An Eco-friendly Method of Purification for Xylanase from Aspergillus niger by Polyelectrolyte Precipitation

Abstract

The separation of xylanase (Xyl) from a fungal extract obtained by submerged fermentation (SmF) was carried out using precipitation with natural polyelectrolytes ɩ-carrageenan (Carr) and chitosan (CHS). Xyl activity determined from the fungal extract at different times was modeled by mixed linear model for longitudinal data considering the factors as fixed effects and the experimental units as random effects. A spline regression was used to model the Xyl activity with knots at days 2 and 3 and interaction between three factors (p < 0.0001 in each section). High purification factors were reached with both polymers. Mass spectrometry analysis and zymogram analysis revealed that the Xyl that precipitated with both polymers had a molecular weight of 24 kDa and an isoelectric point (pI) of 5.45. The purification factor (PF) of Xyl in the precipitate of the fungal extract from SmF with 0.05% w/v CHS (pH 8.00) was around 5.6 for a mass ratio of 1.8 × 10−5 mg CHS/mg protein and with 0.5% w/v Carr (pH 7.00) it was 9.0 for a mass ratio of 0.03 mg Carr/mg protein. These results make this precipitation method suitable to be included in a downstream process, providing such advantages as simplicity, scalability, ability to concentrate the enzyme in a single step process as well as the use non-toxic reagents. Taking into account that the process of purification of macromolecules is a decisive step with a profound impact on their final market cost, the developed methodology has a high potential to be used in the purification of Xyl.