Purification of Aspergillus carbonarius polygalacturonase using polymeric membranes

BACKGROUND:Microfiltration (MF: 70-450 nm) and ultrafiltration (UF: 10-500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes.RESULTS:MF resulted in removal of 2-25% carbohydrates with an enzyme recovery of 69-82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg⁻¹ of dry matter.CONCLUSIONS:The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright