Journal of Materials Science

, Volume 54, Issue 6, pp 4970–4983 | Cite as

Bioinformatics and enzymatics investigation of Trametes laccase for optical biosensing application

  • Yang WangEmail author
  • Zhi-Hui ChenEmail author
Materials for life sciences


Laccase has recently drawn a considerable attention as a promising tool for optical biosensing application, mainly due to its attractive intrinsic optical properties. The discovery of a laccase with great enzyme stability is the principal step for enzyme-based optical biosensing application in in vitro environments. In this paper, we found that Trametes sp. SQ1 laccase retained more than 150% activity after storage in different conditions for 96 h, and the laccase activity was not affected by freeze–thaw. The reduced form of enzyme showed a new maximal increase in absorbance peak at 400 nm, while the fluorescence intensity of the oxidized form of enzyme was much stronger than that of the reduced enzyme. Therefore, Trametes sp. SQ1 laccase with good stability and optical properties will be a competitive candidate for optical biosensing application. Moreover, bioinformatics analysis on Trametes laccases revealed that the enzymes may be modified by N-glycosylation and have intermolecular disulfide bond for forming functional oligomer, which plays a role in stabilizing enzyme function. The results indicate that Trametes laccases are able to be with good stability. It will provide a guide in searching stable laccases for optical biosensing application.



This work was supported by the National Natural Science Foundation of China (31640019, 11674239 and 21601112) and Program for the Top Young Talents of Shanxi Province, China. The authors thank Prof. X. Yang for Trametes sp. SQ01.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Larkin J, Henley RY, Jadhav V, Korlach J, Wanunu M (2017) Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing. Nat Nano.
  2. 2.
    Wilson GS, Gifford R (2005) Biosensors for real-time in vivo measurements. Biosens Bioelectron 20:2388–2403Google Scholar
  3. 3.
    Li Z, Zheng Y, Gao T, Liu Z, Zhang J, Zhou G (2018) Fabrication of biosensor based on core–shell and large void structured magnetic mesoporous microspheres immobilized with laccase for dopamine detection. J Mater Sci 53:7996–8008. Google Scholar
  4. 4.
    Ruggeri FS, Mahul-Mellier A-L, Kasas S, Lashuel HA, Longo G, Dietler G (2017) Amyloid single-cell cytotoxicity assays by nanomotion detection. 3: 17053.
  5. 5.
    Li L, Shi Y, Pan L, Shi Y, Yu G (2015) Rational design and applications of conducting polymer hydrogels as electrochemical biosensors. J Mater Chem B 3:2920–2930Google Scholar
  6. 6.
    Bollella P, Gorton L (2018) Enzyme based amperometric biosensors. Curr Opin Electrochem 10:157–173. Google Scholar
  7. 7.
    Richardson DS, Gregor C, Winter FR, Urban NT, Sahl SJ, Willig KI, Hell SW (2017) SRpHi ratiometric pH biosensors for super-resolution microscopy. Nat Commun 8:577. Google Scholar
  8. 8.
    Akdoğan E, Mutlu M (2010) Basic principles of optical biosensors in food engineering. In: Biosensors in food processing, safety, and quality control. CRC Press, Boca Raton, pp 53–70Google Scholar
  9. 9.
    Yu Z, Li W, Pan L, Zhai D, Yu W, Li L, Wen C, Wei Y, Wang X, Xu JB (2016) ZnO-nanorods/graphene heterostructure: a direct electron transfer glucose biosensor. Sci Rep 6:32327Google Scholar
  10. 10.
    Rakow NA, Suslick KS (2000) A colorimetric sensor array for odour visualization. Nature 406: 710–713.
  11. 11.
    Wang J, Qiu J (2016) A review of carbon dots in biological applications. J Mater Sci 51:4728–4738. Google Scholar
  12. 12.
    Raja MMM, Raja A, Imran MM, Santha AMI (2011) Enzymes application in diagnostic prospects. Biotechnology 10:51–59Google Scholar
  13. 13.
    Feng L, Zhu A, Shi H (2013) Recent advances in optical biosensors for environmental monitoring and early warning. Sensors 13:13928–13948Google Scholar
  14. 14.
    D’Souza SF (2001) Immobilization and stabilization of biomaterials for biosensor applications. Appl Biochem Biotechnol 96:225–238. Google Scholar
  15. 15.
    Chien P-J, Ye M, Suzuki T, Toma K, Arakawa T, Iwasaki Y, Mitsubayashi K (2016) Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH). Talanta 159:418–424Google Scholar
  16. 16.
    Pahurkar V, Tamgadge Y, Gambhire A, Muley G (2015) Glucose oxidase immobilized PANI cladding modified fiber optic intrinsic biosensor for detection of glucose. Sens Actuators B Chem 210:362–368Google Scholar
  17. 17.
    Mohammad R, Ahmad M, Heng LY (2014) Chilli hotness determination based on optical capsaicin biosensor using stacked immobilisation technique. Sens Actuators B Chem 190:593–600Google Scholar
  18. 18.
    Abdullah J, Ahmad M, Heng LY, Karuppiah N, Sidek H (2007) An optical biosensor based on immobilization of laccase and MBTH in stacked films for the detection of catechol. Sensors 7:2238–2250Google Scholar
  19. 19.
    George M, Mussone PG, Alemaskin K, Chae M, Wolodko J, Bressler DC (2016) Enzymatically treated natural fibres as reinforcing agents for biocomposite material: mechanical, thermal, and moisture absorption characterization. J Mater Sci 51:2677–2686. Google Scholar
  20. 20.
    Pecht I, Faraggi M (1971) Reduction of copper (II) in fungal laccase by hydrated electrons. Nature 233:116–118Google Scholar
  21. 21.
    Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26Google Scholar
  22. 22.
    Solomon EI, Sundaram UM, Machonkin TE (1996) Multicopper oxidases and oxygenases. Chem Rev 96:2563–2606. Google Scholar
  23. 23.
    Morozova OV, Shumakovich GP, Gorbacheva MA, Shleev SV, Yaropolov AI (2007) “Blue” laccases. Biochemistry 72:1136–1150. Google Scholar
  24. 24.
    Shleev S, Tkac J, Christenson A, Ruzgas T, Yaropolov AI, Whittaker JW, Gorton L (2005) Direct electron transfer between copper-containing proteins and electrodes. Biosens Bioelectron 20:2517–2554. Google Scholar
  25. 25.
    Miyazawa N, Tanaka M, Hakamada M, Mabuchi M (2017) Molecular dynamics study of laccase immobilized on self-assembled monolayer-modified Au. J Mater Sci 52:12848–12853. Google Scholar
  26. 26.
    Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226Google Scholar
  27. 27.
    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 41:7325–7333Google Scholar
  28. 28.
    Zoppellaro G, Sakurai T, H-w Huang (2001) A novel mixed valence form of Rhus vernicifera laccase and its reaction with dioxygen to give a peroxide intermediate bound to the trinuclear center. J Biochem 129:949–953Google Scholar
  29. 29.
    Sanz J, de Marcos S, Galbán J (2012) Autoindicating optical properties of laccase as the base of an optical biosensor film for phenol determination. Anal Bioanal Chem 404:351–359. Google Scholar
  30. 30.
    Huang J, Fang H, Liu C, Gu E, Jiang D (2008) A Novel Fiber Optic Biosensor for the Determination of Adrenaline Based on Immobilized Laccase Catalysis. Anal Lett 41:1430–1442. Google Scholar
  31. 31.
    Rodríguez-Delgado MM, Alemán-Nava GS, Rodríguez-Delgado JM, Dieck-Assad G, Martínez-Chapa SO, Barceló D, Parra R (2015) Laccase-based biosensors for detection of phenolic compounds. TrAC Trends Anal Chem 74:21–45Google Scholar
  32. 32.
    Bilir K, Weil M-T, Lochead J, Kok FN, Werner T (2012) Development of optical laccase-based biosensors for phenolic compound detection. New Biotechnol 29:S180. Google Scholar
  33. 33.
    Silva LIB, Ferreira FDP, Freitas AC, Rocha-Santos TAP, Duarte AC (2009) Optical fiber biosensor coupled to chromatographic separation for screening of dopamine, norepinephrine and epinephrine in human urine and plasma. Talanta 80:853–857. Google Scholar
  34. 34.
    Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform 5:113. Google Scholar
  35. 35.
    Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44:W242–W245. Google Scholar
  36. 36.
    Krieger E, Vriend G (2014) YASARA view—molecular graphics for all devices—from smartphones to workstations. Bioinformatics 30:2981–2982. Google Scholar
  37. 37.
    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. Google Scholar
  38. 38.
    Crooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190. Google Scholar
  39. 39.
    Nielsen H (2017) In: Kihara D (ed) Protein Function Prediction: Methods and Protocols. Springer, New YorkGoogle Scholar
  40. 40.
    Gupta R, Jung E, Brunak S (2004) Prediction of N-glycosylation sites in human proteins. Accessed 27 Mar 2018
  41. 41.
    Gasteiger E, Hoogland C, Gattiker A, Se Duvaud, Wilkins MR, Appel RD, Bairoch A (2005) In: Walker JM (ed) The proteomics protocols handbook. Humana Press, TotowaGoogle Scholar
  42. 42.
    McGinnis MR, D’Amato RF, Land GA (1982) Pictorial handbook of medically important fungi and aerobic actinomycetes. Praeger, New YorkGoogle Scholar
  43. 43.
    Kiiskinen LL, Rättö M, Kruus K (2004) Screening for novel laccase-producing microbes. J Appl Microbiol 97:640–646Google Scholar
  44. 44.
    Downes F (1992) Compendium of methods for the microbiological examination of foods. American Public Health Association, New YorkGoogle Scholar
  45. 45.
    Tlecuitl-Beristain S, Sánchez C, Loera O, Robson GD, Díaz-Godínez G (2008) Laccases of Pleurotus ostreatus observed at different phases of its growth in submerged fermentation: production of a novel laccase isoform. Mycol Res 112:1080–1084Google Scholar
  46. 46.
    Hou H, Zhou J, Wang J, Du C, Yan B (2004) Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochem 39:1415–1419Google Scholar
  47. 47.
    Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B (2013) Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels 6:41. Google Scholar
  48. 48.
    Madzak C, Mimmi MC, Caminade E, Brault A, Baumberger S, Briozzo P, Mougin C, Jolivalt C (2006) Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis. Protein Eng Des Sel 19:77–84. Google Scholar
  49. 49.
    Huang L, Liu Y, Liu X, Ban L, Wang Y, Li M, Lu F (2016) Functional expression of Trametes versicolor thermotolerant laccase variant in Pichia pastoris. Biotechnol Biotechnol Equip 30:261–269. Google Scholar
  50. 50.
    Jiao X, Li G, Wang Y, Nie F, Cheng X, Abdullah M, Lin Y, Cai Y (2018) Systematic analysis of the Pleurotus ostreatus laccase gene (PoLac) family and functional characterization of PoLac2 involved in the degradation of cotton-straw lignin. Accessed April 2018
  51. 51.
    Uzan E, Nousiainen P, Balland V, Sipila J, Piumi F, Navarro D, Asther M, Record E, Lomascolo A (2010) High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications. J Appl Microbiol 108:2199–2213. Google Scholar
  52. 52.
    Galhaup C, Goller S, Peterbauer CK, Strauss J, Haltrich D (2002) Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148:2159–2169Google Scholar
  53. 53.
    Dedeyan B, Klonowska A, Tagger S, Tron T, Iacazio G, Gil G, Le Petit J (2000) Biochemical and molecular characterization of a laccase from Marasmius quercophilus. Appl Environ Microbiol 66:925–929. Google Scholar
  54. 54.
    Maestre-Reyna M, Liu W-C, Jeng W-Y, Lee C-C, Hsu C-A, Wen T-N, Wang AHJ, Shyur L-F (2015) Structural and functional roles of glycosylation in fungal laccase from Lentinus sp. PLoS ONE 10:e0120601. Google Scholar
  55. 55.
    Christensen NJ, Kepp KP (2013) Stability mechanisms of a thermophilic laccase probed by molecular dynamics. PLoS ONE 8:e61985. Google Scholar
  56. 56.
    Iimura Y, Sonoki T, Habe H (2018) Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae. Protein Expr Purif 141:39–43. Google Scholar
  57. 57.
    Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH (2003) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol 69:987–995Google Scholar
  58. 58.
    Yaver DS, Xu F, Golightly EJ, Brown KM, Brown SH, Rey MW, Schneider P, Halkier T, Mondorf K, Dalboge H (1996) Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot Basidiomycete Trametes villosa. Appl Environ Microbiol 62:834–841Google Scholar
  59. 59.
    Plácido J, Capareda S (2015) Ligninolytic enzymes: a biotechnological alternative for bioethanol production. Biores Bioprocess 2:23. Google Scholar
  60. 60.
    Klonowska A, Gaudin C, Fournel A, Asso M, Petit JL, Giorgi M, Tron T (2002) Characterization of a low redox potential laccase from the Basidiomycete C30. Eur J Biochem 269:6119–6125. Google Scholar
  61. 61.
    Fujihiro S, Higuchi R, Hisamatsu S, Sonoki S (2009) Metabolism of hydroxylated PCB congeners by cloned laccase isoforms. Appl Microbiol Biotechnol 82:853–860. Google Scholar
  62. 62.
    Kojima Y, Tsukuda Y, Kawai Y, Tsukamoto A, Sugiura J, Sakaino M, Kita Y (1990) Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot Basidiomycete Coriolus hirsutus. J Biol Chem 265:15224–15230Google Scholar
  63. 63.
    Lu C, Wang H, Luo Y, Guo L (2010) An efficient system for pre-delignification of gramineous biofuel feedstock in vitro: application of a laccase from Pycnoporus sanguineus H275. Process Biochem 45:1141–1147. Google Scholar
  64. 64.
    Klonowska A, Gaudin C, Asso M, Fournel A, Réglier M, Tron T (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. Google Scholar
  65. 65.
    Hoshida H, Nakao M, Kanazawa H, Kubo K, Hakukawa T, Morimasa K, Akada R, Nishizawa Y (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 92:372–380. Google Scholar
  66. 66.
    Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C (2005) Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol 66:450–456Google Scholar
  67. 67.
    Antorini M, Herpoëlgimbert I, Choinowski T, Sigoillot JC, Asther M, Winterhalter K, Piontek K (2002) Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi. Biochem Biophys Acta 1594:109–114Google Scholar
  68. 68.
    Temp U, Zierold U, Eggert C (1999) Cloning and characterization of a second laccase gene from the lignin-degrading Basidiomycete Pycnoporus cinnabarinus. Gene 236:169–177. Google Scholar
  69. 69.
    Park JW, Kang HW, Ha BS, Kim SI, Kim S, Ro HS (2015) Strain-dependent response to Cu 2+ in the expression of laccase in Pycnoporus coccineus. Arch Microbiol 197:589Google Scholar
  70. 70.
    Dantán-González E, Vite-Vallejo O, Martínez-Anaya C, Méndez-Sánchez M, González MC, Palomares LA, Folch-Mallol J (2008) Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat. Int Microbiol Off J Span Soc Microbiol 11:163Google Scholar
  71. 71.
    Xiao YZ, Hong YZ, Li JF, Hang J, Tong PG, Fang W, Zhou CZ (2006) Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl Microbiol Biotechnol 71:493–501Google Scholar
  72. 72.
    Kandasamy S, Muniraj IK, Purushothaman N, Sekar A, Sharmila DJS, Kumarasamy R, Uthandi S (2016) High level secretion of laccase (LccH) from a newly isolated white-rot Basidiomycete, Hexagonia hirta MSF2. Front Microbiol 7.
  73. 73.
    Bertrand B, Trejo-Hernández MR, Morales-Guzmán D, Caspeta L, Suárez Rodríguez R, Martínez-Morales F (2016) Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology. Fungal Biology 120:1609–1622. Google Scholar
  74. 74.
    Sun J, Peng R-H, Xiong A-S, Tian Y, Zhao W, Xu H, Liu D-T, Chen J-M, Yao Q-H (2012) Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris. Mol Biol Rep 39:3807–3814. Google Scholar
  75. 75.
    Chakroun H, Mechichi T, Martinez MJ, Dhouib A, Sayadi S (2010) Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: application on bioremediation of phenolic compounds. Process Biochem 45:507–513. Google Scholar
  76. 76.
    Wang B, Yan Y, Tian Y, Zhao W, Li Z, Gao J, Peng R, Yao Q (2016) Heterologous expression and characterisation of a laccase from Colletotrichum lagenarium and decolourisation of different synthetic dyes. World J Microbiol Biotechnol 32:40. Google Scholar
  77. 77.
    Hildén K, Hakala T, Lundell T (2009) Thermotolerant and thermostable laccases. Biotechnol Lett 31:1117–1128Google Scholar
  78. 78.
    An H, Xiao T, Fan H, Wei D (2015) Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R. Electron J Biotechnol 18:451–458. Google Scholar
  79. 79.
    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 Enzym 101:1–6. Google Scholar
  80. 80.
    Hildén K, Mäkelä M, Lundell T, Kuuskeri J, Chernykh A, Golovleva L, Archer DB, Hatakka A (2012) Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 97:1589–1599Google Scholar

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Authors and Affiliations

  1. 1.Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of BiotechnologyShanxi UniversityTaiyuanChina
  2. 2.Key Lab of Advanced Transducers and Intelligent Control System, Ministry of Education and Shanxi Province, College of Physics and OptoelectronicsTaiyuan University of TechnologyTaiyuanChina

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