Purification of the Glycomacropeptide from Cheese Whey

치즈 유청으로부터 Glycomacropeptide의 분리.정제

Glycomacropeptide(GMP) was purified from cheese whey which is obtaining as a byproduct in cheese producing. Cheese whey was first concentrated 10 times with a ultrafiltration aparratus, and then heated at 95$^{\circ}C$ for 5 min. The concentrated fraction was centrifuged at 20,000$\times$g for 30 min to remove fat layer. The supernatant layer enriched GMP protein was fractionated by ion exchange chromatography on DEAE-Sepharose Fast Flow column. GMP was bound to DEAE resin and eluted with 0.1~0.25 M NaCl when using a linear NaCl gradient from 0 M to 0.5 M. The purified GMP gave a single band of 24 kDa which seems to be trimer molecular weight in SDS-PAGE, and migrated to the same molecular weight with control GMP obtained commercially. Its amino acid composition were consistent with that of standard GMP. About 0.71 g of GMP was recovered from 1 L of cheese whey. These results indicate that glycomacropeptide could be simply purified from cheese whey by using ultrafiltration and DEAE column chromatography.

Glycomacropeptide(GMP) was purified from cheese whey which is obtaining as a byproduct in cheese producing. Cheese whey was first concentrated 10 times with a ultrafiltration aparratus, and then heated at 95℃ for 5 min. The concentrated fraction was centrifuged at 20,000×g for 30 min to remove fat layer. The supernatant layer enriched GMP protein was fractionated by ion exchange chromatography on DEAE-Sepharose Fast Flow column. GMP was bound to DEAE resin and eluted with 0.1∼0.25 M NaCl when using a linear NaCl gradient from 0 M to 0.5 M. The purified GMP gave a single band of 24 kDa which seems to be trimer molecular weight in SDS-PAGE, and migrated to the same molecular weight with control GMP obtained commercially. Its amino acid composition were consistent with that of standard GMP. About 0.71 g of GMP was recovered from 1 L of cheese whey. These results indicate that glycomacropeptide could be simply purified from cheese whey by using ultrafiltration and DEAE column chromatography. 본 연구에서는 치즈 제조시의 부산물인 치즈 유청으로부터 glycomacropeptide(GMP)를 정제하려고 하였다. 치즈 유청을 한외여과 장치를 이용하여 10배로 농축하여 공시시료로 하였고, 95℃에서 5분간 가열한 후 20,000rpm에서 30분간 원심분리하였다. 원심분리 후 혼입된 지방층을 제거하였고, GMP를 포함하고 있는 상청액은 DEAE-Sepharose Fast Flow 크로마토그래피 칼럼에 주입하였다. GMP는 DEAE 수지의 관응기에 결합하였으며, 0에서 0.5M의 NaCl 직선농도 구배를 실시하였을 때 약 0.1∼0.25M에서 용출되었다. 정제한 GMP는 SDS-PAGE에서 단일한 band를 나타냈으며, 분자량은 24kDa으로 trimer 형태로 상업적으로 제공받은 대조 GMP와 같은 분자량 위치로 영동하였다. 정제한 GMP의 아미노산 조성은 대조GMP의 결과와 비교하였을 때 Ser, Val이 약간 적었으며 Gly은 반대로 2배의 함량을 나타냈으나 전체적인 조성비에서는 거의 일치하였다. 단백질의 회수율은 유청 1L에서 약 0.71g의 GMP를 효과적으로 분리한 것으로 나타났다. 이 결과로부터 한외여과와 DEAE-Sepharose Fast Flow chromatography를 이용하여 GMP를 산업적으로 대량 정제할 수 있는 것이 확인되었다.