Aspergillus niger에서 추출되는 포도당 산화효소의 정제

박현규; 황우성; 김인호; Hyun-Kyu Park; Wu-Sung Whang; In-Ho Kim

HWAHAK KONGHAK, Vol.39, No.4, 512-516, August, 2001

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Aspergillus niger에서 추출되는 포도당 산화효소의 정제

Purification Study of Glucose Oxidase from Aspergillus niger

박현규, 황우성¹, 김인호 ^†

충남대학교 화학공학과, 대전 305-764
¹RMC Korea, 대전 305-701

Hyun-Kyu Park, Wu-Sung Whang¹, and In-Ho Kim^†

Department of Chemical Engineering, Chungnam National University, Daejeon 305-764, Korea
¹RMC Korea, TIC/TBI Center in KAIST, Daejeon 305-701, Korea

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초록

Glucose oxidase를 보다 간단한 방법으로 정제하는 방법을 찾기 위해 Table 1에 근거한 여러 가지 정제법을 도입하여 각 단계를 거칠 때마다 단백질의 농도와 GOD의 역가를 측정하였다. Aspergillus niger를 배양한 후 그 상등액을 원심분리하고 전처리 공정으로 증류수를 이용하여 투석을 시킴으로써 시료의 탁도를 떨어뜨리고 음이온 교환기인 DEAE-Sepharose에서 정제한다. 용리액 조성으로 0.1 M NaCl과 0.1M McIlvane buffer(pH 6.0)를 이용하여 GOD를 분리한다. 이러한 정제 과정을 거치게 되면 높은 Sp. activity(304 μmol/mg·min)와 높은 순도(95%)의 glucose oxidase(GOD)을 얻을 수 있었다. 얻어진 GOD는 보고된 GOD의 분자량 150,000과는 달리 그 분자량이 70,000-81,000 사이로 확인되었다.

Glucose oxidase(GOD) from Aspergillus niger was mainly purified by anion exchange chromatography on DEAE-sepharose. Other purification steps such as salting out, cation exchange chromatography, and dialysis were also tried. The purified enzyme was homogeneous on HPLC and SDS-PAGE. We determined an optimum purification condition for high GOD purity. Activity(304 mmol/mg · min), concentration(5.07 mg/mL) and yield (95%) from the starting enzyme source were obtained.

Keywords:Glucose Oxidase;Aspergillus niger;Purification;HPLC

[References]

Pazur JH, Kleppe K, Biochem., 3, 578 (1964)
Hong SH, Kim TJ, Jung YS, Kim SU, Yun JW, Korean J. Biotechnol. Bioeng., 10(4), 468 (1995)
Sung WJ, Bae YH, Anal. Chem., 72(9), 2177 (2000)
Kushi RT, Monti R, Contiero J, J. Ind. Microbiol. Biotechnol., 25(2), 63 (2000)
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Swoboda BEP, Massey V, Biological Chemistry, 240, 2209 (1965)
Chakraborty S, Schopter LM, Massey V, J. Biol. Chem., 265(7), 3793 (1990)
El-Enshasy H, Hellmuth K, Rinas U, Appl. Biochem. Biotechnol., 81(1), 1 (1999)
Moon IS, Park SK, Lee KY, Korean J. Biotechnol. Bioeng., 8(4), 409 (1993)
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The MERK INDEX 11(th) ed., p4354.
Ye WN, Combes D, Monsan P, Enzyme Microb. Technol., 10, 498 (1988)
Dawson RMC, Elliott DC, Elliott WH, Jones KM, "Data for Biochemical Research," 2(nd) ed., Oxford University (1968)
Markowitq E, Rosenthal, Anal. Chem., 28, 1179 (1956)
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Ye WN, Combes D, Biochim. Biophys. Acta, 999, 86 (1989)

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[1] Pazur JH, Kleppe K, Biochem., 3, 578 (1964)

[2] Hong SH, Kim TJ, Jung YS, Kim SU, Yun JW, Korean J. Biotechnol. Bioeng., 10(4), 468 (1995)

[3] Sung WJ, Bae YH, Anal. Chem., 72(9), 2177 (2000)

[4] Kushi RT, Monti R, Contiero J, J. Ind. Microbiol. Biotechnol., 25(2), 63 (2000)

[5] Garzillo AMV, Dipaolo S, Fenice M, Petruccioli M, Buonocore V, Federici F, Appl. Biochem. Biotechnol., 22, 169 (1995)

[6] Swoboda BEP, Massey V, Biological Chemistry, 240, 2209 (1965)

[7] Chakraborty S, Schopter LM, Massey V, J. Biol. Chem., 265(7), 3793 (1990)

[8] El-Enshasy H, Hellmuth K, Rinas U, Appl. Biochem. Biotechnol., 81(1), 1 (1999)

[9] Moon IS, Park SK, Lee KY, Korean J. Biotechnol. Bioeng., 8(4), 409 (1993)

[10] Bradford MM, Anal. Biochem., 72, 248 (1976)

[11] The MERK INDEX 11(th) ed., p4354.

[12] Ye WN, Combes D, Monsan P, Enzyme Microb. Technol., 10, 498 (1988)

[13] Dawson RMC, Elliott DC, Elliott WH, Jones KM, "Data for Biochemical Research," 2(nd) ed., Oxford University (1968)

[14] Markowitq E, Rosenthal, Anal. Chem., 28, 1179 (1956)

[15] Han SB, Kim KJ, Lim HJ, Kim TN, Choi DS, Korean J. Biotechnol. Bioeng., 9(1), 55 (1994)

[16] Ye WN, Combes D, Biochim. Biophys. Acta, 999, 86 (1989)