- Title
- Transcriptional analysis of the laccase-like gene from Burkholderia cepacia BNS and expression in Escherichia coli
- Creator
- Ma, Yinghui; Li, Lijun; Tian, Haixia; Lu, Meihuan; Megharaj, Mallavarapu; He, Wenxiang
- Relation
- Applied Microbiology and Biotechnology Vol. 103, Issue 2, p. 747-760
- Publisher Link
- http://dx.doi.org/10.1007/s00253-018-9468-5
- Publisher
- Springer
- Resource Type
- journal article
- Date
- 2019
- Description
- 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.
- Subject
- Burkholderia cepacia; laccase-like enzyme; expression; transcription; qRT-PCR
- Identifier
- http://hdl.handle.net/1959.13/1467543
- Identifier
- uon:47840
- Identifier
- ISSN:0175-7598
- Language
- eng
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