Partitioning and purification of polygalacturonases produced by Aspergillus niger URM 5162 using PEG-phosphate in an aqueous two-phase system

摘要

Pectinases, or pectinolytic enzymes, are naturally produced by plants, filamentous fungi, bacteria and yeasts. The pectinases areof great importance to clarify and reduce viscosity in fruit juices, improving and increasing tbe filtration efficiency. When usedin the crushing of grapes or wine must improve juice extraction, reduce the time to clarify and enhance tbe content ofterpenes inwine. The filamentous fungi most frequently used fur industrial purposes because as much as 90% ofthe enzyme can be excretedinto the culture medium. The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM5162 were investigated in aqueous two-phase systems (ATPS), furmed by polyetbylene glycol and phosphate salts(PE(ijlhosphate). To evaluate the effect oftbe 4 independent variables- molar mass ofpolyetbylene glycol (PEG) (400-8000g1nol MPEG), PEG concentration (12.5-17.5%, w/w- CPEG), phosphate concentration (15-25%, ...W, CPHOS) and pH (6.0, 8.0)- on the 3 response variables: partition coefficient (K), activity yield (Y) and purification fàctor (PF), a fuctorial design (24) wasused. The endo-polygalacturonases (endo-PG) were prefurentially partitioned in tbe top phase. For endo-PG, the highest valuesfor the response variables K, Y and PF of 1.23, 74.04% and 8.18, respectively, were obtained for a CPEG of 12.5% (...W), MPEGof8000 g1nol, and CPHOS of25% (w/w) at pH 6.0. Also, exo-polygalacturonases (exo-PG) were preferentially partitioned in thetop phase. ln tbis case, the highest values ofK (2.40), Y (33.33%), and PF (1.98) were obtained with a MPEG of 8000 g1nol,CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0. ln both cases, MPEG had a positive influence on K, Y and PF. Theconditions ofMPEG 8000 g1nol, CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0 were considered the most suitablefor tbe purification of PG produced by A. niger URM 5162. Furtbermore, MPEG and CPHOS were the most importantindependent variables. The PEG/phosphate system is a useful cost-effective altemative for PG purification.