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PLOS ONE  2014 

Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus

DOI: 10.1371/journal.pone.0093912

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Abstract:

The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser?)tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.

References

[1]  Baldrian P (2006) Fungal laccases-occurrence and properties. FEMS Microbiol Rev 30: 215–242. doi: 10.1111/j.1574-4976.2005.00010.x
[2]  Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60: 551–565. doi: 10.1016/s0031-9422(02)00171-1
[3]  Kunamneni A, Camarero S, García-Burgos C, Plou FJ, Ballesteros A, et al. (2008) Engineering and applications of fungal laccases for organic synthesis. Microb Cell Fact 7: 32. doi: 10.1186/1475-2859-7-32
[4]  Polak J, Jarosz-Wilkolazka A (2012) Structure/redox potential relationship of simple organic compounds as potential precursors of dyes for laccase-mediated transformation. Biotechnol Prog 8: 93–102. doi: 10.1002/btpr.713
[5]  Herter S, Michalik D, Mikolasch A, Schmidt M, Wohlgemuth R, et al. (2013) Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and 3-methyl-2,5-bis(alkylamino)-[1,4]-benzo?quinones. J Molecular Catalysis B: Enzymatic 90: 91–97. doi: 10.1016/j.molcatb.2013.01.020
[6]  Kües U, Rühl M (2011) Multiple multicopper oxidase gene families in basidiomycetes - what for? Curr Genomics 12: 72–94. doi: 10.2174/138920211795564377
[7]  Kilaru S, Hoegger PJ, Kues U (2006) The laccase multigene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies. Curr Genet 50: 45–60. doi: 10.1007/s00294-006-0074-1
[8]  Bleve G, Lezzi C, Mita G, Rampino P, Perrotta C, et al. (2008) Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells. Appl Microbiol Biotechnol 79: 731–41. doi: 10.1007/s00253-008-1479-1
[9]  Hildén K, M?kel? MR, Lundell T, Kuuskeri J, Chernykh A, et al. (2013) Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 97: 1589–1599. doi: 10.1007/s00253-012-4011-6
[10]  Kiiskinen LL, Kruus K, Bailey M, Yl?sm?ki E, Siika-Aho M, et al. (2004) Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology 150 (Pt 9): 3065–74. doi: 10.1099/mic.0.27147-0
[11]  Rodríguez E, Ruiz-Due?s FJ, Kooistra R, Ram A, Martínez AT, et al. (2008) Isolation of two laccase genes from the white-rot fungus Pleurotus eryngii and heterologous expression of the pel3 encoded protein. J Biotechnol 134: 9–19. doi: 10.1016/j.jbiotec.2007.12.008
[12]  Yano A, Kikuchi S, Nakagawa Y, Sakamoto Y, Sato T (2009) Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae. Microbiol Res 164: 642–9. doi: 10.1016/j.micres.2008.12.001
[13]  Koschorreck K, Richter SM, Swierczek A, Beifuss U, Schmid RD, et al. (2008) Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs. Arch Biochem Biophys 474: 213–9. doi: 10.1016/j.abb.2008.03.009
[14]  Piscitelli A, Pezzella C, Giardina P, Faraco V, Giovanni S (2010) Heterologous laccase production and its role in industrial applications. Bioeng Bugs 1: 252–62. doi: 10.4161/bbug.1.4.11438
[15]  Wong KS, Cheung MK, Au CH, Kwan HS (2013) A novel Lentinula edodes laccase and its comparative enzymology suggest guaiacol-based laccase engineering for bioremediation. PLoS One 8: e66426. doi: 10.1371/journal.pone.0066426
[16]  Bao S, Teng Z, Ding S (2013) Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus.Mol Biol Rep. 40: 1927–36. doi: 10.1007/s11033-012-2249-9
[17]  Lu X, Ding SJ (2010) Effect of Cu2+, Mn2+ and aromatic compounds on the production of laccase isoforms by Coprinus comatus. Mycoscience 51: 68–74. doi: 10.1007/s10267-009-0002-6
[18]  Hoshida H, Nakao M, Kanazawa H, Kubo K, Hakukawa T, et al. (2001) Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts.J Biosci Bioeng. 92: 372–80. doi: 10.1263/jbb.92.372
[19]  Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599. doi: 10.1093/molbev/msm092
[20]  Zheng F, Huang J, Yin Y, Ding S (2013) A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase. J Ind Microbiol Biotechnol 40: 1083–1093. doi: 10.1007/s10295-013-1312-4
[21]  Childs RE, Bardsley WG (1975) The steady-state kinetics of peroxidase with 2,2′-azino-di-(3-ethyl-benzthiazoline-6-?sulphonicacid) as chromogen. Biochem J 145: 93–103.
[22]  Canters GW, Gilardi G (1993) Engineering type 1 copper sites in proteins. FEBS Lett 325: 39–48. doi: 10.1016/0014-5793(93)81410-2
[23]  Piontek K, Antorini M, Choinowski T (2002) Crystal structure of a laccase from the fungus Trametes versicolor at 0.90 A° resolution containing a full complement of coppers. J Biol Chem 277: 37663–37669. doi: 10.1074/jbc.m204571200
[24]  Koikeda S, Ando K, Kaji H, Inoue T, Murao S, et al. (1993) Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast. J Biol Chem 268: 18801–9.
[25]  Christenson A, Shleev S, Mano N, Heller A, Gorton L (2006) Redox potentials of the blue copper sites of bilirubin oxidases. Biochim Biophys Acta 1757: 1634–41. doi: 10.1016/j.bbabio.2006.08.008
[26]  Uthandi S, Saad B, Humbard MA, Maupin-Furlow JA (2010) LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii. Appl Environ Microbiol 76: 733–43. doi: 10.1128/aem.01757-09
[27]  Klonowska A, Gaudin C, Fournel A, Asso M, Le Petit J, et al. (2002) Characterization of a low redox potential laccase from the basidiomycete C30. Eur J Biochem 269: 6119–25. doi: 10.1046/j.1432-1033.2002.03324.x
[28]  Klonowska A, Gaudin C, Asso M, Fournel A, Reglier M, et al. (2005) LAC3, a new low redox potential laccase from Trametes sp. strain C30 obtained as a recombinant protein in yeast. Enzyme Microb Technol 36: 34–41. doi: 10.1016/j.enzmictec.2004.03.022
[29]  Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, et al. (2005) Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol 66: 450–456. doi: 10.1007/s00253-004-1717-0
[30]  Guo M, Lu FP, Du LX, Pu J, Bai DQ (2006) Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica. Appl Microbiol Biotechnol 71: 848–52. doi: 10.1007/s00253-005-0210-8
[31]  Brown MA, Zhao Z, Mauk AG (2002) Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor. Inorg Chim Acta 331: 232–8. doi: 10.1016/s0020-1693(01)00814-3
[32]  Cassland P, Jonsson LJ (1999) Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature. Appl Microbiol Biotechnol 52: 393–400. doi: 10.1007/s002530051537
[33]  Bohlin C, J?nsson LJ, Roth R, van Zyl WH (2006) Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger. Appl Biochem Biotechnol 129–132: 195–214. doi: 10.1385/abab:129:1:195
[34]  Baker CJO, White TC (2000) Expression of the laccase IV gene from Trametes versicolor in Trichoderma reesei. Abst Papers Am Chem Soc 219: 154.
[35]  Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system.Yeast. 22: 249–70.
[36]  Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, et al. (2003) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol 69: 987–95. doi: 10.1128/aem.69.2.987-995.2003
[37]  Bai Y, Wang J, Zhang Z, Shi P, Luo H, et al. (2010) Expression of an extremely acidic beta-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence. Microb Cell Fact 9: 33. doi: 10.1186/1475-2859-9-33
[38]  Andberg M, Hakulinen N, Auer S, Saloheimo M, Koivula A, et al. (2009) Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure. FEBS J 276: 6285–300. doi: 10.1111/j.1742-4658.2009.07336.x
[39]  Zhao D, Zhang X, Cui D, Zhao M (2012) Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes. PLoS One 7(6): e38817. doi: 10.1371/journal.pone.0038817
[40]  Lin Y, Zhang Z, Tian Y, Zhao W, Zhu B, et al. (2013) Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes. Mol Biol Rep 40: 1487–1494. doi: 10.1007/s11033-012-2191-x
[41]  Murugesan K, Yang IH, Kim YM, Jeon JR, Chang YS (2009) Enhanced transformation of malachite green by laccase of Ganoderma lucidum in the presence of natural phenolic compounds. Appl Microbiol Biotechnol 82: 341–350. doi: 10.1007/s00253-008-1819-1
[42]  Yan KL, Wang HX, Zhang XY, Yu HB (2009) Bioprocess of triphenylmethane dyes decolorization by Pleurotus ostreatus BP under solid-state cultivation. J Microbiol Biotechnol 19: 1421–1430. doi: 10.4014/jmb.0901.0033
[43]  Ciullini I, Tilli S, Scozzafava A, Briganti F (2008) Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes. Bioresour Technol 99: 7003–10. doi: 10.1016/j.biortech.2008.01.019
[44]  Kokol V, Doliska A, Eichlerova I, Baldrian P, Nerud F (2007) Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase. Enzyme Microb Technol 40: 1673–1677. doi: 10.1016/j.enzmictec.2006.08.015

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