NADH 동시재생을 이용한 실관효소반응기에서의 L-leucine생산

이윤영; 장호남

HWAHAK KONGHAK, Vol.30, No.2, 253-260, April, 1992

Export Citation

NADH 동시재생을 이용한 실관효소반응기에서의 L-leucine생산

L-leucine Production with Simultaneous NADH Regeneration in a Hollow Fiber Enzyme Reactor

이윤영, 장호남

초록

실관반응기에 주반응효소(leucine dehydrogenase), 재상반응효소(formate dehydrogenase), 그리고 고분자 조효소 유도체(dextran-NAD⁺)를 고정화시켜 연속적으로 루이신을 생산하였다. 실관반응기의 루이신 생산은 34시간 유지되었고 최고 전화율이 83%, 평균전화율이 76%였다. Space time yield는 78.2g/L/d(또는 596mmol/L/d)의 높은 값을 얻었는데 이러한 높은 space time yield 값은 발효공정으로는 얻기 힘든 효소반응기의 장점이라 하겠다. 효소반응시 조효소 유도체의 retention coefficient는 평균적으로 87%, 최고 91%이었고, resi-dence time은 평균적으로 1시간 정도를 유지하였다. 조효소유도체의 turn over number는 8500을 넘어섰다. 결과적으로 조효소재생방법을 사용하지 않은 경우의 루이신 kg생산당 NAD⁺비용을 재생방법에 의하여 약 10,000배로 줄여, 산화환원효소의 산업적 응용의 가장 큰 문제점이었던 조효소 비용문제를 해결하였다.

L-leucine was produced from α-ketoisocaproate in a hollow fiber enzyme reactor, where leucine degydrogenase(L-leucine:NADH oxidoreductase, EC1.4.1.9), formate dehydrogenase(formate:NAD⁺ oxido-reductase, EC1.2.1.2), and NAD⁺ covalently bound to water-soluble dextran(dextran-NAD⁺) were used as the catalytic enzyme, coenzyme regeneration enzyme, and enlarged coenzyme, respectively. The continuous-production of L-leucine in a hollow fiber enzyme reactor for 34hrs resulted in a space-time yield of 78.2g/L/d(or 596mmol/L/d) with a mean substrate conversion of 76%. After 34hrs, the reaction did not continue due to loss of enzyme activity. The turnover number of cofactor-retio of moles of product to moles of NAD(H)-exceeded 8,500. The cofactor regeneration scheme for NADH had been successful to the extent that cofactor cost was no longer dominant in the L-leucine production.

[References]

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[1] Enzyme Nomenclature: Recommendations of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry, Academic Press, New York (1979)

[2] Sigma price list (1991)

[3] Chenault HW, Whitesides GM, Appl. Biochem. Biotechnol., 14, 147 (1987)

[4] Laval JM, Bourdillon C, Moiroux J, Biotechnol. Bioeng., 30, 157 (1987)

[5] Willner I, Mandler D, Enzyme Microb. Technol., 11, 467 (1989)

[6] Nath PK, Izumi Y, Yamada H, Enzyme Microb. Technol., 12, 28 (1990)

[7] Gu KF, Chang TMS, Biotechnol. Appl. Biochem., 12, 227 (1990)

[8] Campbell J, Chang TMS, Biochem. Biophys. Res. Commun., 69, 562 (1976)

[9] Yamazaki Y, Maeda H, Suzuki H, Biotechnol. Bioeng., 18, 1761 (1976)

[10] Guagliard A, Raia CA, Rella R, Buckmann AF, Dauria S, Rossi M, Bartolucci S, Biotechnol. Appl. Biochem., 13, 25 (1991)

[11] KulaMR, Wandrey C, Methods Enzymol., 136, 9 (1987)

[12] Ohshima T, Wandrey C, Kula MR, Soda K, Biotechnol. Bioeng., 27, 1616 (1985)

[13] Leuchtenberger W, Karrenbauer M, Plocker U, Ann. N.Y. Acad. Sci., 434, 78 (1983)

[14] Kajiwara S, Maeda H, Agric. Biol. Chem., 51, 2873 (1987)

[15] Blatt WF, Membrane Separation Technology, Flin, J.E. (ed.), Plenum Press, New York, p. 47 (1970)

[16] Kim BS, Chang HN, Bioseparation, 2, 23 (1991)

[17] Charm SE, Wong BL, Biotechnol. Bioeng., 12, 1103 (1970)