Characterization of sn-1,3 extracellular lipases of Aspergillus niger and Rhizopus oryzae for the crude palm oil hydrolysis
Keywords:
enzymatic hydrolysis, filamentous fungi, sn-2 palmitate, vegetable oilAbstract
Crude palm oil (CPO) processing for sn-2 palmitate synthesis requires a specific sn-1,3 lipase to catalyze the hydrolysis step. These lipases are known to be derived from filamentous fungal isolates including Aspergillus niger and Rhizopus oryzae. The purpose of this study was to describe extracellular lipases from A. niger and R. oryzae that hydrolyze CPO. Extracellular lipases were successfully recovered from both fungal isolates using CPO-enriched fermentation media. The crude fraction was partially refined using (NH4)2SO4 and dialyzed, yielding two fractions. Lipase fraction I from both fungal species had the highest specific activity, had a molecular mass of ~30 kDa, and was sn-1,3 specific. The best conditions for enzyme activity of both fungal species in CPO hydrolysis were pH 6-7 and 35 ℃. The pH and heat stability of lipase fraction I in both fungi were relatively low. However, this enzyme worked effectively in benzene, ethanol, and methanol solvents.
Downloads
References
Alabdalall, H. A., Al-Anazi, N. A., Aldakheel, L. A., Amer, I. H. F., Aldakheel, A. F., Ababutain, M. I., Alghamdi, I. A., & Al-Khaldi, M. E. 2021. Application and characterization of crude fungal lipases used to degrade fat and oil wastes. Scientific Reports, 11(19670), 1-10. https://doi.org/10.1038/s41598-021-98927-4
Albayati, H. S., Masomia, M., Ishak, H. S., Ali, M. S. M., Thean, L. A., Shariff, Noor, M. D. N., & Rahman, A. R. J. N. 2020. Main structural targets for engineering lipase substrate specificity. Catalysts, 10(747), 1-34. https://doi.org/10.3390/catal10070747
Al-dahlan, Baghdadi, M. A., & Alkattan, O. M. 2024. Microbial lipases: Between production, purification, and their biotechnological applications. Current Science International. 13(1), 75-86. https://doi.org/10.36632/csi/2024.13.1.6
Ali, S., Khan, A. S., Hamayun, M., & Lee, I. 2023. The recent advances in the utility of microbial lipases, 11(540), 1-26. https://doi.org/10.3390/microorganisms11020510
Ayinla, A. Z., Ademakinwa, N. A., & Agboola, K. F. 2017. Studies on the optimization of lipase production by Rhizopus sp. ZAC3 isolated from the contaminated soil of a palm oil processing shed. Journal of Applied Biology amd Biotechnology, 5(2), 30-37. https://doi.org/10.7324/JABB.2017.50205
Barathi, D., Rajalakshmi, G., & Komathi, S. 2019. Optimization and production of lipase enzyme from bacterial strains isolated from petrol spilled soil. Journal of King Saud University, 31, 898-901. https://doi.org/10.1016/j.jksus.2017.12.018
Bomfima, S. R., Velasco, J., Cardoso, A. L., Ribeiro, F. D. C., Marinho, M. Q. L., Ribeiro, R, P., & Almeida, T. D. (2024). Processing practices and quality of crude palm oil produced on a small scale in Valença, Bahia, Brazil. Grasas Y Aceites, 75(2), 1-12, https://doi.org/10.3989/gya.1191231.2084
Dasetty, S., Blenner, A., M., & Sarupria, S. 2017. Engineering lipases: walking the fine line between activity and stability. Materials Research Express, 1-32. https://doi.org/10.1088/2053-1591/aa9946
Du, M., Hou, Z., Liu, L., Xuan, Y., Chen, X., Fan, L., Li, Z., & Xu, B. 2022. Progress, applications, challenges and prospects of protein purification technology. Friontiers in Bioengineering and Biotechnology, 10, 1-26. https://doi.org/10.3389/fbioe.2022.1028691
Emmanuel, B. M., Evans, C. D., Abubakar, A., Labaran, M. L., Ali, V. A., & Zabe, M. 2020. Production, partial purification and characterization of lipase enzyme expressed by Klebsiella pnemoniae of vegetable oil contaminated soil. International Journal of Biochemistry and Biophysics, 8(2), 30-39. https://doi.org/10.13189/ijbb.2020.080202
Fathi, F., Kasra-Kermanshahi, Moosavi-Nejad, Z., & Qamsari, M. E. 2021. Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum. Iranian Journal of Microbiology, 13(6), 871-877. https://doi.org/10.18502/ijm.v13i6.8093
Girelli, G. M., & Chiappini, V. 2023. Renewable, sustainable, and natural lignocellulosic carriers for lipase immobilization: A review. Journal of Biotechnology, 365, 29-47. https://doi.org/10.1016/j.jbiotec.2023.02.003
Goldring, D. P. J. 2018. Measuring protein concentration with absorbance, Lowry, Bradford Coomassie Blue, or the smith bicinchoninic acid assay before electrophoresis. Electrophoretic Separation of Protein: Method and Protocols, 1855, 31-39. https://doi.org/10.1007/978-1-4939-8793-1_3
Hasibuan, A. H. 2022. The synthesis of sn-2 palmitate as human milk fat substitute from palm oil fractions by enzymatic interesterification – a review. Journal of Oil Palm Research, 34(4), 608-621. https://doi.org/10.21894/jopr.2022.0045
Helal, E. S., Abdelhady, M. H., Abou-Taleb, A. K., Hassan, G. M., & Amer, M. M. 2021. Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production. Journal of Genetic Engineering and Biotechnology, 19(1), 1-13. https://doi.org/10.1186/s43141-020-00094-y
Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M. H., Riaz, M., Afzal, M., Kouser, A., & Nadeem, H. 2018. Bacterial lipases: a review on purification and characterization. Progress in Biophysic and Molecular Biology, 132, 23-34. https://doi.org/10.1016/j.pbiomolbio.2017.07.014
Köse, K., Erol, K., Köse, A. D., Evci, E., & Uzun, L. 2016. Affinity purification lipase from wheat germ: comparison of hydrophobic and metal chelation effect. Artificial Cells, Nanomedicine, and Biotechnology, 45(3), 574-583. https://doi.org/10.3109/21691401.2016.1161642
Kumar, A., Mukhia, S., Kumar, N., Acharya, V., Kumar, S., & Kumar, R. 2021. A broad temperature active lipase purified from a psychrotrophic bacterium of sikkim himalaya with potential application in detergent formulation. Frontier in Bioengineering and Biotechnology, 8(642), 1-16. https://doi.org/10.3389/fbioe.2020.00642
Liu, Z., & Smith, R. S. 2021. Enzyme recovery from biological wastewater treatment. Waste and Biomass Valorization, 12, 4185-4211. https://doi.org/10.1007/s12649-020-01251-7
Man, C. B. Y., Haryati, T., Ghazali, M. H., & Asbi, A. B. 1999. Composition and thermal profile of crude palm oil and its products. JAOCS, 76(2), 237-242. https://doi.org/10.1007/s11746-999-0224-y
Nargotra, P., Sharma, V., Sharma, S., Bangotra, R., & Bajaj, K. B. 2022. Purification of an ionic liquid stable cellulase from Aspergillus aculeatus PN14 with potential for biomass refining. Environmental Sustainability 5, 313-323. https://doi.org/10.1007/s42398-022-00232-x
Nuraliyah, A., Perdani, Putri, N. D., Sahlan, M., & Hermansyah, H. 2021. Effect of additional amino group to improve the performance of immobilized lipase from Aspergillus niger by adsorption-crosslinking method. Frontiers in Energy Research, 9, 1-11. https://doi.org/10.3389/fenrg.2021.616945
Pérez, M. M., Gonçalves, S. C., Vici, C. A., Salgado, S. C., & Polizeli, M. T. L. M. 2019. Fungal lipases: versatile tools for white biotechnology. Springer Nature Switzerland, 361-404. https://doi.org/10.1007/978-3-030-10480-1_11
Putra, L., Natadiputri, H. G., Meryandini, A., & Suwanto, A. 2019. Isolation, cloning and co-expression of lipase and foldase genes of Burkholderia territorii GP3 from Mount Papandayan Soil. Journal Microbialogy biotechnology, 29(6), 944-951. https://doi.org/10.4014/jmb.1812.12013
Remonatto, D., Ferrari, R. B., Bassan, C. J., Santos-Ebinuma, C. V., & Paula, V. A. 2021. Utilization of clay materials as support for Aspergillus japonicus lipase: an eco-friendly approach. Catalysts, 11(10), 1-17. https://doi.org/10.3390/catal11101173
Salvatierra NH, Regner LE, Baifori DM, Pera ML. 2021. Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology. AMB Express, 11(42), 1-11. doi:10.1186/s13568-021-01202-y
Tako, M., Katogan, A., Papp, T., Kadaikunnan., Alharbi, N., & Vágvölgyi, C. 2017. Purification and properties of extracellular lipases with transesterification activity and 1,3-regioselectivity from Rhizomucor miehei and Rhizopus oryzae. Journal Microbiology and Biotechnology, 27(2), 277-288. https://doi.org/10.4014/jmb.1608.08005
Thomas, L. S., Thacker, B. J., Schung, A. K., & Maráková, K. 2020. Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis. Journal of Separation Science, 44(1), 211-246. https://doi.org/10.1002/jssc.202000936
Tian M, Yang L, Wang Z, Lv P, Fu J, Changlin M, Li Z, Li L, Liu T, Du W, Luo W. 2022. Improvement of methanol tolerances and catalytic activity of Rhizomucor miehei lipase for one-step synthesis of biodiesel by semi-rational design. Bioresource Technology, 348, 1-31. https://doi.org/10.1016/j.biortech.2022.126769
Tri-Panji, Kresnawaty, I., Dimawarnita, F., Saadah, S., Aminingsih, T., & Miranti, M. 2019. Gliserolisis enzimatik CPO dengan lipase amobil untuk produksi diasil dan monoasil gliserol. Menara Perkebunan, 87(1), 11-19. https://doi.org/10.22302/iribb.jur.mp.v1i87.321
Vidal, L., Dong, Z., Olofsson, K., Karlsson, N. E., & Nicaud, J. 2023. Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system. FEM Yeast Research, 23, 1-14. https://doi.org/10.1093/femsyr/foad037
Wei, W., Sun, C., Wang, X., Jin, Q., Xu, X., Akoh, C.C., & Wang, X. 2020. Lipase-catalyzed synthesis of sn-2 palmitate. Engineering, 6(406), 1-14. https://doi.org/10.1016/j.eng.2020.02.008
Xing, S., Zhu, R., Cheng, K., Cai, Y., Yuedan, Z. X., Zhu, Q., & He, L. 2021. Gene expression, biochemical characterization of sn-1,3 extracellular lipase from Aspergillus niger GZUF36 and its mode-structure analysis. Frontiers in Microbiology. 12, 1-16. https://doi.org/10.3389/fmicb.2021.633489
Xing, S., Zhu, R., Li, C., He, L., Zheng, X., & Zhang, Q. 2020. Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from Aspergillus niger GZUF36. Journal Food Science Technology, 57, 2669-2680. https://doi.org/10.1007/s13197-020-04303-x
Yang, X., Zhang, Y., Pang, H., Yuan, S., Wang, X., Hu, Z., Zhou, Q., He, Y., Yan, Y., & Xu, L. 2021. Codisplay of Rhizopus oryzae and Candida rugosa lipases for biodiesel production. Catalysts, 11(4), 1-13. https://doi.org/10.3390/catal11040421.
Yao, W., Liu, K., Liu, H., Wang, R., Wang, W., & Wang, T. 2021. A valuable product of microbioal cell factories: microbial lipase. Frontiers in Microbiology, 12, 1-16. https://doi.org/10.3389/fmicb.2021.743377
Yuan, T., Wei, W., Wang, X., & Jin, Q. 2020. Biosynthesis of structured lipids enriched with medium and long-chain triacylglycerols of human milk fat substitute. Food Science and Technology, 128, 1-7. https://doi.org/10.1016/j.lwt.2020.109255
Zheng, J., Liang, Y., Li, J., Lin, S., Zhang, Q., Zuo, K., Zhong, & Xu, X. 2023. Enzymatic preparation of mono- and diacylglycerols: A review. Grain and Oil Science and Technology, 6, 185-205. https://doi.org/10.1016/j.gaost.2023.10.002
Zhu, S., Xu, Y., & Yu, X. W. 2020. Improved Homologous Expression of the Acidic Lipase from Aspergillus niger. Journal Microbiology Biotechnology, 30(2), 196-205. https://doi.org/10.4014/jmb.1906.06028
Downloads
Submitted
Accepted
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Ludwinardo Putra, Eneng Nurhasanah, Eti Rohaeti, Irma Kresnawaty

This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.











