Journal of Mushroom (한국버섯학회지)

The Korean Society of Mushroom Science (한국버섯학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and characterization of versatile peroxidase from Pleurotus ostreatus produced in a rotary draft tube bioreactor

회전식 통풍관 생물반응기로부터 생산된 느타리균의 다목적 과산화효소(VP) 정제 및 특성

  • Hyo-Cheol Ha (Department of bio-technology and convergence, Daegu Haany University)
  • 하효철 (대구한의대학교 바이오산업융합학부)
  • Received : 2023.10.04
  • Accepted : 2023.11.07
  • Published : 2023.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

In this study, Pleurotus ostreatus No.42 was cultured in glucose-peptone-yeast-wheat bran medium using a previously reported novel rotary draft tube bioreactor. Versatile peroxidase (VP), a lignin-degrading enzyme, was isolated from a pellet-type mycelium culture grown in the medium for seven days. The VP was purified by sequentially applying ultra-filtration, DEAE-Sepharose CL-6B column, and Mono Q column. SDS-PAGE analysis revealed the molecular weight of VP to be 36.4 KDa with an isoelectric point of 3.65. The amino acid sequence was confirmed as VTCATGQTT. The purified VP was observed to possess the property of not only oxidizing Mn ions but also decomposing veratryl alcohol, a non-phenolic compound. The catalytic ability of VP is a subject for future research.

본 연구에서 Pleurotus ostreatus No.42는 이전에 보고된 새로운 유형의 회전식 통풍관 생물반응기(RTB)를 사용하여 포도당-펩톤-효모-밀기울(GPYW) 배지에서 배양하였다. 이 배지에서 7일 동안 펠렛형 균사체 배양 후, 리그닌 분해효소인 다목적 과산화 효소(VP)를 분리 및 정제하였다. 다목적 과산화 효소의 정제 과정은 한외여과, DEAE-Sepharose CL-6B 컬럼, Mono Q 컬럼을 순차적으로 적용하여 정제하였다. 그 결과, SDS-PAGE상에서 분자량(MW)은 36.4 KDa, 등전점 (IEF)은 3.65로 나타났으며, 아미노산 조성은 VTCATGQTT로 확인되었다. 정제된 다목적 과산화 효소는 Mn 이온을 산화시킬 뿐만 아니라 비페놀성 화합물인 베라트릴 알코올을 분해하는 특성을 갖는 것으로 나타났다.

Keywords

References

  1. Barber-Zucker S, Mindel V, Garcia-Ruiz E, Weinstein JJ, Alcalde M, Fleishman SJ. 2022. Stable and functionally diverse versatile peroxidases designed directly from sequences. J Am Chem Soc 144: 3564-3571. https://doi.org/10.1021/jacs.1c12433
  2. Bradford MM. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254. https://doi.org/10.1006/abio.1976.9999
  3. Camarero S, Sarkar S, Ruiz-Duenas FJ, Martinez MJ, Martinez AT. 1999. Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites. J Biol Chem 274: 10324-10330. https://doi.org/10.1074/jbc.274.15.10324
  4. Cohen R, Hadar Y, Yarden O. 2001. Transcript and activity levels of different Pleurotusostreatus peroxidases are differentially affected by Mn2+.Environ Microbiol 3: 312-322. https://doi.org/10.1046/j.1462-2920.2001.00197.x
  5. Fernandez-Fueyo E, Ruiz-Duenas FJ, Martinez MJ, Romero A, Hammel KE, Medrano FJ, Martinez AT. 2014. Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability. Biotechnol Biofuels 7: 2-24. https://doi.org/10.1186/1754-6834-7-2
  6. Giardina P, Palmieri G, Fontanella B, Rivieccio V, Sannia G. 2000. Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust. Arch Biochem Biophys 376: 171-179. https://doi.org/10.1006/abbi.1999.1691
  7. Gold MH, Alic M. 1993. Molecular biology of the lignin-degrading basidiomycete phanerochaete chrysosporium. Microbiol Rev 57: 605-622. https://doi.org/10.1128/mr.57.3.605-622.1993
  8. Ha HC, Honda Y, Watanabe T, Kuwahara M. 2001. Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete, Pleurotus ostreatus. Appl Microbiol Biotechnol 55: 704-711.
  9. Ha HC. 2019. N-terminal amino acid sequencing analysis of major manganese peroxidase (MnP3) produced by static culture of Pleurotus ostreatus. J Mushrooms 17: 185-190.
  10. Ha HC. 2021. Production and characterization of manganese peroxidase from Pleurotus ostreatus using a rotary draft tube bioreactor (RTB). J Mushrooms 19: 316-321.
  11. Juarez-Hernandez J, Castillo-Hernandez D, Perez-Parada C, Nava-Galicia S, Cuervo-Parra JA, Surian-Cruz E, Diaz-Godinez G, Sanchez C, Bibbins-Martinez M. 2021. Isolation of fungi from a textile industry effluent and the screening of their potential to degrade industrial dyes. J Fungi 7: 805-822. https://doi.org/10.3390/jof7100805
  12. Kamitsuji H, Honda Y, Watanabe T, Kuwahara M. 2004. Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus. Appl Microbiol Biotechnol 65: 287-294. https://doi.org/10.1007/s00253-003-1543-9
  13. Kamitsuji H, Honda Y, Watanabe T, Kuwahara M. 2005. Mn2+ is dispensable for the production of active MnP2 by Pleurotus ostreatus. Biochem Biophys Res Commun 327: 871-876. https://doi.org/10.1016/j.bbrc.2004.12.084
  14. Knop D, Yarden O, Hadar Y. 2015. The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications. Appl Microbiol Biotechnol 99: 1025-1038. https://doi.org/10.1007/s00253-014-6256-8
  15. Knop D, Levinson D, Makovitzki A, Agami A, Lerer E, Mimran A, Yarden O, Hadar Y. 2016. Limits of versatility of versatile Peroxidase. Appl Environ Microbiol 82: 4070-4080. https://doi.org/10.1128/AEM.00743-16
  16. Kong W, Fu X, Wang L, Alhujaily A, Zhang J, Ma F, Zhang X, Yu H. 2017. A novel and efficient fungal delignification strategy based on versatile peroxidase for lignocellulose bioconversion. Biotechnol Biofuels 10: 218-233. https://doi.org/10.1186/s13068-017-0906-x
  17. Martinez AT. 2002. Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microb Technol 30: 425-444. https://doi.org/10.1016/S0141-0229(01)00521-X
  18. Mendez-Hernandez JE, Eibes G, Arca-Ramos A, Lu-Chau TA, Feijoo G, Moreira MT, Lema JM. 2015. Continuous removal of nonylphenol by versatile peroxidase in a two-stage membrane bioreactor. Appl Biochem Biotechnol 175: 3038-3047. https://doi.org/10.1007/s12010-014-1474-1
  19. Nakazawa T, Izuno A, Kodera R, Miyazaki Y, Sakamoto M, Isagi Y, and Honda Y. 2017. Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white-rot agaricomycete Pleurotus ostreatus. Environ Microbiol 19: 261-272. https://doi.org/10.1111/1462-2920.13595
  20. Perez-Boada, M, Ruiz-Duenas, FJ, Pogni, R, Basosi, R, Choinowski, T, Martinez, MJ, Piontek, K, Martinez, AT. 2005. Versatile peroxidase oxidation of high redox potential aromatic compounds: Site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways. J Mol Biol 354: 385-402. https://doi.org/10.1016/j.jmb.2005.09.047
  21. Ruiz-Duenas FJ, Camarero S, Perez-Boada M, Martinez MJ, Martinez AT. 2001. A new versatile peroxidase from Pleurotus. Biochem Soc Trans 29: 116-122. https://doi.org/10.1042/bst0290116
  22. Saez-Jimenez V, Rencoret J, Rodriguez-Carvajal MA, Gutierrez A, Ruiz-Duenas FJ, Martinez AT. 2016. Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin. Biotechnol Biofuels 9: 198-211. https://doi.org/10.1186/s13068-016-0615-x
  23. Salame TM, Knop D, Tal D, Levinson D, Yarden O, Hadar Y. 2012. Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus. Appl Environ Microbiol 78: 5341-5352.
  24. Salame TM, Knop D, Levinson D, Mabjeesh SJ, Yarden O, Hadar Y. 2012. Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation. PLoS ONE 7: e52446.
  25. Sarkar S, Martinez AT, Martinez MJ. 1997, Biochemical and molecular characterization of a manganese peroxidase isoenzyme from Pleurotus ostreatus. Biochem Biophys Acta 1339: 23-30. https://doi.org/10.1016/S0167-4838(96)00201-4
  26. Sellami K, Couvert A, Nasrallah N, Maachi R, Abouseoud M, Amrane A. 2022. Peroxidase enzymes as green catalysts for bioremediation and biotechnological applications: A review. Sci total environ 806: 150500-150518. https://doi.org/10.1016/j.scitotenv.2021.150500
  27. Singh AK, Iqbal HMN, Cardullo N, Muccilli V, Fernandez-Lucas J, Schmidt JE, Jesionowski T, Bilal M. 2023. Structural insights, biocatalytic characteristics, and application prospects of lignin-modifying enzymes for sustainable biotechnology (review). Int J Biol Macromol 242: 124968-124996. https://doi.org/10.1016/j.ijbiomac.2023.124968
  28. Taboada-Puig R, Eibes G, Lloret L, Lu-Chau TA, Feijoo G, Moreira MT, Lema JM. 2016. Fostering the action of versatile peroxidase as a highly efficient biocatalyst for the removal of endocrine disrupting compounds. N Biotechnol 33: 187-195. https://doi.org/10.1016/j.nbt.2015.05.003
  29. Tien M, Kirk TK. 1983. Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds. Science 221: 661-663. https://doi.org/10.1126/science.221.4611.661
  30. Tsukihara T, Honda Y, Watanabe T, Watanabe T. 2006. Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2. Appl Microbiol Biotechnol 71: 114-120. https://doi.org/10.1007/s00253-005-0136-1
  31. Wang X, Yao B, Su X. 2018. Linking enzymatic oxidative degradation of lignin to organics detoxification(review). Int J Mol Sci 19: 3373-3390. https://doi.org/10.3390/ijms19113373
  32. Wang Y, Vazquez-Duhalt R, Pickard MA. 2002. Purification, characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258. Curr Microbiol 45: 77-87.
  33. Warishi H, Dunford HB, Macdonald ID, Gold MH. 1989. Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. J Biol Chem 264: 3335-3340. https://doi.org/10.1016/S0021-9258(18)94070-6