Vegetos
(An International Journal of Plant Research & Biotechnology)
Enhanced production and partial purification of lipase from Rhizopus sp. grown on low-cost deoiled sesame cake
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Research Articles | Published: 05 June, 2025
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Keywords: Lipase, n Rhizopus sp., Deoiled sesame cake, Submerged fermentation, SDS-PAGE
Abstract
Lipases are a group of industrially important enzymes widely used in food processing, pharmaceuticals, fine chemicals, etc., due to their ability to catalyze the hydrolysis of fats and oils into diglycerides, monoglycerides, glycerol, and free fatty acids. However, the high cost of enzyme production remains a significant challenge, leading to the search for economical production methods. One promising approach is using low-cost agro-industrial residues as substrates, which reduces production costs and supports waste valorization. However, data on optimal conditions for lipase production by Rhizopus sp. using such residues remain limited. Therefore, the present study aimed to determine the optimum parameters favorable for lipase production by Rhizopus sp. employing low-cost agro-industrial residues under submerged fermentation and to partially purify the enzyme. Deoiled sesame cake (SC) was found to be the best substrate among four readily available residues evaluated. Optimization was carried out using the one-variable-at-a-time approach by varying temperature, pH, incubation period, inoculum size, and agitation. The optimum conditions for maximum lipase production were 35 °C, pH 8, 96 h of incubation, 1% (v/v) inoculum, and shaking at 100 rpm. Glucose, peptone, Triton X-100, tributyrin, and olive oil were found to be the best carbon source, nitrogen source, surfactant, fat, and oil, respectively. Under the optimum conditions, the strain showed a maximum of 7.84 U/ml of lipase activity. The enzyme was purified by ammonium sulfate precipitation followed by ion exchange chromatography. On SDS PAGE, the purified fraction showed a single band with a molecular weight of ~ 40 kDa.
References
Abdelaziz AA, Abo-Kamar AM, Elkotb ES, Al-Madboly LA (2025) Microbial lipases: advances in production, purification, biochemical characterization, and multifaceted applications in industry and medicine. Microb Cell Fact 24:40. https://doi.org/10.1186/s12934-025-02664-6
Açıkel Ü, Erşan M, Açıkel YS (2010) Optimization of critical medium components using response surface methodology for lipase production by Rhizopus delemar. Food Bioprod Process 88:31–39. https://doi.org/10.1016/j.fbp.2009.08.003
Al-Ghanayem AA, Joseph B (2020) Current prospective in using cold-active enzymes as eco-friendly detergent additive. Appl Microbiol Biotechnol 104:2871–2882. https://doi.org/10.1007/s00253-020-10429-x
Ali U, Anwar Z, Hasan S, Zafar M, Ain Nu, Afzal F, Khalid W, Rahim MA, Mrabti HN, AL-Farga A et al (2023) Bioprocessing and screening of indigenous wastes for hyper production of fungal lipase. Catalysts 13:853. https://doi.org/10.3390/catal13050853
Bapiraju KV, Sujatha P, Ellaiah P, Ramana T (2005) Sequential parametric optimization of lipase production by a mutant strain Rhizopus sp. BTNT-2. J Basic Microbiol 45:257–273. https://doi.org/10.1002/jobm.200410548
Bharathi D, Rajalakshmi G, Komathi SJ (2019) Optimization and production of lipase enzyme from bacterial strains isolated from petrol spilled soil. J King Saud Univ Sci 31:898–901. https://doi.org/10.1016/j.jksus.2017.12.018
Booth C (1971) Introduction to general methods. In: Booth C (ed) Methods in microbiology. Academic Press Inc, London and New York, pp 1–47
Carvalho ASS, RdCPe R, Sales JCS, Souza CECD, Lemes AC, Coelho MAZ (2023) Biotechnological valorization of cupuaçu by-products: Solid-state fermentation for lipase production by Yarrowia lipolytica. Ferment 9:989. https://doi.org/10.3390/fermentation9110989
Colla LM, Rizzardi J, Pinto MH, Reinehr CO, Bertolin TE, Costa JA (2010) Simultaneous production of lipases and biosurfactants by submerged and solid-state bioprocesses. Bioresour Technol 101:8308–8314. https://doi.org/10.1016/j.biortech.2010.05.086
Costa AR, Salgado JM, Belo I (2023) Olive and sunflower cakes as suitable substrates for lipase production by Yarrowia spp: from flasks to bioreactor. Biocatal Agri Biotechnol 51:102783. https://doi.org/10.1016/j.bcab.2023.102783
Costas M, Deive FJ, Longo MA (2004) Lipolytic activity in submerged cultures of Issatchenkia orientalis. Process Biochem 39:2109–2114. https://doi.org/10.1016/j.procbio.2003.11.001
de Almeida AF, Taulk-Tornisielo SM, Carmona EC (2013) Influence of carbon and nitrogen sources on lipase production by a newly isolated Candida viswanathii strain. Ann Microbiol 63:1225–1234. https://doi.org/10.1007/s13213-012-0580-y
Dobrev G, Strinska H, Hambarliiska A, Zhekova B, Dobreva V (2018) Optimization of lipase production in solid-state fermentation by Rhizopus arrhizus in nutrient medium containing agroindustrial wastes. Open Biotech J 12:189–203. https://doi.org/10.2174/1874070701812010189
Elhussieny NI, El-Refai HA, Mohamed SS, Shetaia YM, Amin HA, Klöck G (2023) Rhizopus stolonifer biomass catalytic transesterification capability: optimization of cultivation conditions. Microb Cell Fact 22:154. https://doi.org/10.1186/s12934-023-02141-y
Gulati R, Isar J, Kumar V, Prasad AK, Parmar VS, Saxena RK (2005) Production of a novel alkaline lipase from Fusarium globosum using neem oil, and its applications. Pure Appl Chem 77:251–262. https://doi.org/10.1351/PAC200577010251
Helal S, Abdelhady H, Abou-Taleb K, Hassan M, Amer M (2017) Evaluation of factors affecting the fungal lipase production using one factor at a time approach and response surface methodology. Egypt J Microbiol 52:1–16. https://doi.org/10.21608/ejm.2017.602.1012
Hiol A, Jonzo MD, Rugani N, Druet D, Sarda L, Comeau LC (2000) Purification and characterization of an extracellular lipase from thermophilic Rhizopus oryzae strain isolated from palm fruit, Enzyme Microb Technol 26:421–430. https://doi.org/10.1016/S0141-0229(99)00173-8
Humaid AA, Al-Ghalibi SM, Abdel-Sater MA, Salahaddin RH (2020) Proteases and lipases activity of fungi isolated from local cheese, Republic of Yemen. Assiut Univ Bull Environ Researches 23:1–12. https://doi.org/10.2608/auber.2020.112659
Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53:211–213. https://doi.org/10.1128/aem.53.1.211-213.1987
Kumar A, Verma V, Dubey VK, Srivastava A, Garg SK, Singh VP, Arora PK (2023) Industrial applications of fungal lipases: a review. Front Microbiol 14:1142536. https://doi.org/10.3389/fmicb.2023.1142536
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Mala JG, Edwinoliver NG, Kamini NR, Puvanakrishnan R (2007) Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594. J Gen Appl Microbiol 53:247–253. https://doi.org/10.2323/jgam.53.247
Nahas E (1988) Control of lipase production by Rhizopus oligosporus under various growth conditions. Microbiol 134:227–233. https://doi.org/10.1099/00221287-134-1-227
Pereira EB, De Castro HF, De Moraes FF, Zanin GM (2001) Kinetic studies of lipase from Candida rugosa: a comparative study between free and immobilized enzyme on to porous chitosan beads. Appl Biochem Biotechnol 91–93:739–752. https://doi.org/10.1385/abab:91-93:1-9:739
Prabaningtyas RK, Putri DN, Utami TS, Hermansyah H (2018) Production of immobilized extracellular lipase from Aspergillus Niger by solid state fermentation method using palm kernel cake, soybean meal, and coir pith as the substrate. Energy Procedia 153:242–247. https://doi.org/10.1016/j.egypro.2018.10.010
Putri DN, Khootama A, Perdani MS, Utami TS, Hermansyah H (2020) Optimization of Aspergillus niger lipase production by solid state fermentation of agro-industrial waste. Energy Rep 6:1331–1335. https://doi.org/10.1016/j.egyr.2019.08.064
Ramos EZ, Júnior RH, de Castro PF, Tardioli PW, Mendes AA, Fernandéz-Lafuente R, Hirata DB (2015) Production and immobilization of Geotrichum candidum lipase via physical adsorption on eco-friendly support: Characterization of the catalytic properties in hydrolysis and esterification reactions. J Mol Catal B Enzym 118:43–51. https://doi.org/10.1016/j.molcatb.2015.05.009
Reis WS, Matias AB, Mendes AA, de Castro HF, Pereira EB (2022) Production and characterization of whole-cell Rhizopus oryzae CCT3759 to be applied as biocatalyst in vegetable oils hydrolysis. Catal Lett 152:1–11. https://doi.org/10.1007/s10562-021-03622-8
Rodriguez JA, Mateos JC, Nungaray J, Gonzalez V, Bhagnagar T, Roussos S, Cordova J, Baratti J (2006) Improving lipase production by nutrient source modification using Rhizopus homothallicus cultured in solid state fermentation. Process Biochem 41:2264–2269. https://doi.org/10.1016/j.procbio.2006.05.017
Sahay S, Chouhan D (2018) Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation. J Genet Eng Biotechnol 16:319–325. https://doi.org/10.1016/j.jgeb.2018.04.006
Salihu A, Alam MZ, Abdul Karim MI, Salleh HM (2012) Lipase production: an insight in the utilization of renewable agricultural residues. Resour Conserv Recycling 58:36–44. https://doi.org/10.1016/j.resconrec.2011.10.007
Saranya G, Ramachandra TV (2020) Novel biocatalyst for optimal biodiesel production from diatoms. Renew Energy 153:919–934. https://doi.org/10.1016/j.renene.2020.02.053
Savitha J, Srividya S, Jagat R, Payal P, Priyanki S, Rashmi GW, Roshini KT, Shantala YM (2007) Identification of potential fungal strain(s) for the production of inducible, extracellular and alkalophilic lipase. Afr J Biotechnol 6:564–568. https://doi.org/10.5897/AJB2007.000-2048
Sethi BK, Nanda PK, Sahoo S (2016) Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10. Braz J Microbiol 47:143–149. https://doi.org/10.1016/j.bjm.2015.11.026
Shukla P, Gupta K, Kango N (2007) Production of lipase by hyper-lipolytic Rhizopus oryzae KG-10 on low-value oil emulsions. Res J Microbiol 2:671–677. https://doi.org/10.17311/jm.2007.671.677
Silva JN, Godoy MG, Gutarra ML, Freire DM (2014) Impact of extraction parameters on the recovery of lipolytic activity from fermented babassu cake. PLoS ONE 9:e103176. https://doi.org/10.1371/journal.pone.0103176
Sivaramakrishnan S, Gangadharan D (2009) Edible oil cakes. In: nee’ Nigam PS, Pandey A (eds) Biotechnology for agro-industrial residues utilisation. Springer, Dordrecht, pp 253–271
Skjold-Jørgensen J, Vind J, Svendsen A, Bjerrum MJ (2016) Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan-induced fluorescence quenching. Eur J Lipid Sci Technol 118:1644–1660. https://doi.org/10.1002/ejlt.201600059
Turati DF, Almeida AF, Terrone CC, Nascimento JM, Terrasan CR, Fernandez-Lorente G, Pessela BC, Guisan JM, Carmona EC (2019) Thermotolerant lipase from Penicillium sp. section Gracilenta CBMAI 1583: effect of carbon sources on enzyme production, biochemical properties of crude and purified enzyme and substrate specificity. Biocatal Agric Biotechnol 17:15–24. https://doi.org/10.1016/j.bcab.2018.10.002
Vanleeuw E, Winderickx S, Thevissen K, Lagrain B, Dusselier M, Cammue BP, Sels BF (2019) Substrate-specificity of Candida rugosa lipase and its industrial application. ACS Sustain Chem Eng 7:15828–15844. https://doi.org/10.1021/acssuschemeng.9b03257
Xu L, Li J, Zhang H, Zhang M, Qi C, Wang C (2025) Biological modification and industrial applications of microbial lipases: a general review. Int J Biol Macromol 302:140486. https://doi.org/10.1016/j.ijbiomac.2025.140486
Zhang Q, Xiong Z, Sun L, Tian X, Tian G, Yang Y, Li X, Wang Y, Chu J (2021) Effect of biosurfactant sophorolipids on Rhizomucor miehei lipase fermentation by Aspergillus oryzae. Bioresour Bioprocess 8:84. https://doi.org/10.1186/s40643-021-00433-y
Author Information
Department of Microbiology, M.G.R. College, Hosur, India