42(1)-3(p.34-39).fm

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1 The Korean Journal of Microbiology, Vol. 42, No. 1, March 2006, p Copyright 2006, The Microbiological Society of Korea z³ wz y yw k k Á Á k 1 Á½³ * w w w y w z³ wz lignin peroxidase (LIP), Mn-peroxidase (MNP) laccase w ³ z wù y z w ù x w ³. x w z³ z yw k wš w. w 23 36³ MNP ³ 30³ LIP y laccase ³ ƒƒ 11³ 12³. w z y w. wz y w k z ƒ z³ k w š ³ ƒ ù MNP y ³, poly R-478 polymeric dye anthron-type dye remazol brilliant blue R z y methylene blue, bromophenol blue congo red z q, ³ w k ùkþ. LIP, MNP laccase z y z³ bromophenol blue wš k 10% y x k. Key wordsý decolorization, dye compound, laccase, peroxidase, wood-rot fungi z³ z³, z³ z³ z p xk w f ƒ z³ w w. w z w z ƒ š w» w. w z ƒ z³ ƒ r š w. w w z yz (lignin peroxidase, Mn-peroxidase), y yz (glucose oxidase, glyoxal oxidase, aryl alcohol oxidase ) laccase w. z³ ƒ z ƒ š ƒ š w z y ƒ q w z³ z ww w ƒ. w xk t ù (8) ƒ z³ z y q wš ww w w ƒ j š q w. w w yw wz ù w w yw ù œ,, ƒ t ƒ w w ƒ *To whom correspondence should be addressed. Tel: , Fax: kyujkim@kangnung.ac.kr š (2, 3, 4, 6, 17, 18, 20). z³ w z p wz ù w ƒ yw k z³ wš w. ³ z ƒ l ³ ³ 1 wz Malt (1 l s 20 g, 20 g, rm 1 g, w 20 g) w 25 C 15 w o š ³ w xk, s p w w. œ ³ z w Kirk (12) 10 ml ³ ƒ 7 w 25 C o w. k w 0.2% ƒw š w. w Difco t w. z y d LIP (lignin peroxidase) Kirk (19) w yw 1.0 ml 125 mm tartaric acid buffer (ph4.5) 450 µl,» 40 mm veratryl alcohol 50 µl, z 450 µl ƒw z 8 mm y 50 µl yw g 310nm Ÿ y d w. z y veratryl alcohol l (min) veratryl aldehyde (µmole) 1 unit 34

2 Vol. 42, No. 1 wz y k 35 w. MNP (Mn-dependent peroxidase) Gold (7) w yw 1.0 ml 0.5 M sodium tartrate buffer (ph5.0) 200 µl, H 2 O µl, 10 mm MnSO 4 10 µl, z 100 µl w 8 mm y 12.5 µl yw g 238nm Ÿ y d w. z y (min) 1µmole Mn(II) y j 1 unit w. Laccase Harkin (9) xw syringaldazine» w š yw 0.04 M MES-NaOH buffer (ph 5.5) 2.5 ml 0.5 mm» 0.3 ml, z 0.2 ml ƒw 30 C o 40 z 530 nm Ÿ y d w. Lowry (13) Bradford (1) ww d w t bovine serum albumin (Sigma Chemical Co) w. š Ÿ d UVIKON UV/VIS Spectrophotometer ( 922, Kontron, UK) w d w. z d w t w» veratryl alcohol syringaldazine Aldrich t w. k k z y d w 0.2% (poly-r, remazol brilliant blue, methylene blue, bromophenol blue, congo red)ƒ ƒƒ w š w. ³ ƒ w ùkù k d w k rp» d w. ³ d š w ³ d w rp» w. ³ d w. ³ w 100 o C 90 w d w. š ³ z y x w ³ ƒ sw ³ 9³ sww 36³. Table 1 w ³ z³ ü z y, MNP+LIP+Lac+x, MNP+LIP-Lac+x, š MNP+LIP+ Lac-x xkƒ. w e ³» d ³ ü MNP+LIP-Lac- x MNP-LIP-Lac-x. w MNP z y. Laccase y ³ z y ³ y ƒ w. Hatakka (8) w z³ z y x j elš ù LIP-MNP, MNP-Lac, ƒ LIP-Lac w š w. ³ k Fig. 1 Fig. 5 t poly-r, RBBR (remazol brilliant blue), MB (methylene blue), BB (bromophenol blue) CR (congo red) w ³ 36³ ƒ k ³ ùkü. Fig. 1 poly-r w w k 10-20% Table 1. Enzymatic activity of 36 species of wood-rot fungi No. Species MNP (U/L) LIP (U/L) Laccase (U/L) 1 Coriolus versicolor KR-11W Coriolus versicolor KR-65W Coriolus versicolor KR-22W Coriolus versicolor KR-323W Coriolus versicolor KR-310W Coriolus versicolor KR-317W Coriolus versicolor KR-312W Coriolus versicolor KR-42W Coriolus versicolor KR-61W Coriolus hirsutus KR-412W Coriolus hirsutus KR-21W Coriolus hirsutus KR-1W Hymenochaete intricata KR-2W Hymenochaete intricata KR-45W Schizophyllum commune KR-36W Schizophyllum commune KR-39W Irpex lacteus KR-321W Irpex lacteus KR-35W Pycnoporus coccineus KR-41W Trichaptum abietinum KR-46W Trametes suaveolens KR-47W Tyromyces lacteus KR-48B Schizopora paradoxa KR-49W Lentinus edodes KR-411W Perenniporia medulla KR-51W Fomitopsis rosea KR-62W Inonotus mikadoi KR-314W Cerrena unicolor KR-23W Phanerochaete chrysosporium Unidentified Brown-rot KR-53B Unidentified Brown-rot KR-63B Unidentified White-rot KR-JB Unidentified White-rot KR-JW Unidentified White-rot KR-JW Unidentified White-rot KR-DW Unidentified White-rot KR-DW Total amounts of activity from the 8th day, 12th day, 16th day and 20th day of culture in the presence of 12 mg/l Mn(II). LIP, lignin peroxidase; MNP, Mn-peroxidase.

3 36 Tae-Won Jang et al. Comparison of decolorization of poly-r by 36 strains of woodrot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. Fig. 1 도 낮았다. 구름버섯균은 대체로 poly-r 배지에서 생장과 탈색율 이 높았다. 치마버섯균(KR-36W, KR-39W)과 기계층버섯균(KR321W, KR-35W)은 MNP 활성과 비례하여 탈색율이 좌우되었다. 갈색 부후균(KR-48B, KR-53B, 및 KR-63B)은 생장율에 비해 탈 색이 10% 미만이거나 전혀 일어나지 않았다. 생장과 탈색이 일 어나지 않은 균들(KR-46W, KR-411W, KR-23W, JB-01, JW-01, 및 JW-02)과 생장은 양호하나 탈색이 전혀 일어나지 않거나 미 약한 균들(KR-61W, KR-36W, KR-39W, KR-321W, KR-47W, KR-48B, KR-53B, KR-63B, DW-01, 및 DW-02)이 예상외로 많 았다. Fig. 2는 RBBR 염료에 대한 분해양상으로 구름버섯균의 RBBR 염료에 대한 탈색율이 여전히 높았다. MNP활성이 높은 균들이 전반적으로 탈색율이 높았으나 구름버섯균(KR-323W, KR-310W), 및 치마버섯균(KR-36W, KR-39W)은 측정한 효소활 성도와 관계없이 양호한 탈색율을 보임으로서 다른 효소군의 존 재 가능성을 시사하고 있다. 갈색부후균은 poly-r 염료와 마찬가 지로 생장율은 양호하였으나 탈색율은 10% 미만이었다. 생장과 탈색이 일어나지 않은 균주들(KR-46W, KR-411W, KR-23W, JB01, JW-01 및 JW-02)과 생장은 양호하나 탈색이 일어나지 않은 균주들(KR-321W, KR-48B, KR-53B, 및 KR-63B) 이 다소 있었 다. Fig. 3은 염료 MB에 대한 결과로 BB 및 CR과 함께 생장과 비례하여 전반적으로 탈색율이 높았다. 생장과 탈색이 일어나지 않은 균으로는 KR-412W, KR-321W, KR-46W, KR-47W, KR411W, KR-23W, JW-01, JW-02 및 DW-02였으며 생장이 양호하 Kor. J. Microbiol Comparison of decolorization of remazol brilliant bule R by 36 strains of wood-rot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. Fig. 2 나 탈색이 일어나지 않는 균들로는 갈색 부후균(KR-48B, KR53B 및 KR-63B)과 KR-JB-01이었다. Fig. 4는 염료 BB에 대한 결과로 전반적으로 조사한 전체 균들에 대한 탈색율이 양호하였 다. 생장과 탈색이 일어나지 않은 균으로는 KR-412W, KR321W, KR-46W, KR-47W, KR-411W, KR-23W, KR-JB-01, KRJW-01, KR-JW-02, 및 KR-DW-02 등이다. 생장은 양호하나 탈색 이 되지 않은 균류에는 KR-36W, KR-39W였으며 갈색 부후균의 경우, KR-63B는 생장에 비해 탈색율이 10% 정도였으나 KR48B 및 KR-53B 는 생장과 탈색율이 양호하였으며 이는 다른 염료와는 달리 갈색 부후균의 BB염료 탈색에 다른 효소계의 존 재를 암시하는 것으로 생각된다. Fig. 5는 CR에 대한 것으로 전 균주에 대해 대체로 생장과 비례하여 염료 탈색율이 높았다. 갈 색 부후균의 경우는 생장은 양호하였으나 탈색은 일어나지 않았 다. 생장과 탈색이 전부 일어나지 않은 균들로는 KR-412W, KR- 45W, KR-46W, KR-47W, KR-411W, KR-23W, JB-01, JW-01, JW-02, 및 DW-02 였다. Table 1과 Fig. 1에서 Fig. 5까지의 실험 결과를 근거로 3 종 류의 리그닌 분해효소 활성도의 유형에 따라 36균주를 구분하여 염료탈색 및 생장율을 비교하여 분석한 결과(Table 2), MNP+ LIP+Lac+형 구름버섯균 혹은 MNP+LIP+Lac-형인 부후균에서 대체로 실험한 염료에 대하여 탈색율이 높았다. 그러나 MNP+ LIP-Lac-형이더라도 MNP활성이 높은 균주는 특히 poly-r과

4 Vol. 42, No. 1 wz y k 37 Fig. 3 Comparison of decolorization of methylene blue by 36 strains of wood-rot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. Fig. 4 Comparison of decolorization of bromophenol bule by 36 strains of wood-rot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. RBBR w k ùkþ. MB, BB CR z³ wš ³ w k yw. w MNP-LIP-Lac-x ³ ³ w wù k Table 2. Comparison of decolorization of poly-r, remazol brilliant blue R, methylene blue, bromophenol blue and congo red by 36 strains of wood-rot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. Category Strain Poly-R RBBR MB BB CR D G D G D G D G D G MP+LP+LC+ Coriolus versicolor KR-65W Coriolus versicolor KR-22W Coriolus versicolor KR-312W Coriolus versicolor KR-42W Coriolus versicolor KR-61W Pycnoporus coccineus KR-41W Unidentified White-rot KR-DW MP+LP+LC- Coriolus versicolor KR-11W Coriolus versicolor KR-317W Fomitopsis rosea KR-62W Phanerochaete chrysosporium ME MP+LP-LC+ Coriolus versicolor KR-323W Coriolus versicolor KR-310W Trichaptum abietinum KR-46W Trametes suaveolens KR-47W Unidentified White-rot KR-JW

5 38 Tae-Won Jang et al. Kor. J. Microbiol Table 2. Continued Category Strain Poly-R RBBR MB BB CR D G D G D G D G D G MP+LP-LC- Coriolus hirsutus KR-412W Coriolus hirsutus KR-21W Coriolus hirsutus KR-1W Hymenochaete intricata KR-2W Hymenochaete intricata KR-45W Schizophyllum commune KR-36W Irpex lacteus KR-35W Schizopora paradoxa KR-49W Lentinus edodes KR-411W Perenniporia medulla-paris KR-51W Inonitus mikadoi KR-314W Unidentified Brown-rot KR-63B Unidentified White-rot KR-JW Unidentified White-rot KR-DW MP-LP-LC- Schizophyllum commune KR-39W Irpex lacteus KR-321W Tyromyces lacteus KR-48B Cerrena unicolor KR-23W Unidentified Brown-rot KR-53B Unidentified White-rot KR-JB RBBR: remazol brilliant blue R, MB: methylene blue, BB: bromophenol blue, CR: congo red, D: percentage of decolorization, G: percentage of growth MP: Mn-peroxidase, LP: lignin peroxidase, LC: laccase +: positive, -: negative t 3 wz wz ƒ. t w ew (10, 11, 15, 16). z³ w yw w w x ³ w k w (2, 3, 4, 5, 6, 20) wz ƒƒ w k w w (10, 11, 14, 15, 16, 17, 18) w, w x z w ³ ww x w k z MNP y LIPz ƒ w w w ƒ. w w ( w ) w w. š x Fig. 5. Comparison of decolorization of congo red by 36 strains of wood-rot fungi in the presence of 12 mg/l Mn(II). Decolorization zone of radial growth was measured from the reverse side of plates referring to the growth of mycelium on agar media without dye compound. 1. Bradford, M.M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, Bumpus, J.A., M. Tien, D. Wright, and S.D. Aust Oxidation of persistent environmental pollutants by a white-rot fungus. Science, 228, Bumpus, J.A. and S.D. Aust Biodegradation of DDT(1,1,1-

6 Vol. 42, No. 1 wz y k 39 tetrachloro -2,2-bis(4-chlorophenyl ethane) by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53, Bumpus, J.A. and B.J. Brock Biodegradation of crystal violet by the white-rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54, Cripps, C. and J.A. Bumpus Biodegradation of azo and hetero cyclic dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 56(4), Eichlerova, I., L. Homolka, L. Lisa and F. Nerud Orange G and remazol brilliant blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus. Chemosphere, 60, Gold, M.H. and J.K. Glenn Manganese peroxidase from Phanerochaete chrysosporium. Methods in Enzymol. 161, Hatakka, A Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, Harkin, J.M. and J.R. Obst Syringaldazine, an effective reagent for detecting laccase and peroxidase in fungi. Experientia. 29, Heinfling, A., M.J. Martinez, A.T. Martinez, M. Bergbauer, and U. Szewzyk Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction. Appl. Environ. Microbiol. 64(8), Kim, S.J. and M. Shoda Purification and characterization of a novel peroxidase from Geotrichum candidum Dec 1 involved in decolorization of dyes. Appl. Environ. Microbiol. 65(3), Kirk, T.K. and W.J. Connors Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117(3), Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.L. Randall Protein measurement with the folin phenol reagent. J. Biol. chem. 193, Molitoris, H.P. and B.R.M. Vyas Involvement of an extracellular H 2 O 2 -dependent ligninolytic activity of the white-rot fungus Pleurotus ostreatus in the decolorization of Remazol Brilliant Blue R. Appl. Environ. Microbiol. 61(11), Ollikka, P., K. Alhonmaki, and T. Glumoff Decolorization of azo, triphenyl methane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59(12), Shin, K.S. and C.J. Kim Production and purification of remazol Brilliant Blue R decolorizing peroxidase from the culture filtrate of Pleurotus ostreatus. Appl. Environ. Microbiol. 63(5), Shrivastava, R., V. Christian and B.R.M. Vyas Enzymatic decolorization of sulfonphthalein dyes. Enzyme Microb. Technol. 36, Tauber, M.M., G.M. Gueb itz and A. Rehorek Degradation of azo dyes by laccase and ultrasound treatment. Appl. Environ. Microbiol. 71(5), Tien, M. and T.K. Kirk Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymol Yang, Q., A. Yediler, M. Yang and A. Kettrup Decolorization of an azo dye, reactive black 5 and MnP production by Yeast isolate: Debaryomyces polymorphus. Biochem. Eng. J. 24, (Received March 8, 2006/Accepted March 14, 2006) ABSTRACT : Production of Lignin Degrading Enzymes and Decolorization of Various Dye Compounds by Wood-Rot Fungi Tae-Won Jang, Sang-Cheol Jun, Tae-Seok Ahn 1 and Kyu-Joong Kim* (Dept of Biology, Kangnung National University, Kangnung , Korea, 1 Dept of Environmental Science, Kangwon National University, Chuncheon , Korea) Wood-rot fungi produce extracellular lignin-degrading enzymes, the best known of which are lignin peroxidase, Mn-peroxidase and laccase. In this experiment, some of them produced all of three enzymes. Many other woodrot fungi produced one or two of those enzymes with various combinations. In this experiment, we tried to clarify the relationship between the pattern of enzyme production and degradative activity of several dye compounds. From the 36 strains of 23 species of wood-rot fungi, Mn-peroxidase activity was found in 30 strains of the fungi tested, whereas the activity of lignin peroxidase and laccase was detected in 11 strains and 12 strains of species, repectively, in Kirks low nitrogen media. In relation to the activity of lignin degrading enzymes and degradation of dye compounds, the white-rot fungi with three kinds of enzymes tested showed the best dye decolorizers. The fungi with Mn-peroxidase activity only decolorized poly R-478 and remazol brilliant blue R dye in proportion to the enzyme activity, while methylene blue, bromophenol blue and congo red dye were degraded in regardless of enzyme activity. Those dyes were degraded in relation to the growth rate of mycelium. Brown-rot fungi did not degrade all the dye compounds except bromophenol blue, in spite of moderate growth rate.