The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
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Characterization of Laccase Purified from Korean Pycnoporus cinnabarinus SCH-3

한국산 주걱송편버섯(Pycnoporus cinnabarinus) SCH-3로부터 정제 된 Laccase의 특성

  • Park, Eun-Hye (Department of Life Science, Soonchunhyang University) ;
  • Yoon, Kyung-Ha (Department of Life Science, Soonchunhyang University)
  • 박은혜 (순천향대학교 생명과학부) ;
  • 윤경하 (순천향대학교 생명과학부)
  • Published : 2003.08.30
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Abstract

Laccase produced by Pycnoporus cinnabarinus SCH-3 isolated from Korea was partially purified using ultrafiltration, anion exchange chromatography and affinity chromatography, The laccase was produced as the predominant extracellular phenoloxidase during primary metabolism. Neither lignin peroxidase nor manganese-dependent peroxidase were detected in the culture fluid. In order to examine the effect of inducers in laccase production, 2,5-xylidine was added in the culture of Pycnoporus cinnabarinus SCH-3. Addition of 2,5-xylidine enhanced 25-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 9%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to be $44.4{\mu}M\;and\;56.0{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $60{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 10 min at $80{\circ}C$. 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 P. cinnabarinus SCH-3 laccase showed 94% of homology to the N-terminal sequences of laccases from P. cinnabarinus PB and P. coccineus.

한국산 주걱송편버섯(Pycnoporus cinnabarinus) SCH-3로부터 배지 내로 분비된 laccase를 ultrafiltration과 anion exchange chromatography, adsorption chromatography를 이용하여 분리 정제하고 정제된 호소의 특성을 조사하였다. Laccase는 균주의 일차 대사 과정에서 주로 생산되는 세포외 페놀 산화효소였다. 주걱송편버섯을 기본 배지에서 배양하였을 때 생장은 배양 9일에 최대였고, laccase의 활성은 배양 7일에 최대활성을 나타냈으며 배양액에서 LiP, MnP 그리고 AAO의 활성은 측정되지 않았다. Laccase의 생산에 미치는 유도원의 영향을 조사하기 위하여 배양 중인 주걱 송편버섯에 몇몇 유도원을 첨가한결과, 2,5-xylidine은 대조구에 비하여 laccase의 생산을 25배 증가 시켰다. 정제된 laccase는 SDS 젤 전기영동에서 대략 66 kDa의 분자량을 가지는 단일 폴리펩타이드(single polypeptide)였고, 탄수화물 함량은 9%였다. ABTS [2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)]를 기질로 사용하며 정제된 laccase의 $K_{m}}$과$V_{max}$를 조사한 결과 각각 $44.4{\mu}M\;and\;56.0{\mu}mole$로 측정되었다. Laccase 활성의 최적 pH는 3.0이며, 이 효소는 $60{\circ}C$에서 1시간 동안 처리하였을 때 매우 안정적이었고 $80{\circ}C$에서 10분간 처리하였을 때 효소의 활성이 반감되었다. Laccase의 분광학적 특성을 조사한 결과 구리를 포함하는 단백질로 나타났다. 일반적으로 알려진 laccase의 기질들에 대한 특이성을 조사한 결과 5mM ABTS와 5mM hydroquinone에서 높은 활성을 나타내었으며 tyrosine에서는 laccase의 활성이 나타나지 않았다. 저해제의 영향을 조사한 결과, 일반적으로 구리를 포함하는 단백질의 저해제인 $NaN_{3}$, TGA, DDC를 일정농도로 처리한 실험구에서는 효소의 활성이 완전하게 억제되었으며, p-coumaric acid와 EDTA 처리구에서는 효소의 활성이 억제되지 않았다. 한국산 주걱송편버섯 SCH-3 균주로부터 생산되는 laccase의 N-말단의 아미노산의 서열은 P. coccineus의 laccase와 호주에서 분리 동정된 P.cinnabarinus PB의 laccase와 94%가 같았다.

Keywords

References

  1. Rev. Environ. Contam. T. v.138 Pollutant degradation by white rot fungi Barr, D.P.;Aust, S.D.
  2. Appl. Environ. Microbiol. v.48 Comparative studis of extracellular fungel laccase Bollag, J.M.;Leonowicz, A.
  3. FEBS Lett. v.167 Oxidation of non-phenolic substrates. An expended role for laccase in lignin biodegradation Bourbonnais, R.;Paice, M.G. https://doi.org/10.1016/0014-5793(90)80298-W
  4. Appl. Microbiol. Biotechnol. v.36 Demethylation and delignification of kraft pulp by Tramates versicolor laccase in lignin biodegradation Bourbonnais, R.;Paice, M.G.
  5. J. Biol. Chem. v.274 Description of a versatile peroxidase involved in natural degradation of lignin that has both Mn-per-oxidse and lignin-peroxidase substrate binding site Camarero, S.;Sarkar, S.;Ruiz-Duenas, F.J.;Martinez, M.J.;Martinez, A.T. https://doi.org/10.1074/jbc.274.15.10324
  6. Ann. Rev. Biochem. v.36 Enzyme kinetic Cleland, W.W. https://doi.org/10.1146/annurev.bi.36.070167.000453
  7. Appl. Environ. Microbiol. v.59 Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971) Coll, P.M.;Fernandez-Abalos, J.M.;Villanueva, J.R.;Santamaria, R.;Perez, P.
  8. Appl. Environ. Microbiol. v.62 Oxidation of anthracene and benzo[a]pyrene by lacase from Tramates versicolor Collins, P.J.;Kotterman, M.J.J.;Field, J.A.;Dobson, A.D.W.
  9. J. Gen. Microbiol. v.132 Formation of an extracellular laccase by Schizophyllum commune dicaryon De Vries, O.M.H.;Kooistra, W.H.C.F.;Wessels, J.G.H.
  10. FEBS Letter v.391 A fungal metabolite mediates degradation of nonphenolic lignin structures and synthetic lignin by laccase Eggert, C.;Temp, U.;Dean, J.F.D.;Eriksson, K.E.L. https://doi.org/10.1016/0014-5793(96)00719-3
  11. Appl. Environ. Microbiol. v.62 The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase Eggert, C.;Temp, U.;Eriksson, K.E.L.
  12. Trends Biotechnol. v.11 Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics Field, J.A.;De Jong, E.;Feijoo-Costa, G.;De Bont, J.A.M. https://doi.org/10.1016/0167-7799(93)90121-O
  13. Appl. Microbiol. v.32 Production of hydrogen peroxide arylcohol oxidase from the ligninolytic fungus Pleurotus eryngii Guillen, F.;Martinez, A.T.;Martinez, M.J.
  14. Eur. J. Forest Pathol. v.11 Effect of phenolic compounds and tannin on growth and laccase activity of Fomes annosus Haars, A.;Chet, I.;Huttermann, A. https://doi.org/10.1111/j.1439-0329.1981.tb00072.x
  15. Wood Sci. Technol. v.24 Lignin biochemistry: biosynthesis and biodegradation Higuchi, T. https://doi.org/10.1021/es00071a002
  16. Journal of Bioscience and Bioengineering Isolation of five laccase gene sequences from the white-rot fungus trametes sanguinea by cloning, characterization and expression of the laccase cDNA in yeasts Hoshida, H.;Nakao, M.;Kubo, K.;Hakukawa, T.;Morimasa, K.;Akada, R.;Nishizawa, Y.
  17. Appl. Environ. Microbiol. v.66 Natural mediators in the oxidaton of polycyclic aromatic hydrocarbns by laccase mediator system Johannes, C.;Majcherczyk, A. https://doi.org/10.1128/AEM.66.2.524-528.2000
  18. FEBS Lett. v.267 A novel combination of prosthetic group in a fungal laccase, PQQ and two copper atoms Karhumen, E.;Niku-Paavola, M.E.;Viikari, L.;Haltia, T.;Van Der Meer, R.A.;Duine, J.A. https://doi.org/10.1016/0014-5793(90)80273-L
  19. Arch. Microbiol. v.169 Involvement of a new enzyme, glyoxal oxidases in extracellular $H_2O_2$ production by Phenerochate chrysosporum Kersten, P.J.;Kirk, T.K.
  20. Annu. Rev. Biochem. v.41 Enzymic 'Combustion': the microbial degradation of lignin Kirk, T.K.;Farrell, R.L.
  21. J. Bacteriol. v.265 Cloning, sequence analysis, and expression of lignolytic phenoloxidase gene of the white-rot basidiomycete Coriclus hirsutus Kojima, Y.;Tsukuda, Y.;Kawai, Y.;Tsukamoto, A.;Sugiura, J.;Sakaino, M.;Kita, Y.
  22. Nature v.227 Cleavage of the structural protein during the assembly of the head of bacterophage T4 Laemmli, U.K. https://doi.org/10.1038/227680a0
  23. FEMS Microbiol. Lett. v.156 Blue and yellow alccases of ligninolytic fungi 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.;Hatakka, A. https://doi.org/10.1016/S0378-1097(97)00393-5
  24. J. Biol. Chem. v.193 Protein measurement with the folin phenol reagent Lowry, O.H.;Rosebrough, N.J.;Farr, A.L.;Randall, R.J.
  25. Arch Biophem. Biophys. v.392 Characterization of a novel laccase produced by the wood-rotting group Phellinus ribis Min, K.L.;Kim, Y.H.;Kim, Y.W.;Jung, H.S.;Hah, Y.C. https://doi.org/10.1006/abbi.2001.2459
  26. Biochem. J. v.254 Lignolitic enzymes of the white-rot fungus Phlebia radiata Niku-Paavola;Karhunen, M.L.;Salola, E.P.;Raunio, V. https://doi.org/10.1042/bj2540877
  27. J. Mol. Cat. B Enzymol. v.10 Enzymatic oxidation of alkens Niku-Paavola;Viikari, L. https://doi.org/10.1016/S1381-1177(99)00117-4
  28. Methods Enzymol. v.161 Manganese peroxidase of Phenerochaete chrysosporium: purification Paszczynski, A.;Crawford, R.L.;Huynh, V.B. https://doi.org/10.1016/0076-6879(88)61028-7
  29. P. chrysosporium. Biotechnol. Prog. v.111 Potential for bioremediation of xenobiotic compounds by the white-rot fungus Paszczynski, A.;Crawford, R.L.
  30. Appl. Environ. Microbiol. v.68 Induction, isolation and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida Saparrat, M.C.N.;Guillen, F.;Arambarri, A.M.;Martinez, A.T.;Martinez, M.J. https://doi.org/10.1128/AEM.68.4.1534-1540.2002
  31. Rec. Travaux chim. Pays-bas. v.109 On the chemistry of lignin degradation Schoemaker, H.E.
  32. Appl. Environ. Microbiol. v.61 Demonstration of laccese in white rot basidiomycete Phenerochaete chrysosporium BKM-F1767 Srinivasan, C.;D'Souza, T.M.;Boominathan, K.;Reddy, C.A.
  33. International Seminar on Lignin Enzymic and Microbiol Degradation. International Symposium on Wood and Pulping Chemistry Purification and properties of phenol oxidase produced by white-rot fungi and molecular cloning for phenol oxidase gene Sugiura, J.;Sakaino, M.;Kojima, Y.;Tsujioka, K.;Mutoh, Y.;Shinohara, Y.;Koide, K.
  34. Science v.326 Lignin-degrading enzyme from the hymenomycete Phenerochate chrysosporium Tien, M.;Kirk, T.K.
  35. Proc. Natl. Acad. Sci. USA v.81 Lignin-degrading enzyme from Phanerochaete chrysosporium; purification, characterization, and catelytic properties of a unique $H_2O_2-requiring$ oxygenase Tien, M.;Kirk, T.K. https://doi.org/10.1073/pnas.81.8.2280
  36. Microbiology v.140 The structure and function of fungal laccase Thurston, C.F. https://doi.org/10.1099/13500872-140-1-19
  37. Arch. Microbiol. v.165 Only C-2 specific glouse oxidase activity is expressed in ligninolytic cultures of the white rot fungus Phanerocheate chrysosporium Volc, J.;Kubatova, E.;Daniel, G.;Prikrylova, V. https://doi.org/10.1007/s002030050348
  38. J. Gen. Microbiol. v.117 Production purification and properties of extracellular laccase of Agaricus hirporus Wood, D.A.
  39. Biochem. v.57 The Estimation of carbohy-drates in plant extracts by Anthron Yemm, E.W.;Willis, A.J. https://doi.org/10.1042/bj0570508