Recovery and purification of Aspergillus niger phytase from crude extract using AOT / isooctane reversed micelles

Abstract

This work describes the successful extraction of Aspergillus niger phytase from a crude extract (CE) obtained from solid-state fermentation by reversed micelle system using anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane achieved in two simple steps: forward and backward extractions. The effects of potassium chloride (KCl) concentration, pH of the aqueous solution, and AOT concentration that affect the system were examined. The best result for the forward extraction was obtained with the CE solution at pH 4.0, 50 mM KCl, and 100 mM AOT, while for the backward extraction the best result was achieved with a stripping aqueous solution at pH 5.5 containing 200 mM KCl, achieving a purification factor of 4.03, 1.15 times higher than that reported for the conventional purification process. Phytase purity was demonstrated by SDS-PAGE (89 kDa) and its activity by zymogram, confirming the efficiency of the process with low time consumption (∼40 min).

Keywords: Liquid-liquid extraction; Phytase; Reversed micelles; Triticale.

Graphical abstract

Fig. 1

Effect of pH on phytase forward extraction from an initial aqueous phase (CE), added with 100 mM KCl, with reversed micelles of 100 mM AOT in isooctane, at 25 °C. (▲) % of protein transferred to the micellar phase; (◼) % of Phytase activity in the residual aqueous phase. Each data point is the average of three determinations and the error bars show the standard deviation.

Fig. 2

Effect of AOT concentration on phytase forward extraction from an initial aqueous phase (CE). (▲) % of the protein in the micellar phase, (◼) % of phytase activity in the residual aqueous phase. Extraction condition: 100 mM KCl solution, pH 4.0 stirring at 200 rpm, 25 °C for 10 min. Each data point is the average of three determinations and the error bars show the standard deviation.

Fig. 3

Effect of KCl concentration on the phytase forward extraction from an initial aqueous phase (CE) at pH 4.0. (▲) % of the protein in the micellar phase, (◼) % of phytase activity in the residual aqueous phase. Extraction condition: 100 mM AOT/isooctane, 200 rpm, 25 °C and 10 min. Each data point is the average of three determinations and the error bars show the standard deviation.

Fig. 4

Fig. 4. Effect of pH on the phytase backward extraction of micellar phase from forward extraction. (▲) % of the protein in the aqueous phase of backward extraction (stripping solution), (◼) % of phytase activity in the stripping solution. Backward extraction condition: fresh stripping solution buffered containing 100 mM KCl, 200 rpm, 25 °C, and 10 min. Each data point is the average of three determinations and the error bars show the standard deviation.

Fig. 5

Effect of KCl concentration on the phytase backward extraction of micellar phase from forward extraction. (▲) % of the protein in the aqueous phase of backward extraction (stripping solution), (◼) % of phytase activity in the stripping solution. Backward extraction condition: fresh stripping solution buffered at pH 5.5, 200 rpm, 25 °C, and 10 min. Each data point is the average of three determinations and the error bars show the standard deviation.

Fig. 6

(A) SDS-PAGE of the purification steps of phytase from Aspergillus niger 7A-1 by reversed micelles. The protein bands were stained with Coomassie Brilliant Blue. Lane 1: Molecular weight markers, Lane 2: sample from initial aqueous phase (CE); Lane 3 – sample from the residual aqueous phase of forward extraction; Lane 4: sample from stripping solution (Phytase purified). (B) Zymogram developed for phytase activity purified by reversed micelles. The activity band was visualized by immersing of gel in cobalt chloride/ammonium molybdate/ammonium vanadate (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).