title: Anoxybacillus sp. Strain UARK-01, a New Thermophilic Soil Bacterium with Hyperthermostable Alkaline Laccase Activity.
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.
authors.affiliation: Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA.|Cell and Molecular Biology program, University of Arkansas, Fayetteville, AR, USA.
authors.forename: Thamir H
authors.initials: TH
authors.lastname: Al-Kahem Al-Balawi
authors.affiliation: Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Wood
authors.affiliation: Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA.
authors.forename: Alexis
authors.initials: A
authors.lastname: Solis
authors.affiliation: Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA.
authors.forename: Ted
authors.initials: T
authors.lastname: Cooper
authors.affiliation: Department of Biological Sciences, University of Arkansas, 850 W Dickson Street, SCEN 601, Fayetteville, AR, 72701, USA. barabote@uark.edu.|Cell and Molecular Biology program, University of Arkansas, Fayetteville, AR, USA. barabote@uark.edu.
authors.forename: Ravi D
authors.initials: RD
authors.lastname: Barabote
mesh_terms: D000595:Amino Acid Sequence; D058677:Anoxybacillus / Q000145:classification* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D001482:Base Composition; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D042845:Laccase / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008031:Lignin; D042845:Laccase; D010100:Oxygen