Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 879; no. 32; pp. 3853 - 3858
• Main Authors: Barbosa, José Murillo P., Souza, Ranyere L., Fricks, Alini T., Zanin, Gisella Maria, Soares, Cleide Mara F., Lima, Álvaro S.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.12.2011; Elsevier
• Subjects: ammonium sulfate; Analysis; Analytical, structural and metabolic biochemistry; aqueous solutions; Aqueous two-phase system; Bacillus; Bacillus (bacteria); Bacillus - enzymology; Bacillus - metabolism; Biological and medical sciences; carboxylic ester hydrolases; Chemical Fractionation - methods; chromatography; Electrophoresis, Polyacrylamide Gel; Entropy; Enzymes; Fermentation; Fundamental and applied biological sciences. Psychology; General pharmacology; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; hydrophobicity; Lipase; Lipase - biosynthesis; Lipase - isolation & purification; Lipolytic enzyme; Medical sciences; molecular weight; Partitioning; Pharmacology. Drug treatments; Phosphates - chemistry; polyacrylamide gel electrophoresis; polyethylene glycol; Polyethylene Glycols - chemistry; Potassium Compounds - chemistry; Potassium phosphates; protein content; Purification; Sodium Chloride; submerged fermentation; Temperature; viscosity; Aqueous two-phase system; Purification; Lipolytic enzyme; Enzyme; Triacylglycerol lipase; Esterases; Bacillaceae; Fermentation; Carboxylic ester hydrolases; Biphasic system; Bacillales; Aqueous medium; Bacillus; Bacteria; Hydrolases
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.10.035

► ATPS could be used to selectively purify lipase from the crude culture filtrates. ► The best purification factor was 201.53 fold. ► The enzyme partitioning is governed by entropy. ► Lipase by Bacillus sp. ITP-001 purified in PEG/potassium phosphate ATPS has 54 kDa. This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000 g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4 °C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS–PAGE. According to the results the best combination for purifying the enzyme is PEG 8000 g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4 °C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.