Precipitation Using Organic Solvents for Purifying Lipase from Preussia africana

Eric E. A. Ferreira

Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil.

Brenda C. Pereira

Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil.

Daniela B. Hirata *

Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil.

*Author to whom correspondence should be addressed.


Aims: The present study aimed to purify a new lipase from the endophytic fungus Preussia africana (P. africana).

Study Design: Lipases were produced through fermentation and precipitation using different organic solvents and characterized at optimal temperature and pH conditions.

Place and Duration of Study: Assays were carried out at the Laboratory of Bioprocess at the Federal University of Alfenas between January and August, 2023.

Methodology: Enzymes were produced via submerged fermentation. After fermentation, the broth was filtered and lyophilized so that enzymes were concentrated. Organic solvents (methanol, ethanol, isopropanol and acetone) were used at 10 and 25°C for lipase precipitation. A second precipitation step was investigated in a supernatant: solvent ration ranging from 1:3 to 1:2 (v/v) and temperatures between 10°C and 25°C. Optimal temperature and pH conditions for the hydrolytic activity of precipitated lipase were also found.

Results: Acetone and isopropanol were more efficient in precipitating lipase while maintaining its catalytic activity, as specific activities of 211.34 ± 0.05 and 179.50 ± 0.08 U/mg were achieved, respectively. For the second precipitation step, acetone in a supernatant: solvent ratio of 1:2 (v/v) at 10°C achieved optimal performance, as purification factor of 1.89 ± 0.01 and recovered activity of 93.45% were found. Maximum hydrolytic activity of 72.80 ± 1.29 U/mL was observed using olive oil as substrate at pH between 7 and 8 and 37°C.

Conclusion: The use of acetone at 10°C in two precipitation steps proved to be a more robust strategy for P. africana lipase precipitation in the purification process.

Keywords: Lipaseq, Preussia africana, purification, organic solvents, acetone

How to Cite

Ferreira , E. E. A., Pereira , B. C., & Hirata , D. B. (2023). Precipitation Using Organic Solvents for Purifying Lipase from Preussia africana. Biotechnology Journal International, 27(6), 12–21.


Download data is not yet available.


Jagannath S, Konappa N, Lokesh A, Bhuvaneshwari, Dasegowda T, Udayashankar AC et al. Bioactive compounds guided diversity of endophytic fungi from Baliospermum montanum and their potential extracellular enzymes. Anal Biochem. 2020;614(1):114024-39. Available:

Gunatilaka AAL. Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity and implication of their occurrence. J.Nat.Prod. 2006;69(3):509-26.


Mattoo AJ, Nonzom S. Endophytic fungi: understanding complex cross-talks. Symbiosis. 2021;1(1):1-28.

Adeleke BS, Babalola OO. Pharmacological potential of fungal endophytes associated with medicinal plants: A review. J Fungi. 2021;7:1–16. Available:

Bajaj S, Fuloria S, Subramaniyan V, Meenakshi DU, Wakode S, Kaur A et al. Chemical characterization and anti-inflammatory activity of phytoconstituents from Swertia alata. Plants. 2021:31; 10(6):1109. Available:

Yasser MM, Mousa AM, Marym A, Tagyan AI. Molecular identification, extracellular enzyme production and antimicrobial activity of endophytic fungi isolated from Solanum tuberosum L. in Egypt. Biosci Biotechnol Res Asia. 2019;16(1):135-42. Available:

Seddouk L, Jamai L, Tazi K, Ettayebi M, Alaoui-Mhamdi M, Aleya L et al. Isolation and characterization of a mesophilic cellulolytic endophyte Preussia africana from Juniperus oxycedrus. Environ Sci Pollut Res. 2022;29:45589-600. Available:

Pereira AS, Souza AH, Fraga J, Villeneuve P, Torres AG, Amaral PF. Lipases as effective green biocatalysts for phytosterol esters’ production: a review. Catalysts. 2022;12(1)88: 1-24 Available:

Liu S, Bilal M, Rizwan K, Gul I, Rasheed T, Iqbal H. Smart chemistry of enzyme immobilization using various support matrices – A review. International J Biol Macromol. 2021; 190(1):396-408. Available:

Chandra P, Enespa, Singh R, Arora P.K. Microbial lipases and their industrial applications: a comprehensive review. Microb Cell Fact. 2020;19:1–42. Available:

Kilikian BV, Junior AP. Biotecnologia industrial: Engenharia Bioquímica. 4th ed. São Paulo: Blücher; 2001.

Malviya R, Fuloria S, Verma S, Subramaniyan V, Sathasivam KV, Kumarasamy V, et al. A. Commercial utilities and future perspective of nanomedicines. PeerJ. 2021;19(9):12392. Available:

Bharathi D, Rajalakshmi G. Microbial lipases: an overview of screening, production and purification. Biocatal Agric Biotechnol. 2019;22(1)101368:1-7. Available:

Ferreira MM, Santiago F, Silva N, Luiz J, Fernadéz-Lafuente R, Mendes A et al. Different strategies to immobilize lipase from Geotrichum candidum: kinetic and thermodynamic studies. Process Biochem. 2018;67(1):55-63. Available:

Preczeski KP, Dalastra C, Czapela FF, Kubeneck S, Scapini T, Camargo AF et al. Fusarium oxysporum and Aspergillus sp. as keratinase producers using swine hair from agroindustrial residues. Front Bioeng Biotechnol. 2020;8(1): 327-45. Available:

Souza IM, Bassi GJ, Luiz JHH, Hirata DB. Isolation and screening of extracellular lipase-producing endophytic fungi from Handroanthus impetiginosus. Asian J Biotechnol Bioresour Technol 2018;4(2):1-10. Available:

Preczeski K, Kamanski A, Scapini T, Camargo AF, Modkoski T, Rossetto V et al. Efficient and low-cost alternative of lipase concentration aiming at the application in the treatment of waste cooking oils. Bioprocess Biosyst Eng. 2018; 41(6):851-57. Available:

Ota Y, Sawamoto T, Hasuo M. Tributyrin specifically induces a lipase with a preference for the sn-2 position of triglyceride in Geotrichum sp. FO401B. Biosci Biotechnol Biochem. 2000;64(11):2497-99. Available:

Menegotto AL, Fernande IA, Steffens J, Valduga E. Protein purification of Arthrospira platensis using aqueous two-phase system composed of polyethylene glycol and potassium phosphate/sodium citrate. J Appl Phycol. 2021;34(1):311-20. Available:

Bradford MM. A Rapid and Sensitive Method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1):248-54. Available:

Castro PF, Moreira NC, Esperança MN, Oliveira LM, Badino AC, Tavano OL, et al. High lipase production from Geotrichum candidum in reduced time using cottonseed oil: optimization, easy purification and specificity characterization. J Chem Eng Res Updates 2016(3):60–69. Available:

Janson JC. Protein Purification: Principles, High Resolution Methods, and Applications. New Jersey: John Wiley & Sons, Inc; 2011.

Ramos EZ, Júnior RH, Castro PF, Tardioli PW, Mendes AA, Fernanés-Lafuente R et al. 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. 2015; 118(89):43-51. Available:

Nimkande VD, Sivanesan S, Bafana A. Screening, identification, and characterization of lipase-producing halotolerant Bacillus altitudinis Ant19 from Antarctic soil. Arch Microbiol. 2023;205(4): 327-45.


Yan J, Liu S, Hu J, Gui X, Wang G, Yan Y. Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment. Biores Technol. 2011;102(14):7154–58. Available:

Ward OP, Singh A. Omega-3/6 fatty acids: Alternative sources of production. Process Biochem. 2005;40(12): 3627– 52. Available: