Nguyen Hoang Loc, Ho Thi Thu Lien, Do Van Giap and Hoang Tan Quang
A study was made of the partition and purification of recombinant neutral protease (NPRC10) from a culture supernatant of E. coli BL21 (DE3) in the polyethylene glycol (PEG)/potassium phosphate aqueous two-phase systems. Factors that influenced the partition of the enzyme in these systems, including the PEG molecular weight and concentration, potassium phosphate concentration, incubation time and temperature, and pH were investigated. The optimum condition of the primary aqueous two-phase extraction was 35% PEG 6000/30% potassium phosphate (pH 6.5) at 30oC for 25 min. The partitioning coefficient for protease (Kprotease) was 4.69 with a partitioning yield (Y) of 94.93%, a purification fold (PF) of 1.7, and protease specific activity (SAprotease T) of 694.61 unit/mg protein in top phase. The protease, which was concentrated in the top phase, was further mixed with 35% potassium phosphate in combination with 3% potassium chloride at room temperature to elute the bottom phase (salt-rich phase). Using this step, the PF of enzyme achieved a higher value of 2.38 with a recovery yield of 76.17% and SAprotease B of 983.04 unit/mg protein.
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