Purification of xylanase from Aspergillus niger NRRL3 extract by an integrated strategy based on aqueous two-phase systems followed by ion exchange chromatography

Abstract

Xylanases are enzymes that degrade the linear polysaccharide xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Xylanases are applied in a wide range of industrial processes, such as in the pulp and paper industry, which demands a higher purity degree compared to other industries. Processes for the purification of this enzyme has been widely reported using different techniques. However, cases involving the use of a simple combination of unit operations are not common. The objective of this work was to design an integrated bioseparation strategy for the purification of a fungal cellulase-free xylanase. The design consisted of a liquid-liquid extraction using two-combined aqueous two-phase system (ATPS) with an ion exchange chromatography. The effect of different parameters such as polymer type (ethylene oxide-propylene oxide copolymer (UCON) and polyethylene glycol (PEG)), PEG molecular mass (600, 1450, 2000 and 4600 g/mol), volume ratio and the addition of sodium chloride on the enzymes -from the Aspergillus niger NRRL3 extract- partition coefficient were analyzed. The partition behavior of the target product was affected for the type and molecular mass of the polymer used. Extraction using UCON/Citrate system combined with a second extraction using PEG1450/Citrate system proved to be the most suitable for the extraction of xylanase and natural fungal pigments. The enzymatic activity recovery and purification factor achieved were 80% and 4, respectively. The proposed systems have the ability to recycle the UCON and Citrate salt for their reuse in new ATPS. The phases of the combined extraction by ATPS and the elution obtained from ion exchange chromatography were characterized by nano-LC–MS/MS, providing a complete profile of the proteins present. The combination of ATPS with ion exchange chromatography using a Q-Sepharose XL column reached a total enzymatic activity recovery of 79.9% and a purification factor of 11. This is the first time that high-quality xylanase free of cellulolytic enzymes is reported using an integrated bioprocess strategy with potential for industrial adoption.