Production, Gene Cloning, and Overexpression of a Laccase in the Marine-Derived Yeast Aureobasidium melanogenum Strain 11-1 and Characterization of the Recombinant Laccase

  • Thu Aung
  • Hong Jiang
  • Cheng-Cheng Chen
  • Guang-Lei Liu
  • Zhong Hu
  • Zhen-Ming ChiEmail author
  • Zhe ChiEmail author
Original Article


Aureobasidium melanogenum strain 11-1 with a high laccase activity was isolated from a mangrove ecosystem. Under the optimal conditions, the 11-1 strain yielded the highest laccase activity up to 3120.0 ± 170 mU/ml (1.2 U/mg protein) within 5 days. A laccase gene (LAC1) of the yeast strain 11-1 contained two introns and encoded a protein with 570 amino acids and four conserved copper-binding domains typical of the fungal laccase. Expression of the LAC1 gene in the yeast strain 11-1 made a recombinant yeast strain produce the laccase activity of 6005 ± 140 mU/ml. The molecular weight of the recombinant laccase after removing the sugar was about 62.5 kDa. The optimal temperature and pH of the recombinant laccase were 40 °C and 3.2, respectively, and it was stable at a temperature less than 25 °C. The laccase was inhibited in the presence of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and dl-dithiothreitol (DTT). The Km and Vmax values of the laccase for 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was 6.3 × 10−2 mM and 177.4 M/min, respectively. Many synthetic dyes were greatly decolored by the laccase.


Laccase Aureobasidium melanogenum LAC1 gene Overexpression Decolorizing ability 


Funding Information

This study was financially supported by the National Natural Science Foundation of China (Grant No. 31561163001).

Supplementary material

10126_2018_9860_MOESM1_ESM.doc (384 kb)
ESM 1 (DOC 384 kb)


  1. Ademakinwa AN, Femi Kayode Agboola FK (2016) Biochemical characterization and kinetic studies on a purified yellow laccase from newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decayed plant matter. J Genet Eng Biotechnol 14:143–151CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Campos PA, Levin LN, Wirth SA (2016) Heterologous production, characterization and dye decolorizationability of a novel thermostable laccase isoenzyme from Trametes trogii BAFC 463. Process Biochem 51:895–903CrossRefGoogle Scholar
  4. Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179PubMedPubMedCentralGoogle Scholar
  5. Chi ZM, Liu J, Zhang W (2001) Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzym Microb Technol 38:240–246CrossRefGoogle Scholar
  6. Chi Z, Wang XX, Ma ZC, Buzdar MA, Chi ZM (2012) The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2. Biomet 25:219–230CrossRefGoogle Scholar
  7. Eggert C, Temp U, Eriksson K-EL (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158PubMedPubMedCentralGoogle Scholar
  8. Fernández-Fernández M, Sanromán MA, Moldes D (2013) Recent developments and applications of immobilized laccase. Biotechol Adv 31:1808–1825CrossRefGoogle Scholar
  9. Gostinčar C, Ohm RA, Kogej T, Sonjak S, Turk M, Zajc J, Zalar P, Grube M, Sun H, Han J, Sharma A, Chiniquy J, Yee CY, Ngan A, Lipzen K, Barry I, Grigoriev V, Gunde-Cimerman N (2014) Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species. BMC Genomics 15:549–550CrossRefGoogle Scholar
  10. Jiang H, Ma Y, Chi Z, Liu GL, Chi ZM (2016) Production, purification, and gene cloning of a β-fructofuranosidase with a high inulin-hydrolyzing activity produced by a novel yeast Aureobasidium sp. P6 isolated from a mangrove ecosystem. Mar Biotechnol 18:500–510CrossRefGoogle Scholar
  11. Kumar R, Kaur J, Jain S, Kumar A (2016) Optimization of laccase production from Aspergillus flavus by design of experiment technique: partial purification and characterization. J Genet Eng Biotechnol 14:125–131CrossRefGoogle Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  13. Leathers TD, Rich JO, Anderson AM (2013) Lipase production by diverse phylogenetic clades of Aureobasidium pullulans. Biotechnol Lett 35(10):1701–1706CrossRefGoogle Scholar
  14. Liu GL, Wang DS, Wang LF, Zhao SF, Chi ZM (2011) Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11. Fung Genet Biol 48:904–913CrossRefGoogle Scholar
  15. Liu YY, Zhe Chi Z, Wang ZP, Liu GL, Chi ZM (2014) Heavy oils, principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system. J Ind Microbiol Biotechnol 41:1329–1337CrossRefGoogle Scholar
  16. Ma ZC, Fu WJ, Liu GL, Wang ZP, Chi ZM (2014) High-level pullulan production by Aureobasidium pullulans var. melanogenum P16 isolated from mangrove system. Appl Microbiol Biotechnol 98:4865–4873CrossRefGoogle Scholar
  17. Ning YJ, Wang DD, Chen QJ, Ling ZR, Wang SN, Wang WP, Zhang GQ, Zhu MJ (2016) An extracellular yellow laccase with potent dye decolorizing ability from the fungus Leucoagaricus naucinus LAC-04. Int J Biol Macromol 93:837–842CrossRefGoogle Scholar
  18. Richa JO, Leathersa TD, Andersona AM, Bischoffa KM, Manitchotpisit P (2013) Laccases from Aureobasidium pullulans. Enzym Microb Technol 53:33–37CrossRefGoogle Scholar
  19. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:119–226CrossRefGoogle Scholar
  20. Sannia G, Giardina P, Luna M, Rossi M, Buonocore V (1986) Laccase from Pleurotus ostreatus. Biotechnol Lett 8:797–800CrossRefGoogle Scholar
  21. Santo M, Weitsman R, Alex Sivan A (2013) The role of the copper-binding enzyme-laccase-in the biodegradation of polyethylene by the actinomycete Rhodococcus ruber. Int Biodeterior Biodegrad 84:204–210CrossRefGoogle Scholar
  22. Shin KS, Lee YJ (2000) Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. Arch Biochem Biophys 384:109–115CrossRefGoogle Scholar
  23. Sun J, Zheng M, Lu Z, Lu F, Zhang C (2017) Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application. Process Biochem 55:77–84CrossRefGoogle Scholar
  24. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  25. Tong P, Hong Y, Xiao Y, Zhang M, Tu X, Cui T (2007) High production of laccase by a new basidiomycete, Trametes sp. Biotechnol Lett 29:295–301CrossRefGoogle Scholar
  26. Varghese G, Diwan AM (1983) Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250. Anal Biochem 132:481–483CrossRefGoogle Scholar
  27. Vasina DV, Mustafaev ON, Moiseenko KV, Sadovskaya NS, Glazunova OA, Tyurin AA, Fedorova TV, Pavlov AR, Tyazhelova TV, Goldenkova-Pavlova IV, Olga V, Koroleva OV (2015) The Trametes hirsuta 072 laccase multigene family: genes identification and transcriptional analysis under copper ions induction. Biochemie 116:154–164CrossRefGoogle Scholar
  28. Wang CL, Li Y, Xin FH, Liu YY, Chi ZM (2014a) Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production. Process Biochem 49:725–731CrossRefGoogle Scholar
  29. Wang F, Hu JH, Guo C, Liu CZ (2014b) Enhanced laccase production by Trametes versicolor using corn steep liquor as both nitrogen source and inducer. Bioresour Technol 166:602–605CrossRefGoogle Scholar
  30. Wang HW, Zhu H, Liang XF, Du W, Dai C (2014c) Molecular cloning and expression of a novel laccase showing thermo- and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants. Biotechnol Lett 36:167–173CrossRefGoogle Scholar
  31. Wang J, Lu L, Feng F (2017a) Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris. In: Electron J Biotechnol, vol 28, pp 7–13Google Scholar
  32. Wang SS, Ning YJ, Wang SN, Zhang J, Zhang GQ, Chen QJ (2017b) Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01. Int J Biol Macromol 95:920–927CrossRefGoogle Scholar
  33. Wei Y, Pu J, Zhang H, Liu Y, Zhou F, Zhang K, Liu X (2017) The laccase gene (LAC1) is essential for Colletotrichum gloeosporioides development and virulence on mango leaves and fruits. Physiol Mol Plant Pathol:1–10Google Scholar
  34. Zhang FL, Chi ZM, Zhu KL, Li J, Li MJ, Liang LK, Wu LF (2007) Expression in Escherichia coli of the recombinant Vibrio anguillarum metalloprotease and its purification and characterization. World J Microbiol Biotechnol 23:331–337CrossRefGoogle Scholar
  35. Zhang F, Wang ZP, Chi Z, Raoufi Z, Abdollahi S, Chi ZM (2013) The changes in Tps1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17-1 grown at different temperatures. Extremophiles 17:241–249CrossRefGoogle Scholar
  36. Zheng F, An Q, Meng G, Wu XJ, Dai YC, Si J, Cui BK (2017) A novel laccase from white rot fungus Trametes orientalis: purification, characterization, and application. Int J Biol Macromol 102:758–770CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Marine Genetics and Breeding (Ocean University of China)Ministry of EducationQingdaoChina
  2. 2.Laboratory for Marine Biology and BiotechnologyQingdao National Laboratory for Marine Science and TechnologyQingdaoChina
  3. 3.Department of BiologyShantou UniversityShantouChina

Personalised recommendations