Biotechnology and Bioengineering, Vol.44, No.6, 674-681, 1994
Protein Extraction by Reverse Micelles - Studies on the Recovery of Horseradish-Peroxidase
Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 220.127.116.11) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110 mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in well-stirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in a pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80 mM phosphate buffer and 1 M KCl. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH < 4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 M NaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25 degrees C. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios.