The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
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Characterization of Laccase Purified from Korean Trametes hirsuta S1

한국산 흰구름버섯(Trametes hirsuta S1)으로부터 정제된 Laccase의 특성

  • 임형선 (순천향대학교 생명과학부) ;
  • 윤경하 (순천향대학교 생명과학부)
  • Published : 2004.12.30
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Abstract

Laccase produced by Trametes hirsuta S1 isolated from Korea was partially purified and characterized using ultrafiltration, anion exchange chromatography and affinity chromatography. The laccase was produced as the predominant extracellular enzyme during primary metabolism. Neither lignin peroxidase nor veratryl alcohol oxidase (VAO) were detected in the culture fluid. Addition of 2,5-xylidine enhanced 4-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 12%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to $51.2\;{\mu}M\;and\;56.8\;{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $50^{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 20 min at $70^{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the T. hirsuta S1 laccase showed 100% of homology to those of laccase from C. hirsutus.

한국산 흰구름버섯(Trametes hirsuta S1)로부터 배지 내로 분비된 laccase를 ultrafiltration과 anion exchange chromatography, adsorption chromatography를 이용하여 분리 정제하고 정제된 효소의 특성을 조사하였다. Laccase는 균주의 일차 대사 과정에서 주로 생산되는 세포의 페놀 산화효소였다. 흰구름버섯을 기본 배지에서 배양하였을 때 생장은 배양 6일까지 급속히 이루어졌고, laccase의 활성은 배양 5일에 최대활성을 나타냈으며 배양액에서 LiP와 VAO의 활성은 측정되지 않았다. Laccase의 생산에 미치는 유도원의 영향을 조사하기 위하여 배양 중인 흰구름버섯에 몇몇 유도원을 첨가한 결과, 2,5-xylidine은 대조구에 비하여 laccase의 생산을 약4배 증가 시켰다. 정제된 laccase는 SDS 젤 전기영동에서 대략 66 kDa의 분자량을 가지는 단일 폴리펩타이드(single polypeptide)였고, 탄수화물 함량은 12%였다. 정제된 laccase의 $K_m$과 $V_{max}$를 ABTS[2,2-azino-bis(3-ethylbenzthiazo line-6-sulfonic acid)]를 기질로 사용하여 조사한 결과 각각 $51.2\;{\mu}M$과 $56.8\;{\mu}mole{\cdot}min^1{\cdot}mg^{-1}$로 측정되었다. Laccase 활성의 최적 pH는 3.0이며, 이 효소는 $50^{\circ}C$ 미만에서 1시간 동안 처리하였을 때 안정적이었고 $70^{\circ}C$에서 20분간 처리하였을 때 효소의 활성이 반감되었다. Laccase의 분광학적 특성을 조사한 결과 구리를 포함하는 단백질로 나타났다. 일반적으로 알려진 laccase의 기질들에 대한 특이성을 조사한 결과, 5 mM ABTS에서 가장 높은 활성을 나타내었으며 tyrosine에서는 laccase의 활성이 나타나지 않았다. 저해제의 영향을 조사한 결과, 일반적으로 구리를 포함하는 단백질의 저해제인 $NaN_3$, TGA, DDC를 일정 농도로 처리한 실험구에서는 효소의 활성이 완전하게 억제되었으며, EDTA 처리구에서는 효소의 활성이 억제되지 않았다. 한국산 흰구름버섯 S1 균주로부터 생산되는 laccase의 N-말단의 아미노산의 서열은 Coriolus hirsutus의 laccase와 100%의 상동성을 나타냈고, T. versicolor의 laccase I과는 68%의 상동성을 나타냈다.

Keywords

References

  1. Barr, D. P. and Aust. S. D. 1994. Pollutant degradation by whiterot fungi. Rev. Environ. Contam. T. 138: 49-72. https://doi.org/10.1007/978-1-4612-2672-7_3
  2. Bollag, J. M. and Leonowicz, A. 1984. Comparative studies of extracellular fungal laccase. Appl. Environ. Microbiol. 48: 849- 854.
  3. Bourbonnais, R. and Paice, M. G. 1990. Oxidation of non-phenolic substrates. An expended role for laccase in lignin biodegradation. FEBS Lett. 167: 99-102.
  4. Bourbonnais, R. and Paice, M. G. 1992. Demethylation and delignification of kraft pulp by Tramates versicolor laccase in lignin biodegradation. Appl. Microbiol. Biotechnol. 36: 823-827.
  5. Bourbonnais, R., Paice, M. G., Reid, I. A., Lanthier, P. and Yaguchi, M. 1995. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3- ethylbenzthiazolin-6-sulfonate) in kraft lignin depolymerization. Appl. Environ. Microhiol. 61: 1876-1880.
  6. Camarero, S., Sarkar, S., Ruiz-Duenas, F. J., Martinez, M. J. and Martinez, A. T. 1999. Description of a versatile peroxidase involved in natural degradation of lignin that has both Mn-peroxidase and lignin-peroxidase substrate binding sites. J. Biol. Chem. 274: 10324-10330. https://doi.org/10.1074/jbc.274.15.10324
  7. Cleland, W. W. 1967. Enzyme kinetic. Ann. Rev. Biochem. 36: 77- 122. https://doi.org/10.1146/annurev.bi.36.070167.000453
  8. Coll, P. M., Fernandez-Abalos, J. M., Villanueva, J. R., Santamaria, R. and Perez, P. 1993. Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl. Environ. Microbiol. 59: 2607-2613.
  9. Collins, P. J., Kotterman, M. J. J., Field, J. A. and Dobson, A. D. W. 1996. Oxidation of anthracene and benzo[a]pyrene by laccase from Tramates versicolor. Appl. Environ. Microbiol. 62: 4563-4567.
  10. De Vries, O. M. H., Kooistra, W. H. C. F. and Wessels, J. G. H. 1986. Formation of an extracellular laccase by Schizophyllum commune dicaryon. J. Gen. Microbiol. 132: 2817-2826.
  11. Eggert, C., Temp, U., Dean, J. F. D. and Eriksson, K. E. L. 1996a. A fungal metabolite mediates degradation of nonphenolic lignin structures and synthetic lignin by laccase. FEBS Letter. 391: 144-148. https://doi.org/10.1016/0014-5793(96)00719-3
  12. Eggert, C., Temp, U. and Eriksson, K. E. L, 1996b. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus : purification and characterization of the laccase. Appl. Environ. Microbiol. 62: 1151-1158.
  13. Field, J. A., De Jong, E., Feijoo-Costa, G. and De Bont, J. A. M. 1993. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol. 11: 44-49. https://doi.org/10.1016/0167-7799(93)90121-O
  14. Guillen, F., Martinez, A. T. and Martinez, M. J. 1990. Production of hydrogen peroxide aryalcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Appl. Microbiol. 32: 465-469.
  15. Haars, A., Chet, I. and Huttermann, A. 1981. Effect of phenolic compounds and tannin on growth and laccase activity of Fomes annosus. Eur. J. Forest Pathol. 11: 67-76. https://doi.org/10.1111/j.1439-0329.1981.tb00072.x
  16. Higuchi, T. 1990. Lignin biochemistry : biosynthesis and biodegradation. Wood Sci. Technol. 24: 23-63. https://doi.org/10.1021/es00071a002
  17. Hoshida, H., Nakao, M., Kubo, K., Hakukawa, T., Morimasa, K., Akada, R. and Nishizawa, Y. 2001. Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea. J. Biosci. Bioeng. In press.
  18. Johannes, C. and Majcherczyk, A. 2000. Natural mediators in the oxidation of polycyclic aromatic hydrocarbns by laccase mediator system. Appl. Environ. Microbiol. 66: 524-528. https://doi.org/10.1128/AEM.66.2.524-528.2000
  19. Karhumen, E., Niku-Paavola, M.-E., Viikari, L., Haltia, T., Van Der Meer, R. A. and Duine, J. A. 1990. A novel combination of prosthetic group in a fungal laccase, PQQ and two copper atoms. FEBS Lett. 267: 6-8. https://doi.org/10.1016/0014-5793(90)80273-L
  20. Kersten, P. J. and Kirk, T. K. 1987. Involvement of a new enzyme, glyoxal oxidases in extracellular H2O2 production by Phanerochaete chrysosporium. Arch. Microbiol. 169: 2195-2201.
  21. Kirk, T. K. and Farrell, R. L. 1987. Enzymic “Combustion” : the microbial degradation of lignin. Annu. Rev. Biochem. 41: 465-505.
  22. Kojima, Y., Tsukuda, Y., Kawai, Y., Tsukamoto, A., Sugiura, J., Sakaino, M. and Kita, Y. 1990. Cloning, sequence analysis, and expression of lignolytic phenoloxidase gene of the white-rot basidiomycete Coriolus hirsutus. J. Bacteriol. 265: 15224-15230.
  23. Laemmli, U. K. 1970. Cleavage of the structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  24. Leontievsky, A. A., Vares, T., Lankinen, P., Shergill, J. K., Pozdnyakova, N. N., Myasoedova, N. M., Kalkkinen, N., Golovelva, L. A., Cammack, R., Thurston, C. F. and Hatakka, A. 1997. Blue and yellow laccases of ligninolytic fungi. FEMS Microbiol. Lett. 156: 9-14. https://doi.org/10.1016/S0378-1097(97)00393-5
  25. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-267.
  26. Min, K.-L., Kim, Y.-H., Kim, Y. W., Jung, H. S. and Hah, Y. C. 2001. Characterization of a novel laccase produced by the wood-rotting group Phellinus ribis. Arch Biophem. Biophys. 392: 279-286. https://doi.org/10.1006/abbi.2001.2459
  27. Niku-Paavola, Karhunen, M.-L., Salola, E. P. and Raunio, V. 1988. Lignolitic enzymes of the white-rot fungus Phlebia radiata. Biochem. J. 254: 877-884.
  28. Niku-Paavola, Karhunen, M.-L. and Viikari, L. 2000. Enzymatic oxidation of alkens. J. Mol. Cat. B Enzymol. 10: 435-444. https://doi.org/10.1016/S1381-1177(99)00117-4
  29. Paszczynski, A., Crawford, R. L. and Huynh, V. B., 1988. Manganese peroxidase of Phanerochaete chrysosporium : purification. Methods Enzymol. 161: 264-270. https://doi.org/10.1016/0076-6879(88)61028-7
  30. Paszczynski, A. and Crawford, R. L., 1995. Potential for bioremediation of xenobiotic compounds by the white-rot fungus P. chrysosporium. Biotechnol. prog. 111: 368-379.
  31. Saparrat, M. C. N., Guillen, F., Arambarri, A. M., Martinez, A. T. and Martinez, M. J. 2002. Induction, isolation and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida. Appl. Environ. Microbiol. 68: 1534-1540. https://doi.org/10.1128/AEM.68.4.1534-1540.2002
  32. Sarkanen, K. V. 1971. Occuirrence, formation, structure and reaction. Pp 95-163. In Lignin. Sarkanen, K. V. and Ludwig, C. H. Eds. Wiley-Interscience. New York.
  33. Schoemaker, H. E. 1990. On the chemistry of lignin degradation. Rec. Travaux Chim. Pays-bas. 109: 255-272.
  34. Srinivasan, C., D'Souza, T. M., Boominathan, K. and Reddy, C. A. 1995. Demonstration of laccase in white rot basidiomycete Phanerochaete chrysosporium BKM-F1767. Appl. Environ. Microbiol. 61: 4274-4277.
  35. Sugiura, J., Sakaino, M., Kojima, Y., Tsujioka, K., Mutoh, Y., Shinohara, Y. and Koide, K. 1987. Purification and properties of phenol oxidase produced by white-rot fungi and molecular cloning of phenol oxidase gene, Pp 317-320. In International Seminar on Lignin Enzymic and Microbiol Degradation. International Symposium on Wood and Pulping Chemistry, Paris. INRA Publication, Versailles, France.
  36. Tien, M. and Kirk, T. K. 1983. Lignin-degrading enzyme from the hymenomycete Phanerochate chrysosporium. Science 326: 520-523.
  37. Tien, M. and Kirk, T. K. 1984. Lignin-degrading enzyme from Phanerochaete chrysosporium : purification, characterization, and catalytic properties of a unique $H_2O_2$<\TEX>-requiring oxygenase. Proc. Natl. Acad. Sci. USA 81: 2280-2284. https://doi.org/10.1073/pnas.81.8.2280
  38. Volc, J., Kubatova, E., Daniel, G. and Prikrylova, V. 1996. Only C-2 specific glouse oxidase activity is expressed in ligninolytic cultures of the white rot fungus Phanerocheate chrysosporium. Arch. Microbiol. 165: 421-424. https://doi.org/10.1007/s002030050348
  39. Thurston, C. F. 1994. The structure and function of fungal laccase. Microbiology 140: 19-26. https://doi.org/10.1099/13500872-140-1-19
  40. Wood, D. A. 1980. Production, purification and properties of extracellular laccase of Agaricus birporus. J. Gen. Microbiol. 117: 327-338.
  41. Yemm, E. W. and Willis, A. J. 1954. The estimation of carbohydrates in plant extracts by anthron. Bi ochem. 57: 508-514.