The Study Of Isolation And Purification Of Immunoglobulin From Hen Egg Yolk

In the present work, the parameters of water dilution method are optimized to remove the lipids from egg yolk using chloroform-methanol means. The optimal conditions are followings: the yolk content is mixed with six volumes of distilled water, adjust the solution pH to 5.0 and frozen at -20oC over 8 h. The mixture is thawed and centrifuged at 4 oC, 1200×g for 5 minutes. The rate of removed lipids could arrive 100% under optimal conditions.Two different membrane methods were studied to isolate and purify IgY (immunoglobulin from egg yolk) from the crude enzyme solution using parameter scanning ultrafiltration. One method is the single-stage ultrafiltration using 100-kDa regenerated cellulose (RC) membrane. The optimized conditions are followings: 10 mM phosphate buffer containing 30 mM NaCl (pH 6.0); stirring speed, 600 rpm; permeate flux, 13.3 L/m2h. Under these conditions, the purity and recovery of IgY are 85% and 90%, respectively. The other method is the two-stage ultrafiltration using 100-kDa polyethersulfone (PES) membrane and 100-kDa RC membrane. The optimized conditions for 100-kDa PES membrane are followings: 10 mM phosphate buffer containing 70 mM NaCl (pH 8.0); stirring speed, 1200 rpm; permeate flux, 26.5 L/m2h; The optimized conditions for 100-kDa RC membrane are followings: 10 mM phosphate buffer containing 70 mM NaCl (pH 4.5); stirring speed, 600 rpm; permeate flux, 13.3 L/m2h. Under the optimized two-stage conditions, the purity and recovery of IgY are 93% and 85%, respectively.The final ultrafiltration product, IgY, are characterized and analyzed by IEF, SDS-PAGE, CD, FT-IT and DLS. On the basis of experimental results, the following information is obtained: the IgY molecular is consisted of two identical heavy chains (68 kDa) and two identical light chains (28 kDa). Therefore, the molecular weight of IgY is about 190 kDa. The isoelectric point of IgY is about 6.2 and there are 48% ofβ-sheet in IgY secondary structure. The Ultrafiltration product IgY has a higher activity than that from sodium sulphate precipitation method.