Structural and functional analysis of Aspergillus niger xylanase to be employed in polyethylenglycol/salt aqueous two-phase extraction

The structure and enzymatic activity of Aspergillus niger xylanase were evaluated in different media to establish an appropriate protocol for the extraction of the enzyme in polymer/salt aqueous two-phase systems. Different factors were studied: the concentration and molecular weight (1000, 2000, 4600 and 8000) of polyethyleneglycol, the concentration and type of salt (sodium citrate and potassium phosphate) and pH, time and temperature. Xylanase was stable for 5 h at pH between 2.7 and 9.0 and at temperatures up to 50 °C. Fluorescence spectroscopy and circular dichroism experiments showed that neither the secondary/tertiary structure of the enzyme nor its catalytic activity were significantly altered in the presence of either salt or PEG. Xylanase partitioned into the PEG-rich phase driven by the excluded volume effect. Partitioning was more favorable to the polymer phase in the PEG1000/NaCit system, where Kp was 12 times higher than in the others aqueous two-phase systems. These results demonstrate the potential application of the PEG1000/NaCit system as a first step for the extraction of Aspergillus niger xylanase.