyme was then purified using ammonium sulphate precipitation and column chromatography, and its activity was assayed by measuring the releasing sugar group from citrus pectin using a 3, 5-dinitrosalicylic acid (DNSA) reagent assay. The crude extract obtained from solid-state fermentation had an activity of 94.6 U/mL. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Sephadex G-200 was used to purify the enzyme 3.58 times, and its specific activity was determined to be 24.66 U/mg. The Sephacryl S-100 column achieved a final fold purification of 9.93 times and a specific activity of 68.41 U/mg. The purified enzyme performed best when polygalacturonic acid was used as a substrate. The enzyme's optimum temperature and pH were 55°C and 5, respectively. CaCl2 was found to be the best chelating ion for the enzyme. This enzyme is recommended for use in a variety of industrial applications as the enzyme was found to be stable at acidic pH and high temperature.
References
Acuña-Argüelles, M. E., Gutiérrez-Rojas, M., Viniegra-González, G., & Favela-Torres, E. (1995). Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation. Applied Microbiology and Biotechnology, 43(5), 808–814. https://doi.org/10.1007/BF02431912 DOI: https://doi.org/10.1007/BF02431912
Adedayo, M. R., Mohammed, M. T., Ajiboye, A. E., & Abdulmumini, S. A. (2021). Pectinolytic activity of Aspergillus niger and Aspergillus flavus grown on grapefruit (citrus Parasidis) peel in solid state fermentation. Global Journal of Pure and Applied Sciences, 27(2), 93–105. https://doi.org/10.4314/gjpas.v27i2.2 DOI: https://doi.org/10.4314/gjpas.v27i2.2
Adeyefa, O., & Ebuehi, O. (2020). Isolation, Identification and Characterization of Pectinase Producers from Agro Wastes (Citrus sinensis and Ananas comosus). World Journal of Agriculture and Soil Science, 4(3). https://doi.org/10.33552/WJASS.2020.04.000589 DOI: https://doi.org/10.33552/WJASS.2020.04.000589
Ahmed, J., Thakur, A., & Goyal, A. (2021). Emerging trends on the role of recombinant pectinolytic enzymes in industries- an overview. Biocatalysis and Agricultural Biotechnology, 38, 102200. https://doi.org/10.1016/j.bcab.2021.102200 DOI: https://doi.org/10.1016/j.bcab.2021.102200
Ahmed Olaitan, Y. (2019). Characterization and Partial Purification of Pectinase Produced by Aspergillus niger Using Banana Peel as Carbon Source [Kwara State University]. Retrieved from https://www.proquest.com/openview/474b6ae1c0692a0a4f9d6ecdae26e885/1?pq-origsite=gscholar&cbl=18750&diss=y
Alavi, A., Nia, F. T., & Shariati, F. P. (2020). Polygalacturonase Production by Aspergillus niger Solid State Fermentation on Barley Bran and Sugar Beet Pulp Mixture. Advanced Journal of Chemistry-Section A, 3(3), 350–357. https://doi.org/10.33945/ SAMI/AJCA.2020.3.13 DOI: https://doi.org/10.33945/SAMI/AJCA.2020.3.13
Almowallad, S. A., Alshammari, G. M., Alsayadi, M. M., Aljafer, N., Al-Sanea, E. A., Yahya, M. A., & Al-Harbi, L. N. (2022). Partial Purification and Characterization of Exo-Polygalacturonase Produced by Penicillium oxalicum AUMC 4153. Life, 12(2), 284. https://doi.org/10.3390/life12020284 DOI: https://doi.org/10.3390/life12020284
Alves, M. H., Campos-Takaki, G. M., Porto, A. L. F., & Milanez, A. I. (2002). Screening of Mucor spp. For the production of amylase, lipase, polygalacturonase and protease. Brazilian Journal of Microbiology, 33(4). https://doi.org/10.1590/S1517-83822002000400009 DOI: https://doi.org/10.1590/S1517-83822002000400009
Aminzadeh, S., Naderimanesh, H., Khajeh, K., & Sooudi, M. (2007). Isolation and Characterization of Polygalacturonase produced by Tetracoccosporium Sp. Iranian Journal of Chemistry and Chemical Engineering, 26(1), 47–54.
Anand, G., Yadav, S., & Yadav, D. (2016). Purification and characterization of polygalacturonase from Aspergillus fumigatus MTCC 2584 and elucidating its application in retting of Crotalaria juncea fiber. 3 Biotech, 6(2), 201. https://doi.org/10.1007/s13205-016-0517-4 DOI: https://doi.org/10.1007/s13205-016-0517-4
Badhan, A., Huang, J., Wang, Y., Abbott, D. W., Di Falco, M., Tsang, A., & McAllister, T. (2018). Saccharification efficiencies of multienzyme complexes produced by aerobic fungi. New Biotechnology, 46, 1–6. https://doi.org/10.1016/j.nbt.2018.05.003 DOI: https://doi.org/10.1016/j.nbt.2018.05.003
Balabanova, L., Slepchenko, L., Son, O., & Tekutyeva, L. (2018). Biotechnology Potential of Marine Fungi Degrading Plant and Algae Polymeric Substrates. Frontiers in Microbiology, 9, 1527. https://doi.org/10.3389/fmicb.2018.01527 DOI: https://doi.org/10.3389/fmicb.2018.01527
Bentouhami, N. E., Asehraou, A., Mechri, S., Hasnaoui, I., Moumnassi, S., et al. (2024). Purification and biochemical characterization of a novel thermostable endo-polygalacturonase from Aspergillus niger strain HO32 and its suitability for clarification of orange juice. Process Biochemistry, 145, 63–73. https://doi.org/10.1016/j.procbio.2024.06.013 DOI: https://doi.org/10.1016/j.procbio.2024.06.013
Buga, M. L., Ibrahim, S., & Nok, A. J. (2010). Partially purified polygalacturonase from Aspergillus niger (SA6). African Journal of Biotechnology, 9(52), 8944–8954.
Cheng, Z., Chen, D., Lu, B., Wei, Y., Xian, L., Li, Y., Luo, Z., & Huang, R. (2016). A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry. Journal of Microbiology and Biotechnology, 26(6), 989–998. https://doi.org/10.4014/jmb.1511.11045 DOI: https://doi.org/10.4014/jmb.1511.11045
Chinedu, S. N., Dayo-Oduko, O. P., & Iheagwam, F. N. (2016). Partial Purification and Kinetic Properties of Polygalacturonase from Solanum macrocarpum L. Fruit. Biotechnology(Faisalabad), 16(1), 27–33. https://doi.org/10.3923/biotech.2017.27.33 DOI: https://doi.org/10.3923/biotech.2017.27.33
Cocok, A. M. B., Fahrurrozi, F., & Anja, M. (2017). Pectinase Production and Clarification Treatments of Apple (Malus Domestica) Juice. Annales Bogorienses, 21(2), 63–68. DOI: https://doi.org/10.14203/ann.bogor.2017.v21.n2.63-68
Gams, W., Christensen, M., Onions, A. H., Pitt, J. I., & Samson, R. A. (1986). Infrageneric Taxa of Aspergillus. In R. A. Samson & J. I. Pitt (Eds.), Advances in Penicillium and Aspergillus Systematics (pp. 55–62). Springer US. https://doi.org/10.1007/978-1-4757-1856-0_5 DOI: https://doi.org/10.1007/978-1-4757-1856-0_5
Garcia, N. F. L., Santos, F. R. D. S., Bocchini, D. A., Paz, M. F. D., Fonseca, G. G., & Leite, R. S. R. (2018). Catalytic properties of cellulases and hemicellulases produced by Lichtheimia ramosa: Potential for sugarcane bagasse saccharification. Industrial Crops and Products, 122, 49–56. https://doi.org/10.1016/ j.indcrop.2018.05.049 DOI: https://doi.org/10.1016/j.indcrop.2018.05.049
Gomes, E., Leite, R. S. R., Da Silva, R., & Silva, D. (2009). Purification of an Exopolygalacturonase from Penicillium viridicatum RFC3 Produced in Submerged Fermentation. International Journal of Microbiology, 2009, 1–8. https://doi.org/10.1155/2009/631942 DOI: https://doi.org/10.1155/2009/631942
Haile, S., & Ayele, A. (2022). Pectinase from Microorganisms and Its Industrial Applications. The Scientific World Journal, 2022, 1–15. https://doi.org/10.1155/2022/1881305 DOI: https://doi.org/10.1155/2022/1881305
Ikram, M., Ali, N., Jan, G., Jan, F. G., & Khan, N. (2020). Endophytic Fungal Diversity and their Interaction with Plants for Agriculture Sustainability Under Stressful Condition. Recent Patents on Food, Nutrition & Agriculture, 11(2), 115–123. https://doi.org/10.2174/2212798410666190612130139 DOI: https://doi.org/10.2174/2212798410666190612130139
Jayani, R. S., Saxena, S., & Gupta, R. (2005). Microbial pectinolytic enzymes: A review. Process Biochemistry, 40(9), 2931–2944. https://doi.org/10.1016/j.procbio.2005.03.026 DOI: https://doi.org/10.1016/j.procbio.2005.03.026
Jayani, R. S., Shukla, S. K., & Gupta, R. (2010). Screening of Bacterial Strains for Polygalacturonase Activity: Its Production by Bacillus sphaericus (MTCC 7542). Enzyme Research, 2010, 1–5. https://doi.org/10.4061/2010/306785 DOI: https://doi.org/10.4061/2010/306785
Karimi, F., Mazaheri, D., Saei Moghaddam, M., Mataei Moghaddam, A., Sanati, A. L., & Orooji, Y. (2021). Solid-state fermentation as an alternative technology for cost-effective production of bioethanol as useful renewable energy: A review. Biomass Conversion and Biorefinery. https://doi.org/10.1007/ s13399-021-01875-2 DOI: https://doi.org/10.1007/s13399-021-01875-2
Kaur, G., Kumar, S., & Satyanarayana, T. (2004). Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis. Bioresource Technology, 94(3), 239–243. https://doi.org/10.1016/j.biortech.2003.05.003 DOI: https://doi.org/10.1016/j.biortech.2003.05.003
Khatri, B. P., Bhattarai, T., Shrestha, S., & Maharjan, J. (2015). Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal. Springer Plus, 4(1), 488. https://doi.org/10.1186/s40064-015-1286-y DOI: https://doi.org/10.1186/s40064-015-1286-y
Klich, M. A. (2002). Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Lamounier, K. F. R., Rodrigues, P. O., Pasquini, D., & Baffi, M. A. (2018). Saccharification of Sugarcane Bagasse Using an Enzymatic Extract Produced by Aspergillus fumigatus. Journal of Renewable Materials, 6(2), 169–175. https://doi.org/10.7569/ JRM.2017.634151 DOI: https://doi.org/10.7569/JRM.2017.634151
Li, S., Yang, X., Yang, S., Zhu, M., & Wang, X. (2012). Technology Prospecting On Enzymes: Application, Marketing and Engineering. Computational and Structural Biotechnology Journal, 2(3), e201209017. https://doi.org/10.5936/csbj.201209017 DOI: https://doi.org/10.5936/csbj.201209017
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193(1), 265–275. DOI: https://doi.org/10.1016/S0021-9258(19)52451-6
Madamwar, D., Patel, S., & Parikh, H. (1989). Solid state fermentation for cellulases and β-glucosidase production by Aspergillus niger. Journal of Fermentation and Bioengineering, 67(6), 424–426. https://doi.org/10.1016/0922-338X(89)90150-5 DOI: https://doi.org/10.1016/0922-338X(89)90150-5
Majumdar, D. R., Singh, R., Dondilkar, S., Shaikh, N., Pawale, G., Shinde, P., & Sakate, P. (2018). Enzyme array from thermophilic fungal isolate—RSND. World Journal of Pharmaceutical Research, 7(4), 60–70.
Martins, E. D. S., Leite, R. S. R., Da Silva, R., & Gomes, E. (2012). Production and characterization of polygalacturonase from thermophilic Thermoascus aurantiacus on submerged fermentation. Annals of Microbiology, 62(3), 1199–1205. https://doi.org/10.1007/s13213-011-0360-0 DOI: https://doi.org/10.1007/s13213-011-0360-0
Munir, M., Abdullah, R., Haq, I., Kaleem, A., & Iqtedar, M. (2019). Isolation and identification of multi stress tolerant polygalacturonase producing fungi from various fruits. The Journal of Animal & Plant Sciences, 29(3), 825–832.
Oumer, O. J. (2017). Pectinase: Substrate, Production and their Biotechnological Applications. International Journal of Environment, Agriculture and Biotechnology, 2(3), 1007–1014. https://doi.org/10.22161/ijeab/2.3.1 DOI: https://doi.org/10.22161/ijeab/2.3.1
Paolo, D., Bianchi, G., Scalzo, R. L., Morelli, C. F., Rabuffetti, M., & Speranza, G. (2018). The Chemistry behind Tomato Quality. Natural Product Communications, 13(9), 1934578X1801300. https://doi.org/10.1177/1934578X1801300927 DOI: https://doi.org/10.1177/1934578X1801300927
Pedrolli, D. B., Monteiro, A. C., Gomes, E., & Carmona, E. C. (2009). Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes. The Open Biotechnology Journal, 3(1), 9–18. https://doi.org/10.2174/1874070700903010009 DOI: https://doi.org/10.2174/1874070700903010009
Pitt, J. I., & Hocking, A. D. (2009). Fungi and Food Spoilage. Springer US. https://doi.org/10.1007/978-0-387-92207-2 DOI: https://doi.org/10.1007/978-0-387-92207-2
Rahman, Md. S., Choi, Y. S., Kim, Y. K., Park, C., & Yoo, J. C. (2019). Production of Novel Polygalacturonase from Bacillus paralicheniformis CBS32 and Application to Depolymerization of Ramie Fiber. Polymers, 11(9), 1525. https://doi.org/10.3390/polym11091525 DOI: https://doi.org/10.3390/polym11091525
Ramesh, A., Harani Devi, P., Chattopadhyay, S., & Kavitha, M. (2020). Commercial Applications of Microbial Enzymes. In N. K. Arora, J. Mishra, & V. Mishra (Eds.), Microbial Enzymes: Roles and Applications in Industries (Vol. 11, pp. 137–184). Springer Singapore. https://doi.org/10.1007/978-981-15-1710-5_6 DOI: https://doi.org/10.1007/978-981-15-1710-5_6
Ramos-Ibarra, J., Miramontes, C., Arias, A., Arriola, E., Guatemala, G., & Corona-González, R. (2017). Production of hydrolytic enzymes by solid-state fermentation with new fungal strains using orange by-products. Revista Mexicana de Ingeniería Química, 16(1), 19–31. DOI: https://doi.org/10.24275/rmiq/Bio697
Raper, K., & Fennell, D. (1965). The Genus Aspergillus. Williams & Wilkins.
Raveendran, S., Parameswaran, B., Ummalyma, S. B., Abraham, A., Mathew, A. K., Madhavan, A., Rebello, S., & Pandey, A. (2018). Applications of Microbial Enzymes in Food Industry. Food Technology and Biotechnology, 56(1), 16–30. https://doi.org/10.17113/ftb.56.01.18.5491 DOI: https://doi.org/10.17113/ftb.56.01.18.5491
Ravi, K., & Raghu, R. (2017). Isolation and Screening of Industrially Important Polygalcturonase producing Fungi from the Mangrove Soils of Krishna District Andhra Pradesh. International Journal of Agricultural Sciences, 9(19), 4193–4195.
Reddy, P., & Sreeramulu, A. (2012). Isolation, identification and screening of pectinolytic fungi from different soil samples of Chittoor district. International Journal of Life Sciences Biotechnology and Pharma Research, 1(3), 186–193.
Ribeiro, D. S., Henrique, S. M. B., Oliveira, L. S., Macedo, G. A., & Fleuri, L. F. (2010). Enzymes in juice processing: A review. International Journal of Food Science & Technology, 45(4), 635–641. https://doi.org/10.1111/j.1365-2621.2010.02177.x DOI: https://doi.org/10.1111/j.1365-2621.2010.02177.x
Samal, I., Bhoi, T. K., Majhi, P. K., Murmu, S., Pradhan, A. K., et al. (2023). Combatting insects mediated biotic stress through plant associated endophytic entomopathogenic fungi in horticultural crops. Frontiers in Plant Science, 13, 1098673. https://doi.org/10.3389/fpls.2022.1098673 DOI: https://doi.org/10.3389/fpls.2022.1098673
Samson, R., Houbraken, J., Thrane, U., Frisvad, J., & Anderson, B. (2010). Food and indoor fungi: Second Edition. (2nd ed.) CBS-KNAW Fungal Biodiversity Centre. C B S Laboratory Manual Series No. 2, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands
Satapathy, S., Soren, J. P., Mondal, K. C., Srivastava, S., Pradhan, C., Sahoo, S. L., Thatoi, H., & Rout, J. R. (2021). Industrially relevant pectinase production from Aspergillus parvisclerotigenus KX928754 using apple pomace as the promising substrate. Journal of Taibah University for Science, 15(1), 347–356. https://doi.org/10.1080/16583655.2021.1978833 DOI: https://doi.org/10.1080/16583655.2021.1978833
Shamly, V., Kali, A., Srirangaraj, S., & Umadevi, S. (2014). Comparison of Microscopic Morphology of Fungi Using Lactophenol Cotton Blue (LPCB), Iodine Glycerol and Congo Red Formaldehyde Staining. Journal of Clinical and Diagnostic Research, 8(7), DL01-02. https://doi.org/10.7860/JCDR/2014/ 8521.4535
Siddiqui, Mohd. A., Pande, V., & Arif, M. (2012). Production, Purification, and Characterization of Polygalacturonase from Rhizomucor pusillus Isolated from Decomposting Orange Peels. Enzyme Research, 2012, 1–8. https://doi.org/10.1155/2012/138634 DOI: https://doi.org/10.1155/2012/138634
Soares, I., Távora, Z., Barcelos, R., & Baroni, S. (2012). Microorganism-produced enzymes in the food industry. In B. Valdez, M. Schorr, & R. Zlatev (Eds.), Scientific, health and social aspects of the food industry (pp. 83–94). IntechOpen. DOI: https://doi.org/10.5772/31256
Tai, E.S., Hsieh, P.C., & Sheu, S.C. (2013). Purification and Characterization of Polygalacturonase from Screened Aspergillus tubingensis for Coffee Processing. Food Science and Technology Research, 19(5), 813–818. https://doi.org/10.3136/fstr.19.813 DOI: https://doi.org/10.3136/fstr.19.813
Thakur, A., Pahwa, R., Singh, S., & Gupta, R. (2010). Production, Purification, and Characterization of Polygalacturonase from Mucor circinelloides ITCC 6025. Enzyme Research, 2010, 1–7. https://doi.org/10.4061/2010/170549 DOI: https://doi.org/10.4061/2010/170549
Wang, S., Lian, Z., Wang, L., Yang, X., & Liu, Y. (2015). Preliminary investigations on a polygalacturonase from Aspergillus fumigatus in Chinese Pu'er tea fermentation. Bioresources and Bioprocessing, 2(1), 33. https://doi.org/10.1186/s40643-015-0061-9 DOI: https://doi.org/10.1186/s40643-015-0061-9
Yoon, L. W., Ang, T. N., Ngoh, G. C., & Chua, A. S. M. (2014). Fungal solid-state fermentation and various methods of enhancement in cellulase production. Biomass and Bioenergy, 67, 319–338. https://doi.org/10.1016/j.biombioe.2014.05.013 DOI: https://doi.org/10.1016/j.biombioe.2014.05.013
Zhang, S., Amin, F., Xiong, M., Bhatti, H. N., & Bilal, M. (2021). High-yield intracellular production of an exo-polygalacturonase enzyme via heterologous expression of Penicillium notatum gene in Saccharomyces cerevisiae. International Food Research Journal, 28(4), 664–671. https://doi.org/10.47836/ifrj.28.4.03 DOI: https://doi.org/10.47836/ifrj.28.4.03
Downloads
Published
How to Cite
License
Copyright (c) 2024 Journal of Experimental Biology and Agricultural Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
