Precipitation Using Organic Solvents for Purifying Lipase from Preussia africana

Abstract

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.