TY - JOUR
T1 - Separation and purification of laccases from two different fungi using aqueous two-phase extraction
AU - Prinz, Axel
AU - Hönig, Jacqueline
AU - Schüttmann, Ina
AU - Zorn, Holger
AU - Zeiner, Tim
PY - 2014/2
Y1 - 2014/2
N2 - Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)-phosphate aqueous two-phase system at 25 C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g-1 of PEG3000, 0.063 g g-1 of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g-1 of PEG3000, 0.063 g g-1 of phosphate and 0.013 g g-1 of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.
AB - Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)-phosphate aqueous two-phase system at 25 C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g-1 of PEG3000, 0.063 g g-1 of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g-1 of PEG3000, 0.063 g g-1 of phosphate and 0.013 g g-1 of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.
KW - Aqueous two-phase system
KW - Enzyme activity
KW - Laccase
KW - Separation
KW - Superposition
UR - http://www.scopus.com/inward/record.url?scp=84895070200&partnerID=8YFLogxK
U2 - 10.1016/j.procbio.2013.11.006
DO - 10.1016/j.procbio.2013.11.006
M3 - Article
AN - SCOPUS:84895070200
SN - 1359-5113
VL - 49
SP - 335
EP - 346
JO - Process Biochemistry
JF - Process Biochemistry
IS - 2
ER -