Molecular cloning, expression and characterization of poxa1b gene from Pleurotus ostreatus

  • Mahnaz Mohtashami
  • Jamshid Fooladi
  • Aliakbar Haddad-Mashadrizeh
  • Mohammadreza Housaindokht
Original Article


In recent decades, fungus laccases (p-diphenol-dioxygen oxidoreductases; EC have attracted the attention of researches due to their wide range of biotechnological and industrial applications. In the present study, we have cloned a gene encoding laccase (poxa1b) from Pleurotus ostreatus and then heterologously expressed in Escherichia coli BL21. The biochemical properties of POXA1b were characterized using ABTS as a typical substrate of laccases. Moreover, the in vitro oxidation of the benzo[a]pyrene was investigated in the presence or absence of ABTS. The codon-optimized poxa1b showed higher expression yields and efficiency in comparison with the wild-type (p < 0.01). The maximum activity of POXA1b (2075 UL-1) was observed after incubation at 50 °C for 0.5 h and the enzyme retained more than 85% of its initial activity after 2 h incubation at 25–45 °C. The optimum pH of the enzyme was pH4 and the enzyme was stable when being incubated at pH range from 2.5 to 4.5 for 2 h in the absence of ABTS, the enzyme oxidized a little amount of benzo[a]pyrene, whereas its oxidation enhanced following the ABTS addition. These findings indicate POXA1b of P. ostreatus as a promising candidate for further biotechnological approaches.


Laccases POXA1b Codon optimization Cloning and expression ABTS Benzo[a]pyrene 



The authors wish to thank Khayam Bioeconomy Institute (KBI) and Alzahra University for supporting this work. This publication represents a section of one PhD thesis by Ms. Mahnaz Mohtashami at the faculty of Biological Science of Alzahra University, Tehran, Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.


  1. 1.
    Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140(1):19–26Google Scholar
  2. 2.
    Jeon JR, Baldrian P, Murugesan K, Chang YS (2012) Laccase-catalysed oxidations of naturally occurring phenols: from in vivo biosynthetic pathways to green synthetic applications. Microb Biotechnol 5(3):318–332PubMedPubMedCentralGoogle Scholar
  3. 3.
    Mate D, García-Burgos C, García-Ruiz E, Ballesteros AO, Camarero S, Alcalde M (2010) Laboratory evolution of high-redox potential laccases. Chem Biol 17(9):1030–1041PubMedGoogle Scholar
  4. 4.
    Piscitelli A, Pezzella C, Giardina P, Faraco V, Sannia G (2010) Heterologous laccase production and its role in industrial applications. Bioengineered Bugs 1(4):254–264Google Scholar
  5. 5.
    Upadhyay P, Shrivastava R, Agrawal PK (2016) Bioprospecting and biotechnological applications of fungal laccase. 3 Biotech 6(1):15PubMedPubMedCentralGoogle Scholar
  6. 6.
    Mate DM, Alcalde M (2017) Laccase: a multi-purpose biocatalyst at the forefront of biotechnology. Microb Biotechnol 10(6):1457–1467PubMedGoogle Scholar
  7. 7.
    Piscitelli A, Pezzella C, Lettera V, Giardina P, Faraco V, Sannia G (2013) Fungal laccases: structure, function and applications. CRC Press, Boca RatonGoogle Scholar
  8. 8.
    Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 6(1):9PubMedPubMedCentralGoogle Scholar
  9. 9.
    Santhanam N, Vivanco JM, Decker SR, Reardon KF (2011) Expression of industrially relevant laccases: prokaryotic style. Trends Biotechnol 29(10):480–489PubMedGoogle Scholar
  10. 10.
    Pezzella C, Guarino L, Piscitelli A (2015) How to enjoy laccases. Cell Mol Life Sci 72(5):923–940PubMedGoogle Scholar
  11. 11.
    Macellaro G, Baratto MC, Piscitelli A, Pezzella C, De Biani FF, Palmese A, Piumi F, Record E, Basosi R, Sannia G (2014) Effective mutations in a high redox potential laccase from Pleurotus ostreatus. Appl Microbiol Biotechnol 98(11):4949–4961PubMedGoogle Scholar
  12. 12.
    Pezzella C, Giacobelli VG, Lettera V, Olivieri G, Cicatiello P, Sannia G, Piscitelli A (2017) A step forward in laccase exploitation: recombinant production and evaluation of techno-economic feasibility of the process. J Biotechnol 259:175–181PubMedGoogle Scholar
  13. 13.
    Cohen R, Persky L, Hadar Y (2002) Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol 58(5):582–594PubMedGoogle Scholar
  14. 14.
    Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, Sannia G (1999) Protein and gene structure of a blue laccase from Pleurotus ostreatus1. Biochem J 341(Pt 3):655PubMedPubMedCentralGoogle Scholar
  15. 15.
    Garzillo AM, Colao MC, Buonocore V, Oliva R, Falcigno L, Saviano M, Santoro AM, Zappala R, Bonomo RP, Bianco C (2001) Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii. J Protein Chem 20(3):191–201PubMedGoogle Scholar
  16. 16.
    Piscitelli A, Giardina P, Mazzoni C, Sannia G (2005) Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae. Appl Microbiol Biotechnol 69(4):428–439PubMedGoogle Scholar
  17. 17.
    Pezzella C, Giacobbe S, Giacobelli VG, Guarino L, Kylic S, Sener M, Sannia G, Piscitelli A (2016) Green routes towards industrial textile dyeing: a laccase based approach. J Mol Catal B 134:274–279Google Scholar
  18. 18.
    Miele A, Giardina P, Sannia G, Faraco V (2010) Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation. J Appl Microbiol 108(3):998–1006PubMedGoogle Scholar
  19. 19.
    Lettera V, Pezzella C, Cicatiello P, Piscitelli A, Giacobelli VG, Galano E, Amoresano A, Sannia G (2016) Efficient immobilization of a fungal laccase and its exploitation in fruit juice clarification. Food Chem 196:1272–1278PubMedGoogle Scholar
  20. 20.
    Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72(2):211PubMedGoogle Scholar
  21. 21.
    Swartz J (1996) Escherichia coli recombinant DNA technology. In: Escherichia coli and Salmonella: cellular and molecular biology, vol 2. ASM Press, Washington, DC, pp 1693–1711Google Scholar
  22. 22.
    Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O (2008) Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 59(1):94–102PubMedGoogle Scholar
  23. 23.
    Wu X, Jörnvall H, Berndt KD, Oppermann U (2004) Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance. Biochem Biophys Res Commun 313(1):89–96PubMedGoogle Scholar
  24. 24.
    Montazeri-Najafabady N, Ghasemi Y, Mobasher MA, Ghasemian A, Rasoul-Amini S, Ebrahimi S (2013) Codon optimization, cloning and expression of interleukin 11 in two different E. coli systems. J Pure Appl Microbiol 7:2717–2722Google Scholar
  25. 25.
    Yadava A, Ockenhouse CF (2003) Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 71(9):4961–4969PubMedPubMedCentralGoogle Scholar
  26. 26.
    Tokuoka M, Tanaka M, Ono K, Takagi S, Shintani T, Gomi K (2008) Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl Environ Microbiol 74(21):6538–6546PubMedPubMedCentralGoogle Scholar
  27. 27.
    Fei D, Zhang H, Diao Q, Jiang L, Wang Q, Zhong Y, Fan Z, Ma M (2015) Codon optimization, expression in Escherichia coli, and immunogenicity of recombinant chinese sacbrood virus (CSBV) structural proteins VP1, VP2, and VP3. PLoS ONE 10(6):e0128486PubMedPubMedCentralGoogle Scholar
  28. 28.
    Zhou Z, Schnake P, Xiao L, Lal AA (2004) Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization. Protein Expr Purif 34(1):87–94PubMedGoogle Scholar
  29. 29.
    Zylicz-Stachula A, Zolnierkiewicz O, Sliwinska K, Jezewska-Frackowiak J, Skowron PM (2014) Modified ‘one amino acid-one codon’engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase. Microb Cell Fact 13(1):7PubMedPubMedCentralGoogle Scholar
  30. 30.
    Guan Z-B, Song C-M, Zhang N, Zhou W, Xu C-W, Zhou L-X, Zhao H, Cai Y-J, Liao X-R (2014) Overexpression, characterization, and dye-decolorizing ability of a thermostable, pH-stable, and organic solvent-tolerant laccase from Bacillus pumilus W3. J Mol Catal B 101:1–6Google Scholar
  31. 31.
    More SS, PS R, Malini S (2011) Isolation, purification, and characterization of fungal laccase from Pleurotus sp. Enzyme Res 2011:248735PubMedPubMedCentralGoogle Scholar
  32. 32.
    Linke D, Bouws H, Peters T, Nimtz M, Berger RG, Zorn H (2005) Laccases of Pleurotus sapidus: characterization and cloning. J Agric Food Chem 53(24):9498–9505PubMedGoogle Scholar
  33. 33.
    Osma JF, Toca-Herrera JL, Rodríguez-Couto S (2010) Biodegradation of a simulated textile effluent by immobilised-coated laccase in laboratory-scale reactors. Appl Catal A 373(1–2):147–153Google Scholar
  34. 34.
    Bhattacharya S, Das A, Palaniswamy M, Angayarkanni J (2017) Degradation of benzo[a]pyrene by Pleurotus ostreatus PO-3 in the presence of defined fungal and bacterial co-cultures. J Basic Microbiol 57(2):95–103PubMedGoogle Scholar
  35. 35.
    Othman AM, Elshafei AM, Hassan MM, Haroun BM, Elsayed MA, Farrag AA (2014) Purification, biochemical characterization and applications of Pleurotus ostreatus ARC280 laccase. Br Microbiol Res J 4(12):1418–1439Google Scholar
  36. 36.
    Xiao Y, Tu X, Wang J, Zhang M, Cheng Q, Zeng W, Shi Y (2003) Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. Appl Microbiol Biotechnol 60(6):700–707PubMedGoogle Scholar
  37. 37.
    Petroski RJ, Peczynska-Czoch W, Rosazza JP (1980) Analysis, production, and isolation of an extracellular laccase from Polyporus anceps. Appl Environ Microbiol 40(6):1003–1006PubMedPubMedCentralGoogle Scholar
  38. 38.
    Mansur M, Arias ME, Copa-Patino JL, Flärdh M, González AE (2003) The white-rot fungus Pleurotus ostreatus secretes laccase isozymes with different substrate specificities. Mycologia 95(6):1013–1020PubMedGoogle Scholar
  39. 39.
    Sadhasivam S, Savitha S, Swaminathan K, Lin F-H (2008) Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem 43(7):736–742Google Scholar
  40. 40.
    Forootanfar H, Faramarzi MA, Shahverdi AR, Yazdi MT (2011) Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile. Biores Technol 102(2):1808–1814Google Scholar
  41. 41.
    Younes SB, Sayadi S (2011) Purification and characterization of a novel trimeric and thermotolerant laccase produced from the ascomycete Scytalidium thermophilum strain. J Mol Catal B 73(1–4):35–42Google Scholar
  42. 42.
    Jung S, Park S (2008) Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis. Biotechnol Lett 30(4):717–722PubMedGoogle Scholar
  43. 43.
    Ng I-S, Zhang X, Zhang Y, Lu Y (2013) Molecular cloning and heterologous expression of laccase from Aeromonas hydrophila NIU01 in Escherichia coli with parameters optimization in production. Appl Biochem Biotechnol 169(7):2223–2235PubMedGoogle Scholar
  44. 44.
    Grandes-Blanco AI, Tlecuitl-Beristain S, Díaz R, Sánchez C, Téllez-Téllez M, Márquez-Domínguez L, Santos-López G, Díaz-Godínez G (2017) Heterologous expression of laccase (LACP83) of Pleurotus ostreatus. BioResources 12 (2):3211–3221Google Scholar
  45. 45.
    Ihssen J, Reiss R, Luchsinger R, Thöny-Meyer L, Richter M (2015) Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli. Sci Rep 5:10465PubMedPubMedCentralGoogle Scholar
  46. 46.
    Wang C, Cui D, Lu L, Zhang N, Yang H, Zhao M, Dai S (2016) Cloning and characterization of CotA laccase from Bacillus subtilis WD23 decoloring dyes. Ann Microbiol 66(1):461–467Google Scholar
  47. 47.
    Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G (1997) A novel white laccase from Pleurotus ostreatus. J Biol Chem 272(50):31301–31307PubMedGoogle Scholar
  48. 48.
    Patel H, Gupte S, Gahlout M, Gupte A (2014) Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1. 3 Biotech 4(1):77–84PubMedGoogle Scholar
  49. 49.
    Adamafio N, Sarpong N, Mensah C, Obodai M (2012) Extracellular laccase from Pleurotus ostreatus strain EM-1: thermal stability and response to metal ions. Asian J Biochem 7:143–150Google Scholar
  50. 50.
    Palmeiri G, Giardina P, Marzullo L, Desiderio B, Nittii G, Cannio R, Sannia G (1993) Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus. Appl Microbiol Biotechnol 39(4–5):632–636Google Scholar
  51. 51.
    Tinoco R, Pickard M, Vazquez-Duhalt R (2001) Kinetic differences of purified laccases from six. Lett Appl Microbiol 32:331–335PubMedGoogle Scholar
  52. 52.
    Xu L, Wang H, Ng T (2012) A laccase with HIV-1 reverse transcriptase inhibitory activity from the broth of mycelial culture of the mushroom Lentinus tigrinus. BioMed Res Int. PubMedPubMedCentralGoogle Scholar
  53. 53.
    Guo W, Yao Z, Zhou C, Li D, Chen H, Shao Q, Li Z, Feng H (2012) Purification and characterization of three laccase isozymes from the white rot fungus Trametes sp. HS-03. Afr J Biotech 11(31):7916–7922Google Scholar
  54. 54.
    Zou Y-J, Wang H-X, Ng T-B, Huang C-Y, Zhang J-X (2012) Purification and characterization of a novel laccase from the edible mushroom Hericium coralloides. J Microbiol 50(1):72–78PubMedGoogle Scholar
  55. 55.
    Asgher M, Iqbal HMN, Asad MJ (2012) Kinetic characterization of purified laccase produced from Trametes versicolor IBL-04 in solid state bio-processing of corncobs. BioResources 7(1):1171–1188Google Scholar
  56. 56.
    Yoshitake A, Katayama Y, Nakamura M, Iimura Y, Kawai S, Morohoshi N (1993) N-linked carbohydrate chains protect laccase III from proteolysis in Coriolus versicolor. Microbiology 139(1):179–185Google Scholar
  57. 57.
    Soden D, O’Callaghan J, Dobson A (2001) Molecular cloning of a laccase isozyme gene in the heterologous Pichia pastoris host. Microbiology 148:4003–4014Google Scholar
  58. 58.
    Madhavi V, Lele S (2009) Laccase: properties and applications. BioResources 4(4):1694–1717Google Scholar
  59. 59.
    Baldrian P (2006) Fungal laccases–occurrence and properties. FEMS Microbiol Rev 30(2):215–242PubMedGoogle Scholar
  60. 60.
    Pozdnyakova N, Turkovskaya O, Yudina E, Rodakiewicz-Nowak Y (2006) Yellow laccase from the fungus Pleurotus ostreatus D1: purification and characterization. Appl Biochem Microbiol 42(1):56–61Google Scholar
  61. 61.
    Hu X, Wang C, Wang L, Zhang R, Chen H (2014) Influence of temperature, pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis. World J Microbiol Biotechnol 30(4):1285–1290PubMedGoogle Scholar
  62. 62.
    Ike PTL, Moreira AC, de Almeida FG, Ferreira D, Birolli WG, Porto ALM, Souza DHF (2015) Functional characterization of a yellow laccase from Leucoagaricus gongylophorus. SpringerPlus 4(1):654PubMedPubMedCentralGoogle Scholar
  63. 63.
    Daroch M, Houghton CA, Moore JK, Wilkinson MC, Carnell AJ, Bates AD, Iwanejko LA (2014) Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa. Enzyme Microb Technol 58:1–7PubMedGoogle Scholar
  64. 64.
    Haibo Z, Yinglong Z, Feng H, Peiji G, Jiachuan C (2009) Purification and characterization of a thermostable laccase with unique oxidative characteristics from Trametes hirsuta. Biotechnol Lett 31(6):837–843PubMedGoogle Scholar
  65. 65.
    Tamayo-Ramos J, van Berkel WJ, de Graaff LH (2012) Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger. Microb Cell Fact 11(1):165PubMedPubMedCentralGoogle Scholar
  66. 66.
    Mukhopadhyay M, Banerjee R (2015) Purification and biochemical characterization of a newly produced yellow laccase from Lentinus squarrosulus MR13. 3 Biotech 5(3):227–236PubMedGoogle Scholar
  67. 67.
    Juhasz AL, Naidu R (2000) Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene. Int Biodeterior Biodegrad 45(1–2):57–88Google Scholar
  68. 68.
    Morozova O, Shumakovich G, Shleev S, Yaropolov YI (2007) Laccase-mediator systems and their applications: a review. Appl Biochem Microbiol 43(5):523–535Google Scholar
  69. 69.
    Bhattacharya S, Das A, Prashanthi K, Palaniswamy M, Angayarkanni J (2014) Mycoremediation of benzo[a]pyrene by Pleurotus ostreatus in the presence of heavy metals and mediators. 3 Biotech 4(2):205–211PubMedGoogle Scholar
  70. 70.
    Dodor DE, Hwang H-M, Ekunwe SI (2004) Oxidation of anthracene and benzo [a] pyrene by immobilized laccase from Trametes versicolor. Enzyme Microb Technol 35(2–3):210–217Google Scholar
  71. 71.
    Collins PJ, Kotterman M, Field JA, Dobson A (1996) Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor. Appl Environ Microbiol 62(12):4563–4567PubMedPubMedCentralGoogle Scholar
  72. 72.
    Majcherczyk A, Johannes C, Hüttermann A (1998) Oxidation of polycyclic aromatic hydrocarbons (PAH) by laccase of Trametes versicolor. Enzyme Microb Technol 22(5):335–341Google Scholar
  73. 73.
    Potthast A, Rosenau T, Chen C-L, Gratzl JS (1995) Selective enzymic oxidation of aromatic methyl groups to aldehydes. J Organ Chem 60(14):4320–4321Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Mahnaz Mohtashami
    • 1
  • Jamshid Fooladi
    • 2
  • Aliakbar Haddad-Mashadrizeh
    • 3
    • 4
  • Mohammadreza Housaindokht
    • 5
  1. 1.Department of Microbiology, Faculty of Biological ScienceAlzahra UniversityTehranIran
  2. 2.Department of Biotechnology, Faculty of Biological ScienceAlzahra UniversityTehranIran
  3. 3.Recombinant Proteins Research Group, Institute of BiotechnologyFerdowsi University of MashhadMashhadIran
  4. 4.Department of Biology, Faculty of SciencesFerdowsi University of MashhadMashhadIran
  5. 5.Department of Chemistry, Faculty of SciencesFerdowsi University of MashhadMashhadIran

Personalised recommendations