Partial purification of Bacillus subtilis Xylanase using Aqueous Two-Phase System
The present project will focus on the partial purification of Bacillus subtilis xylanase from the fermentation broth using polymer-salt aqueous two-phase system (ATPS) integrated with electrophoresis. Several ATPS parameters are investigated to determine the optimum parameters which lead to the highest xylanase yield and purity.
Xylanases are classes of enzymes which degrade the hemicellulose in plant cells walls into xylose sugars. Conventional xylanase purification methods such as ion exchange chromatography (IEC) and gel filtration have produced low yields of xylanase. In this study, the optimum parameters of polymer/salt ATPS integrated with electrophoresis will be investigated to purify xylanase from the Bacillus subtilis fermentation broth. The effects of several ATPS parameters such as types and compositions of phase-forming components, crude load, pH, time, neutral salt concentration, and position of electrode will be investigated via single-factor experiment. Firstly, the ATPS partitioning experiment will be conducted using different types of ATPS phase-forming components namely various types of polyethylene glycol (PEG), di-potassium hydrogen phosphate, sodium citrate, and ammonium sulphate. The type and composition (12-30% w/w) of ATPS phase-forming components, and crude feedstock loading (10-40% w/w) that gives the highest partition coefficient, yield, and purification factor will be determined. Then, electrophoresis will be integrated into the ATPS. The pH (5-9), electrophoresis time (0-120 minutes), sodium chloride concentration (0-8% w/w), and electrode position that gives maximum xylanase separation will be determined. Sample will be collected from each phase to quantify the xylanase activity and total protein concentration. The purity of the extracted xylanase at the optimized ATPS system and process parameters will be validated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Xylanase produced by Bacillus subtilis is expected to be separated to the polymer-rich top phase while impurities will be separated to the salt-rich bottom phase. A high yield and purity of xylanase is expected at the optimum parameters of the polymer-salt ATPS integrated with electrophoresis.