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Anoxybacillus sp. Strain UARK-01, a New Thermophilic Soil Bacterium with Hyperthermostable Alkaline Laccase Activity

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Abstract

We describe the isolation and characteristics of a novel thermophilic bacterium from soil. The organism is a member of the Anoxybacillus genus based on phylogenetic analysis of the 16S rRNA gene. The 16S rRNA of the organism shares >99% sequence identity with those of two species, Anoxybacillus rupiensis and A. geothermalis. We named this isolate as Anoxybacillus sp. strain UARK-01. UARK-01 grows optimally in the presence of oxygen at 55 °C and pH 8. It grew excellently in the presence of lignin as the sole carbon source. Culture supernatant from UARK-01 grown on lignin was rich in laccase activity. The laccase activity was optimal at 90 °C and pH 9, and there was comparable activity at 80 and 100 °C. The crude laccase decolorized approximately 75% of Congo Red in 7 h under optimal conditions. A single laccase gene was identified from the draft genome sequence of Anoxybacillus sp. UARK-01. The UARK-01 laccase (Anox_Lacc) was cloned and overexpressed in Escherichia coli and was partially purified. The partially purified Anox_Lacc decolorized approximately 1.64+/0.21 nanomoles of Congo Red per microgram protein in 30 min at 90 °C and pH 9. Anox_Lacc is a member of the multicopper polyphenol oxidoreductase laccase family (pfam02578 Cu-oxidase_4) and has novel characteristics. Multiple sequence analysis of Anox_Lacc with six homologs from the family revealed four conserved copper ligands and several new residues that are fully conserved. Anox_Lacc is enriched in leucine, glutamine, and lysine, and it contains fewer alanine, arginine, glycine, and serine residues. Skewed amino acid composition of Anox_Lacc likely contributes to the exceptional thermochemical properties of the laccase activity from UARK-01. Both lignin utilization and production of hyperthermostable alkaline laccase are new findings in the Anoxybacillus genus.

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Acknowledgements

This research was supported by startup funds provided to RDB by the University of Arkansas. We thank Prof. Julie Carrier for providing the switchgrass. THAA acknowledges stipend support from Prince Sattam University. Undergraduate students, AS and TC, acknowledge support from the National Science Foundation Research Experience for Undergraduates program through the University of Arkansas REU Site (DBI-1063067).

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Authors and Affiliations

  1. Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA

    Thamir H. Al-kahem Al-balawi, Adam L. Wood, Alexis Solis, Ted Cooper & Ravi D. Barabote

  2. Cell and Molecular Biology program, University of Arkansas, Fayetteville, AR, USA

    Thamir H. Al-kahem Al-balawi & Ravi D. Barabote

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  1. Thamir H. Al-kahem Al-balawi
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  2. Adam L. Wood
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Correspondence to Ravi D. Barabote.

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Additional information

The 16S and laccase gene sequences from the Anoxybacillus sp. strain UARK-01 have been deposited in GenBank under the accession numbers KX784766 and KY679089, respectively.

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Al-kahem Al-balawi, T.H., Wood, A.L., Solis, A. et al. Anoxybacillus sp. Strain UARK-01, a New Thermophilic Soil Bacterium with Hyperthermostable Alkaline Laccase Activity. Curr Microbiol 74, 762–771 (2017). https://doi.org/10.1007/s00284-017-1239-5

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  • DOI: https://doi.org/10.1007/s00284-017-1239-5

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