Purification of low‐abundance lysozyme in egg white via free‐flow electrophoresis with gel‐filtration chromatography

Abstract

As an effective separation tool, free‐flow electrophoresis has not been used for purification of low‐abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low‐content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free‐flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide‐ co‐acrylic acid) (P(AM‐co‐AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris‐Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM‐co‐AA) gel and co‐removing of high‐abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Functional Properties and Extraction Techniques of Chicken Egg White Proteins

“…The advantage of isoelectric focusing is high resolution, which can separate proteins whose isoelectric points differ by 0.01–0.02 pH units. Free-flow electrophoresis combined with gel filtration chromatography was successfully employed to isolate low-abundance LYS [ 76 ]. However, a special polyacrylamide-co-acrylic acid gel electrophoresis combined with mass spectrometry was used to identify the prepared LYS.…”

The Impact of Processing and Extraction Methods on the Allergenicity of Targeted Protein Quantification as Well as Bioactive Peptides Derived from Egg

“…Isoelectric focusing was found to have high resolution and be capable of separating proteins whose isoelectric points differ by 0.01–0.02 pH units. Free-flow electrophoresis combined with gel filtration chromatography successfully isolated low-abundance LYS [ 143 ]. A special polyacrylamide-co-acrylic acid gel electrophoresis combined with mass spectrometry was used to identify prepared LYS.…”

Study of the inhibitory effect of Lactobacillus strains and lysozyme on growth of Clostridium spp. responsible for cheese late blowing defect

“…On the other hand, some studies report that lysozyme present higher inhibitory activity in the presence of organic acids. In addition, compared to neutral pH, the enzymatic activity of lysozyme is more stable under acidic conditions (Johansen et al 1994;Oh et al 2016;Sheng et al 2017;Dong et al 2020).…”