No abstract

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.

Aqueous two-phase systems (ATPSs) composed by UCON (ethylene oxide/propylene oxide copolymer) and potassium phosphate salts were for the first time evaluated in the recovery of Peniophora cinerea laccase from complex fermented medium. The ATPSs were obtained by combining the random copolymer UCON with KH2PO4, potassium phosphate buffer pH 7 or K2HPO4. According to the results, protein partition occurred predominantly toward the saline phase (bottom phase) of the ATPSs, while some contaminants such as pigments partitioned mainly to the top phase. In preliminary tests, it was found that the salt with the lowest pH value (KH2PO4, pH 4.6) stimulated the enzyme activity, while the other salts (pH between 7.0 and 9.5) caused a strong inhibition. However, the salt inhibition was not observed in the equilibrium phases of the UCON-Potassium phosphate ATPSs. The laccase recovery was high for all the biphasic systems, but the highest value (134%) was obtained when using UCON combined with KH2PO4. When compared to conventional concentration and purification methods (lyophilization, ammonium sulfate precipitation, ultrafiltration, and ion exchange chromatography), ATPS was demonstrated to be an efficient alternative for P. cinerea laccase recovery from fermented medium.

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.