Transcriptional analysis of the laccase-like gene from Burkholderia cepacia BNS and expression in Escherichia coli
- 130 Downloads
Bacterial laccases have received considerable attention because of several advantages associated with the higher environmental stability of these enzymes compared with fungal laccases. In this study, a laccase-like gene from Burkholderia cepacia BNS was successfully cloned. This gene was found to encode a mature protein of 279 amino acids that exhibited laccase activity in dimer form. The mature protein was found to contain approximately 4 mol of copper per monomer, and the metal ion-binding sites were predicted. BC_lacL gene transcription levels were analyzed by qRT-PCR to study expression patterns in the presence of different putative inducers (copper ions, guaiacol, veratryl alcohol, vanillin, coniferaldehyde, p-coumaric acid, sinapic acid, and ferulic acid). Copper ions had a positive effect on both transcription levels and intracellular laccase activity. Interestingly, upon induction with sinapic acid, BC_lacL gene transcription was lower than in the presence of copper ions, but laccase activity was highest under these conditions. The BC_lacL protein expressed in Escherichia coli exhibited a specific activity of 7.81 U/mg with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate and 12.3 U/mg with 2,6-dimethoxyphenol (2,6-DMP) as the substrate after purification through Ni-affinity chromatography. The optimal activity and kinetic parameters of the recombinant BC_lacL protein were observed (kcat/Km = 3.96 s−1 μM−1) at a pH of 4.0 at 55 °C for ABTS oxidization and (kcat/Km = 11.6 s−1 μM−1) at a pH of 10.0 at 75 °C for 2,6-DMP oxidization. The protein exhibited high stability in an alkaline environment, with a half-life of more than 12 h. The same results were obtained via decolorization of eight dyes. Hence, this laccase-like enzyme may have potential industrial applications.
KeywordsBurkholderia cepacia Laccase-like enzyme Expression Transcription qRT-PCR Enzyme characterization
This work was supported by the Applied Basic Research Fund of Shaanxi Province Academy of Sciences of China (2015 k14) and the Agriculture Technological Innovation Program of Shaanxi Province Science and Technology Department of China (2016NY197). We thank American Journal Experts (AJE) for English language editing.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflicts of interest.
- Ausec L, Berini F, Casciello C, Cretoiu MS, van Elsas JD, Marinelli F, Mandic-Mulec I (2017) The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance. Appl Microbiol Biotechnol 101:6261–6276. https://doi.org/10.1007/s00253-017-8345-y CrossRefGoogle Scholar
- Beck S, Berry E, Duke S, Milliken A, Patterson H, Prewett DL, Rae TC, Sridhar V, Wendland N, Gregory BW, Johnson CM (2018) Characterization of Trametes versicolor laccase-catalyzed degradation of estrogenic pollutants: substrate limitation and product identification. Int Biodeterior Biodegrad 127:146–159. https://doi.org/10.1016/j.ibiod.2017.11.020 CrossRefGoogle Scholar
- Beloqui A, Pita M, Polaina J, Martínez-Arias A, Golyshina OV, Zumárraga M, Yakimov MM, García-Arellano H, Alcalde M, Fernández VM, Elborough K, Andreu JM, Ballesteros A, Plou FJ, Timmis KN, Ferrer M, Golyshin PN (2006) Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen. J Biol Chem 281:22933–22942. https://doi.org/10.1074/jbc.m600577200 CrossRefGoogle Scholar
- Berini F, Verce M, Ausec L, Rosini E, Tonin F, Pollegioni L, Mandić-Mulec I (2018) Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens. Appl Microbiol Biotechnol 102:2425–2439. https://doi.org/10.1007/s00253-018-8785-z CrossRefGoogle Scholar
- Chen K, Zhu Q, Qian Y, Song Y, Yao J, Choi MMF (2013) Microcalorimetric investigation of the effect of non-ionic surfactant on biodegradation of pyrene by PAH-degrading bacteria Burkholderia cepacia. Ecotoxicol Environ Saf 98:361–367. https://doi.org/10.1016/j.ecoenv.2013.08.012 CrossRefGoogle Scholar
- De Souza CGM, Zilly A, Peralta RM (2002) Production of laccase as the sole phenoloxidase by a Brazilian strain of Pleurotus pulmonarius in solid state fermentation. J Basic Microbiol 42:83–90. https://doi.org/10.1002/1521-4028(200205)42:2<83::aid-jobm83>3.0.co;2-z CrossRefGoogle Scholar
- Fang Z-M, Li T-L, Chang F, Zhou P, Fang W, Hong Y-Z, Zhang X-C, Peng H, Xiao Y-Z (2012) A new marine bacterial laccase with chloride-enhancing, alkaline-dependent activity and dye decolorization ability. Bioresour Technol 111:36–41. https://doi.org/10.1016/j.biortech.2012.01.172 CrossRefGoogle Scholar
- Granja-Travez RS, Wilkinson RC, Felix Persinoti G, Squina FM, Fülöp V, Bugg TDH (2018) Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate. FEBS J 285:1684–1700. https://doi.org/10.1111/febs.14437 CrossRefGoogle Scholar
- Kılıç N, Nasiri F, Cansaran-Duman D (2016) Fungal laccase enzyme applications in bioremediation of polluted wastewater. Phytoremediation:201–209. https://doi.org/10.1007/978-3-319-41811-7_11
- Liu Y, Huang L, Guo W, Jia L, Fu Y, Gui S, Lu F (2017) Cloning, expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization. Process Biochem 53:125–134. https://doi.org/10.1016/j.procbio.2016.11.015 CrossRefGoogle Scholar
- Lu L, Zeng G, Fan C, Zhang J, Chen A, Chen M, Jiang M, Yuan Y, Wu H, Lai M, He Y (2014) Diversity of two-domain laccase-like multicopper oxidase genes in Streptomyces spp.: identification of genes potentially involved in extracellular activities and lignocellulose degradation during composting of agricultural waste. Appl Environ Microbiol 80:3305–3314. https://doi.org/10.1128/aem.00223-14 CrossRefGoogle Scholar
- Magalhães VC, Barbosa LDO, Andrade JP, Soares ACF, de Souza JT, Marbach PAS (2017) Burkholderia isolates from a sand dune leaf litter display biocontrol activity against the bole rot disease of Agave sisalana. Biol Control 112:41–48. https://doi.org/10.1016/j.biocontrol.2017.06.005 CrossRefGoogle Scholar
- 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. https://doi.org/10.1007/s11274-015-1999-7 CrossRefGoogle Scholar
- Yang Y, Wei F, Zhuo R, Fan F, Liu H, Zhang C, Ma L, Jiang M, Zhang X (2013) Enhancing the laccase production and laccase gene expression in the white-rot fungus Trametes velutina 5930 with great potential for biotechnological applications by different metal ions and aromatic compounds. PLoS One 8:e79307. https://doi.org/10.1371/journal.pone.0079307 CrossRefGoogle Scholar
- Zhu M, Zhang G, Meng L, Wang H, Gao K, Ng T (2016) Purification and characterization of a white laccase with pronounced dye decolorizing ability and HIV-1 reverse transcriptase inhibitory activity from Lepista nuda. Molecules 21:415–430. https://doi.org/10.3390/molecules21040415 CrossRefGoogle Scholar