Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • 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 14:143–151

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 72:248–254

    Article  CAS  Google Scholar 

  • 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 51:895–903

    Article  CAS  Google Scholar 

  • Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chi ZM, Liu J, Zhang W (2001) Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzym Microb Technol 38:240–246

    Article  Google Scholar 

  • Chi Z, Wang XX, Ma ZC, Buzdar MA, Chi ZM (2012) The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2. Biomet 25:219–230

    Article  CAS  Google Scholar 

  • 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 62:1151–1158

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fernández-Fernández M, Sanromán MA, Moldes D (2013) Recent developments and applications of immobilized laccase. Biotechol Adv 31:1808–1825

    Article  CAS  Google Scholar 

  • 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 15:549–550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 18:500–510

    Article  CAS  PubMed  Google Scholar 

  • 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 14:125–131

    Article  PubMed  PubMed Central  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Leathers TD, Rich JO, Anderson AM (2013) Lipase production by diverse phylogenetic clades of Aureobasidium pullulans. Biotechnol Lett 35(10):1701–1706

    Article  CAS  PubMed  Google Scholar 

  • 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 48:904–913

    Article  CAS  Google Scholar 

  • 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 41:1329–1337

    Article  CAS  PubMed  Google Scholar 

  • 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 98:4865–4873

    Article  CAS  PubMed  Google Scholar 

  • 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 93:837–842

    Article  CAS  PubMed  Google Scholar 

  • Richa JO, Leathersa TD, Andersona AM, Bischoffa KM, Manitchotpisit P (2013) Laccases from Aureobasidium pullulans. Enzym Microb Technol 53:33–37

    Article  CAS  Google Scholar 

  • Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:119–226

    Article  CAS  Google Scholar 

  • Sannia G, Giardina P, Luna M, Rossi M, Buonocore V (1986) Laccase from Pleurotus ostreatus. Biotechnol Lett 8:797–800

    Article  CAS  Google Scholar 

  • 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 84:204–210

    Article  CAS  Google Scholar 

  • 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 384:109–115

    Article  CAS  PubMed  Google Scholar 

  • 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 55:77–84

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 29:295–301

    Article  CAS  PubMed  Google Scholar 

  • 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 132:481–483

    Article  CAS  PubMed  Google Scholar 

  • 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 116:154–164

    Article  CAS  Google Scholar 

  • 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 49:725–731

    Article  CAS  Google Scholar 

  • 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 166:602–605

    Article  CAS  Google Scholar 

  • 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 36:167–173

    Article  CAS  PubMed  Google Scholar 

  • 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 7–13

    Google Scholar 

  • 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 95:920–927

    Article  CAS  Google Scholar 

  • 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:1–10

  • 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 23:331–337

    Article  CAS  Google Scholar 

  • 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 17:241–249

    Article  CAS  PubMed  Google Scholar 

  • 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 102:758–770

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

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

Author information

Author notes

  1. Thu Aung, Hong Jiang and Cheng-Cheng Chen contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Yushan Road, No. 5, Qingdao, China

    Thu Aung, Hong Jiang, Cheng-Cheng Chen, Guang-Lei Liu, Zhen-Ming Chi & Zhe Chi

  2. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Shi, 266003, Shandong Sheng, Qingdao, China

    Hong Jiang, Guang-Lei Liu, Zhen-Ming Chi & Zhe Chi

  3. Department of Biology, Shantou University, Shantou, China

    Zhong Hu

Authors
  1. Thu Aung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hong Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheng-Cheng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guang-Lei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhong Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhen-Ming Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhe Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Zhen-Ming Chi or Zhe Chi.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

ESM 1

(DOC 384 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aung, T., Jiang, H., Chen, CC. et al. Production, Gene Cloning, and Overexpression of a Laccase in the Marine-Derived Yeast Aureobasidium melanogenum Strain 11-1 and Characterization of the Recombinant Laccase. Mar Biotechnol 21, 76–87 (2019). https://doi.org/10.1007/s10126-018-9860-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-018-9860-2

Keywords

Search

Navigation