난백으로부터 리소짐 분리를 위한 이온교환 크로마토그래피와 침전법의 비교

김형원; 김인호; Hyoung Won Kim; In Ho Kim

HWAHAK KONGHAK, Vol.41, No.3, 332-336, June, 2003

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난백으로부터 리소짐 분리를 위한 이온교환 크로마토그래피와 침전법의 비교

Comparison of Lysozyme Purification from Egg White Between Ion Exchange Chromatography and Precipitation

김형원, 김인호 ^†

충남대학교 화학공학과, 305-764 대전시 유성구 궁동 220

Hyoung Won Kim, and In Ho Kim^†

Department of Chemical Engineering, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, Korea

E-mail:

초록

단백질을 분리하는 방법 중에서 이온교환 크로마토그래피와 침전법을 이용하여 난백으로부터 lysozyme를 분리하였다. 특히, 이온교환 크로마토그래피에서는 gradient system을 이용하여 용출용액의 농도변화와 유속에 따른 분리정도를 알아 보았다. 그 결과 농도는 0 M에서 1 M NaCl로 농도구배를 걸어주었을 때 lysozyme 분리에 충분하다는 것과 유속의 영향은 적다는 것을 SDS-PAGE를 통해 알 수 있었다. 크로마토그래피와 비교하기 위해 황산암모늄을 이용하여 난백 단백질을 침전시켰다. 황산암모늄의 농도를 변화시켜보았지만, lysozyme을 난백에 있는 다른 단백질과 선택적으로 분리시키지 못했다. 그리고 침전시간과 침전온도를 변화시켜서 lysozyme의 수율변화를 조사하였다.

Ion exchange chromatography(IEC) with gradient and precipitation were used to purify lysozyme from egg white. In IEC, elution of lysozyme from 0 M to 1 M NaCl gradient was performed and SDS-PAGE showed that lysozyme was selectively purified from other egg white proteins. In addition to IEC, egg white proteins were salted out with ammonium sulfate to compare the effectiveness of precipitation method with IEC. The concentrations of ammonium sulfate were varied from 25% to 85%. Precipitation was not able to purify solely lysozyme from egg white. Recovery of lysozyme in the precipitation was improved by changing precipitation temperature and aging time.

Keywords:Chromatography;Precipitation;Lysozyme

[References]

Regel R, Matioli SR, Terra WR, Insect Biochem. Molec. Biol., 28(5-6), 309 (1998)
Kim WK, Chung BH, Korean J. Biotechnol. Bioeng., 14(3), 279 (1999)
Yoshitaka M, J. Jpn. Soc. Food Nutr., 24(6), 311 (1971)
Awade AC, Efstathiou T, J. Chromatogr. B, 723(1), 69 (1999)
Alderton G, Fevold HL, J. Biol. Chem., 164, 1 (1946)
Junowicz E, Charm SE, Febs Lett., 57(2), 219 (1975)
Li-Chan E, Nakai S, Sim J, Bragg DB, Lo KV, J. Food Sci., 51(1), 1032 (1986)
Chiang BH, Su CK, Tsai GJ, Tsao GT, J. Food Eng., 58(2), 303 (1993)
Ghosh R, Silva SS, Cui Z, Biochem. Eng. J., 6, 19 (2000)
Gu Z, Su Z, Janson JC, J. Chromatogr. A, 918, 311 (2001)
Bollag DM, Ededstein SJ, Protein Methods, Wiley-Liss, New York, NY (1991)
Park DH, Lee HJ, Lee EK, Korean J. Chem. Eng., 14(1), 64 (1997)
Forsythe EL, Snell EH, Malone CC, Pusey ML, J. Cryst. Growth, 196, 332 (1999)

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[1] Regel R, Matioli SR, Terra WR, Insect Biochem. Molec. Biol., 28(5-6), 309 (1998)

[2] Kim WK, Chung BH, Korean J. Biotechnol. Bioeng., 14(3), 279 (1999)

[3] Yoshitaka M, J. Jpn. Soc. Food Nutr., 24(6), 311 (1971)

[4] Awade AC, Efstathiou T, J. Chromatogr. B, 723(1), 69 (1999)

[5] Alderton G, Fevold HL, J. Biol. Chem., 164, 1 (1946)

[6] Junowicz E, Charm SE, Febs Lett., 57(2), 219 (1975)

[7] Li-Chan E, Nakai S, Sim J, Bragg DB, Lo KV, J. Food Sci., 51(1), 1032 (1986)

[8] Chiang BH, Su CK, Tsai GJ, Tsao GT, J. Food Eng., 58(2), 303 (1993)

[9] Ghosh R, Silva SS, Cui Z, Biochem. Eng. J., 6, 19 (2000)

[10] Gu Z, Su Z, Janson JC, J. Chromatogr. A, 918, 311 (2001)

[11] Bollag DM, Ededstein SJ, Protein Methods, Wiley-Liss, New York, NY (1991)

[12] Park DH, Lee HJ, Lee EK, Korean J. Chem. Eng., 14(1), 64 (1997)

[13] Forsythe EL, Snell EH, Malone CC, Pusey ML, J. Cryst. Growth, 196, 332 (1999)