MORILLA Esteban Amador
congresos y reuniones científicas
PURIFICATION OF XYLANASE FROM ASPERGILLUS NIGER USING AQUEOUS TWO PHASE SYSTEMS FOLLOWED BY IONIC CHROMATOGRAPHY
TADDIA, ANTONELA; MORILLA, ESTEBAN AMADOR; TUBIO, GISELA
Conferencia; BIOPARTITIONING & PURIFICATION CONFERENCE; 2019
The upstream and downstream processing of a biotechnological project are very important steps since the costs for gathering, concentrating and purifying the final product are significant. Thus, there is a growing interest in the production, recovery, and purification of enzymes. In a previous work, the production of xylanase (EX) by Aspergillus niger was optimized using the response surface methodology. The substrate employed was wheat husk, a lignocelulosic by-product very abundant in the Pampas of Argentina, with an enormous biotechnological potential, for the production of this enzyme. Then, the use of aqueous two-phase system (ATPS) combined with chromatography was used as a purification strategy, which could offer several technical and economical advantages to avoid the use of expensive and time-consuming purifications. For the first ATPS, enzyme extract (EX activity of 5 U / ml) was used in a system composed of UCON and Sodium Citrate (15%w/w UCON/ 7%w/w NaCit) and Vr = 1. The pigments present in the enzyme extract (EE) were obtained in the top phase rich in UCON and the proteins in the lower phase with a yield (RFb) of 100%. Then, the botton phase rich in proteins and free of pigments was used to form a new ATPS composed of PEG1450 and NaCit (15.53 %w/w PEG1450/ 13.63%w/w NaCit) and Vr = 3, where a KrEX = 4.11, and RFT = 81.3 % were obtained. The top phase of the PEG1450/NaCit system was then loaded onto a HiTrap Q Sepharose XL FPLC column, which was previously equilibrated with buffer A (20 mM Tris?HCl, pH 8.0). Proteins were eluted using a linear gradient of NaCl (0?1.0 M) in the same buffer at a flow rate of 1 mL/min. Fractions exhibiting EX activity were collected and concentrated by ultrafiltration with Vivaflow 200 ultrafiltration membrane of 3-kDa molecular weight cut-off. Then they were subjected to sodium dodecyl sulfate?polyacrylamide gel electrophoresis (SDS?PAGE) analysis. The crude enzyme was purified to apparentelectrophoretic homogeneity with a final yield of 43% and three-fold purification by anion exchange chromatography and it allowed the separation of the EX from the PEG. The specific activity of the purified EX towards beechwood xylan was 290 U/mg. The results revealed that the combination of two ATPE systems composed by: UCON/NaCit and PEG1450/NaCit followed by an anion exchange chromatography was an efficient design for the recovery and purification of EX from A. niger.