An Economic and Simple Purification Procedure for the Large-Scale Production of Ovotransferrin from Egg White

Abstract

The objective of this study was to develop a simple and economical protocol for separating ovotransferrin from egg white. Egg white was separated from the yolk and diluted with the same volume of distilled water. To prevent denaturation during the separation process, ovotransferrin in 2x-diluted egg white was converted to its holo-form by adding 20 mM FeCl3.6H2O solution (0.25 to 3 mL/100 mL). The pH of egg white was adjusted to pH 7.0, 8.0, or 9.0, and NaHCO3 and NaCl were added to 50 mM and 0.15 M, respectively (final concentrations) to facilitate iron binding to ovotransferrin. The iron-bound ovotransferrin was separated from the egg white using different concentrations of ethanol (30 to 50%). Ethanol at 43% (final concentration) and pH at 9.0 were the best conditions for separating iron-bound ovotransferrin from 2x-diluted egg white solution. Almost all egg white proteins including ovalbumin were precipitated at 43% ethanol, but most of the iron-bound ovotransferrin remained in the supernatant. Holo-ovotransferrin in the 43% ethanol solution started to precipitate as the concentration of ethanol increased, but the optimal condition for precipitating ovotransferrin was when the ethanol concentration reached 59% (final). The precipitated holo-ovotransferrin was dissolved with distilled water, and AG1-X(2) ion exchange resin (at 3x iron content in ovotransferrin) was used to remove iron bound to ovotransferrin after pH adjustment to 4.7 using 500 mM citric acid. The apo-ovotransferrin obtained using this protocol was >80% in purity and around 99% in yield. The protocol developed is simple, economical, and appropriate for a large-scale production of ovotransferrin from egg white. Also, the isolated ovotransferrin can be applied in human foods, because the only solvent used in this process is ethanol. Furthermore, the AG1-X2 ion exchange resin and ethanol used in this process can be regenerated and recovered.

PC2 Ovotransferrin: Characterization and Alternative Immunotherapeutic Activity

“…Fractions from albumen obtained on DEAE Affi-Gel Blue columns are further purified by liquid chromatography using Q Sepharose Fast Flow methodology [34]. In the study performed by Ko and Ahn, OTf was obtained by diluting the albumen twice with deionized water and, to prevent distortion during the separation process, the apo -form was converted into the holo one, by adding the solution of FeCl 3   ×  6H 2 O. OTf with iron was obtained by using various concentrations of ethanol at pH 9 [35]. The resulting precipitate was dissolved in deionized water and iron was removed by passing through the ion exchange using resin AG 1 -X 2 [36, 37].…”

“…This was performed as described by Ko and Ahn [35]. The albumen diluted in the ratio 1 : 1 with deionized water was adjusted to pH 9.0 and a solution of NaHCO 3 and FeCl 3   × 6H 2 O, 0.5 M, was added to reach the concentration of 20 mM.…”

Enzyme Inhibitory Activity of Ovomucoid Extracted Using a Carboxypeptidase Y-Immobilized Membrane

“…After the collected solution was diluted with 20 L of H 2 O, the concentration of KCl in the collected solution was reduced by ultrafiltration (Microcon, 3,000 cut-off filter, Millipore, Darmstadt, Germany) to obtain the CPY fraction. For further purification of ovomucoid from the CPY fraction according to a previously published method [18,19], the proteins in the CPY fraction were precipitated by slowly adding 100 % ethanol to the final concentration of 43 % , and centrifuged at 10,000 g for 20 min at 4 C to obtain the supernatant (S1) and the precipitate (P1). The ethanol concentration of the extract was increased to 61 % (final) by slowly adding 100 % ethanol to S1.…”

Antioxidant effects of ovotransferrin and its hydrolysates

“…Apo-ovotransferrin (iron-free) was prepared from fresh egg white using the method of Ko and Ahn (2008 (EG.3.4.22.2,Bision). A 2% (wt/ vol) OTF solution was incubated with the enzymes at 45°G for 3 h and the enzyme-to-substrate ratio of 1:25 (wt/wt) was used for all enzymes.…”