Partitioning and purification of the cellulolytic complex produced by Aspergillus japonicus URM5620 using PEG–citrate in an aqueous two-phase system

Abstract

The partitioning and purification of cellulases from the cellulolytic complexes produced by Aspergillus japonicus URM5620 were investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1000–8000g/mol), PEG concentration (20.0–24.0%, w/w), sodium citrate (15–20%, w/w) concentration and pH (6.0–8.0) on the differential partitioning and purification of the cellulolytic complex composed of β-glucosidase (βG), endoglucanase (CMCase) and total cellulase (FPase). The βG preferentially partitioned to the salt phase; however, when the molar mass and concentration of PEG were simultaneously increased, the partition coefficient increased (K=3.0). For βG, 20% (w/w) PEG 8000 (g/mol) and 10% (w/w) sodium citrate at pH 8.0 produced the best results; the partition coefficient was approximately 2.9, the activity yield was 77%, and the purification factor was 1.9. The FPase and CMCase preferentially partitioned to the top phase. The highest K values (2.9 and 3.0) were obtained when the top phase consisted of PEG 1000 (g/mol). The best activity yield for FPase (2.67%) was obtained with 24% (w/w) PEG 1000 (g/mol) and 15% (w/w) sodium citrate at pH 8.0. However, the highest purification factor (64.8) was observed with 20% (w/w) PEG 8000 (g/mol). For CMCase, the highest activity yield (1.64%) and purification factor (45.9) were obtained with 22% (w/w) PEG 3350 (g/mol) and 17.5% (w/w) sodium citrate at pH 7.0. The PEG/sodium citrate system resulted in separations characteristic of the cellulases of cellulolytic complexes, where βG partitions to the salt phase and FPase and CMCase partition to the top phase. This process provides an efficient and attractive increase in the purification factor.