한국버섯학회지 (Journal of Mushroom)

  • 제19권4호
  • /
  • Pages.285-293
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  • 2021
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  • 1738-0294(pISSN)
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  • 2288-8853(eISSN)
한국버섯학회 (The Korean Society of Mushroom Science)
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Flammulina velutipes var. lupinicola의 유전체 정보기반 laccase 유전자 동정 및 특성 규명

Identification and characterization of laccase genes in the Flammulina velutipes var. lupinicola genome

  • 유혜원 (건국대학교 글로컬캠퍼스 의료생명대학 바이오의약학과) ;
  • 박영진 (건국대학교 글로컬캠퍼스 의료생명대학 바이오의약학과)
  • Yu, Hye-Won (Department of Medicinal Biosciences, Konkuk University) ;
  • Park, Young-Jin (Department of Medicinal Biosciences, Konkuk University)
  • 투고 : 2021.12.03
  • 심사 : 2021.12.27
  • 발행 : 2021.12.31
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초록

본 연구에서는 Flammulina velutipes var. lupinicola의 laccase 유전자를 동정하고 최적 활성 pH, 온도, 시간을 분석하고 하였다. F. velutipes var. lupinicola 유전체에서 선별된 laccase 유전자 서열을 바탕으로 구리 결합 부위 및 신호 펩타이드 분석을 수행한 결과 5종의 laccase 유전자(g1934, g1937, g2415, g2539, g5858)를 동정하였다. 5종의 선별된 laccase 유전자 크기는 1,488~1,662 bp로 확인되었고, cDNA 염기서열 분석 결과 14~17개의 인트론이 확인되었다. Laccase 유전자의 신호펩타이드로 예측된 절단 부위는 N-말단으로부터 20~34 bp 사이에 위치하는 것으로 확인되었다. F. velutipes var. lupinicola laccase의 활성 특성을 규명하기 위해 분리 정제를 수행하였으며, Zymogram을 수행하여 0.2 M 및 0.3 M NaCl과 1.6 M 및 1.7 M의 ammonium sulfate로 정제된 단백질에서 5개의 laccsae 활성 밴드를 확인하였다. pH, 온도 및 시간별로 분리 정제된 단백질의 최적 활성을 분석한 결과, 반응의 최적 pH는 5.5이고 최적 온도는 40℃로 확인되었다. 따라서 본 연구를 통하여 확인된 F. velutipes var. lupinicola 유전체의 laccase 유전자 구조 및 활성에 대한 특성은 laccase를 이해하는 데 도움이 될 것이며 추가 연구를 통하여 향후 다양한 산업적 활용이 가능할 것으로 사료된다.

The purpose of this study was to identify and characterize the laccase genes of Flammulina velutipes var. lupinicola. Five laccase genes (g1934, g1937, g2415, g2539, g5858) were selected based on the copper binding site and signal peptide analysis results using the laccase gene selected from the F. velutipes var. lupinicola genome. The size of the laccase genes of F. velutipes var. lupinicola were 1,488 bp~1,662 bp. As a result of cDNA sequence analysis, 14 to 17 introns were identified in the laccase genes. The cleavage site predicted as the signal peptide of the laccase gene was found to be located between 20 bp and 34 bp from the N-terminus. In addition, separation and purification were performed to characterize the F. velutipes var. lupinicola laccases, and the optimal activity of the separated and purified proteins were analyzed by pH, temperature and time. Five bands with laccase activity were found from zymogram analysis. The optimal pH of the reaction was 5.5, the optimal temperature was found to be 40℃. Therefore, characterization of the laccase genes identified in this study should help in better understanding the biomass decomposition of F. velutipes var. lupinicola.

키워드

과제정보

이 논문은 2021년도 건국대학교 KU학술연구비 지원에 의한 결과임.

참고문헌

  1. Altschul SF and Erickson BW. 1985. Significance of nucleotide sequence alignments: a method for random sequence permutation that preserves dinucleotide and codon usage. Mol Biol Evol 2: 526-538.
  2. Baldrian P. 2006. Fungal laccase: occurrence and properties. FEMS Microbiol Rev 30: 215-242. https://doi.org/10.1111/j.1574-4976.2005.00010.x
  3. Bento I, Carrondo MA, Lindley PF. 2006. Reduction of dioxygen by enzymes containing copper. J Biol Inorg Chem 11: 539-547. https://doi.org/10.1007/s00775-006-0114-9
  4. Bolger A.M, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120. https://doi.org/10.1093/bioinformatics/btu170
  5. Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C. 2002. Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41: 7325-7333. https://doi.org/10.1021/bi0201318
  6. Buchfink B, Xie C, Huson D. 2015. Fast and sensitive protein alignment using DIAMOND Nat Methods 12: 59-60. https://doi.org/10.1038/nmeth.3176
  7. Cho NS, Chow HY, Shin SJ, Choi YJ, Leonowicz A, Ohga S. 2008. Production of Fungal Laccase and Its Immobilization and Stability. J Fac Agr 53: 13-18.
  8. Christopher FT. 1994. The structure and function of fungal laccase. Microbiology 140: 19-26. https://doi.org/10.1099/13500872-140-1-19
  9. Eduardo B, Liliana M, Juan MS. 2015. Inconsistencies and ambiguities in calculating enzyme activity: The case of laccase. J Microbiol Methods 119: 126-131. https://doi.org/10.1016/j.mimet.2015.10.007
  10. Edward IS, Uma MS, Timothy EM. 1996. Multicopper Oxidases and Oxygenases. Chem Rev 96: 2563-2606. https://doi.org/10.1021/cr950046o
  11. Eriksson K, Blanchette RA, Ander P. 1990. Morphological aspects of wood degradation by fungi and bacteria. In Microbial and Ezymatic Degradation of Wood and Wood Components. Germany: Spinge. ISBN 978-3-642-46687-8.
  12. Haberle V, Stark A. 2018. Eukaryotic core promoters and the functional basis of transcription initiation. Nat Rev Mol Cell Biol 19: 621-637. https://doi.org/10.1038/s41580-018-0028-8
  13. Kim HI, Kwon OC, Kong WS, Lee CS, Park YJ. 2014. Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes. Mycobiology 42: 322-330. https://doi.org/10.5941/MYCO.2014.42.4.322
  14. Kumar A, Singh D, Sharma KK, Arora S, Singh AK, Gill SS, Singhal B. 2017. Gel-Based Purification and Biochemical Study of Laccase Isozymes from Ganoderma sp. and Its Role in Enhanced Cotton Callogenesis. Front Microbiol 8: 674. https://doi.org/10.3389/fmicb.2017.00674
  15. Kumar SV, Phale PS, Durani S, Wangikar PP. 2003. Combined sequence and structure analysis of the fungal laccase family. Biotechnol Bioeng 83: 386-394. https://doi.org/10.1002/bit.10681
  16. Lee CS, Kong WS, Park YJ. 2018. Genome sequencing and genome-wide identification of carbohydrate-active enzymes (CAZymes) in the white rot fungus Flammulina fennae. Microbiol Biotechnol Lett 46: 300-312. https://doi.org/10.4014/mbl.1808.08012
  17. Lipman DJ, Wilbur WJ, Smith TF, Waterman MS. 1984. On the statistical significance of nucleic acid similarities. Nucleic Acids Res 12: 215-226. https://doi.org/10.1093/nar/12.1Part1.215
  18. Lin Y, Zhang Z, Tian Y, Zhao W, Zhu B, Xu Z, Peng R, Yao Q. 2013. Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes. Mol Biol Rep 40: 1487-1494. https://doi.org/10.1007/s11033-012-2191-x
  19. Lombard V, Golaconda Ramulu H, Drual E, Coutinho PM, Henrissat B. 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42: D490-D495. https://doi.org/10.1093/nar/gkt1178
  20. Ning YJ, Wang SS, Chen QJ, Ling ZR, Wang SN, Wang WP, Zhang GQ, Zhe MJ. 2016. An extracellular yellow laccase with potent dye decolorizing ability from the fungus Leucoagaricus naucinus LAC-04. Biological Mactromolecules 93: 837-842. https://doi.org/10.1016/j.ijbiomac.2016.09.046
  21. Palmer JM and Stajich JE. 2020. Funannotate: Eukaryotic Genome Annotation Pipeline. Available online: http://funannotate.readthedocs.io.
  22. Park YJ,Kong WS. 2018. Genome-wide comparison of carbohydrate-active enzymes (CAZymes) repertoire of Flammulina ononidis. Mycobiology 46: 349-360. https://doi.org/10.1080/12298093.2018.1537585
  23. Park YJ, Baek JH, Lee S, Kim C, Rhee H, Kim H, Seo JS, Park HR, Yoon DE, Nam JY, Kim HI, Kim JG, Yoon HJ, Kang HW, Cho JY, Song ES, Sung GH, Yoo YB, Lee CS. 2014. Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation. PLoS ONE 9: e93560. https://doi.org/10.1371/journal.pone.0093560
  24. Park YJ, Jeong YU, Kong WS. 2018. Genome sequencing and carbohydrate-active enzyme (CAZyme) repertoire of the white rot fungus Flammulina elastica. Int J Mol Sci 19: 2379. https://doi.org/10.3390/ijms19082379
  25. Petr B. 2006. Fungal laccases-occurrence and properties. FEMS Microbiol Rev 30: 215-242. https://doi.org/10.1111/j.1574-4976.2005.00010.x
  26. Redhead SA, Petersen RH. 1999. New species, varieties and combinations in the genus Flammulina. Mycotaxon 71: 285-294.
  27. Rytioja J, Hilden K, Yuzon J, Hatakka A, de Vries RP, Makela MR. 2014. Plant-polysaccharide-degrading enzymes from basidiomycetes. Microbiol Mol Biol Rev 78: 614-649. https://doi.org/10.1128/MMBR.00035-14
  28. Scott CD, Davison BH, Scott TC, Woodward J, Dees C, Rothrock DS. 1994. An advanced bioprocessing concept for the conversion of waste paper to ethanol. Appl Biochem Biotechnol 45: 641-653. https://doi.org/10.1007/BF02941836
  29. Simpson JT, Kim W, Shaun DJ, Jacqueline ES, Steven JMJ, Inanc B. 2009. ABySS: a parallel assembler for short read sequence data. Genome Res 19: 1117-1123. https://doi.org/10.1101/gr.089532.108
  30. Sista Kameshwar AK, Qin W. 2018. Comparative study of genome-wide plant biomass-degrading CAZymes in white rot, brown rot and soft rot fungi. Mycology 9: 93-105. https://doi.org/10.1080/21501203.2017.1419296
  31. Smale ST, Kadonaga JT. 2003. The RNA polymerase II core promoter. Annu Rev Biochem 72: 449-479. https://doi.org/10.1146/annurev.biochem.72.121801.161520
  32. Strange RW, Reinhammer B, Murphy LM, Hasnain SS. 1995. Structural and spectroscopic studies of the copper site of stellacyanin. Biochemistry 34: 220-231. https://doi.org/10.1021/bi00001a026
  33. Sun J, Chen QJ, Cao QQ, Wu YY, Xu LJ, Zhu MJ, Ng TB, Wang HX, Zhang GQ. 2012. A laccase with antiproliferative and HIV-I reverse transcriptase inhibitory activities from the mycorrhizal fungus agaricus placomyces. J Biopmed Biotech 2012: 736472.
  34. Sun J, Chen QJ, Zhu MJ, Wang HX, Zhang GQ. 2014. An extracellular laccase with antiproliferative activity from the sanghuang mushroom Inonotus baumii. J Biopmed Biotech 99: 20-25.
  35. Tien M, Kirk TK. 1988. Lignin peroxidase of Phanerochaete chtysosporium. Method Enzymol 46: 220-225.
  36. Tomoko M, Hitomi I, TaKanori F, Wataru O, Koji T, Satoshi K. 2013. Ethanol prodution from high cellulose concentration by the basidiomycete fungus Flammulina velutipes. Fungal Biol 117: 220-226. https://doi.org/10.1016/j.funbio.2013.02.002
  37. Upadhyay P, Shrivastava R, Agrawal PK. 2016. Bioprospecting and biotechnological applications of fungal laccase. 3 Biotech 6(1): 15. https://doi.org/10.1007/s13205-015-0316-3
  38. Zhang H, Zhang Y, Huang F, Gao P, Chen J. 2009. Purification and characterization of a thermostable laccase with unique oxidative characterisitcs from Trametes hirusta. Biotechnol Letters 31: 837-843. https://doi.org/10.1007/s10529-009-9945-0
  39. Zhou YJ, Wang HX, Ng TB, Huang CY, Zhang JX. 2012. Purification and characterization of a novel laccase from the edible mushroom Hericium coralloides. J Microbiol 50: 72-78. https://doi.org/10.1007/s12275-012-1372-6