Abstract
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.
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Acknowledgments
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.
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Ma, Y., Li, L., Tian, H. et al. Transcriptional analysis of the laccase-like gene from Burkholderia cepacia BNS and expression in Escherichia coli. Appl Microbiol Biotechnol 103, 747–760 (2019). https://doi.org/10.1007/s00253-018-9468-5
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DOI: https://doi.org/10.1007/s00253-018-9468-5