Al-Kahem Al-Balawi Thamir H, Wood Adam L, Solis Alexis, Cooper Ted, Barabote Ravi D
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.
Curr Microbiol. 2017 Jun;74(6):762-771. doi: 10.1007/s00284-017-1239-5. Epub 2017 Apr 8.
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.
我们描述了从土壤中分离出的一种新型嗜热细菌及其特性。基于16S rRNA基因的系统发育分析，该生物体是嗜热栖热放线菌属的成员。该生物体的16S rRNA与鲁氏嗜热栖热放线菌和嗜热栖热放线菌两个物种的16S rRNA序列同一性>99%。我们将该分离株命名为嗜热栖热放线菌菌株UARK-01。UARK-01在55°C和pH 8的有氧条件下生长最佳。它在以木质素作为唯一碳源的情况下生长良好。在木质素上生长的UARK-01的培养上清液富含漆酶活性。漆酶活性在90°C和pH 9时最佳，在80°C和100°C时也有相当的活性。在最佳条件下，粗漆酶在7小时内使约75%的刚果红脱色。从嗜热栖热放线菌菌株UARK-01的基因组草图序列中鉴定出一个单一的漆酶基因。UARK-01漆酶（Anox_Lacc）在大肠杆菌中克隆并过量表达，并进行了部分纯化。在90°C和pH 9条件下，部分纯化的Anox_Lacc在30分钟内每微克蛋白质可使约1.64±0.21纳摩尔的刚果红脱色。Anox_Lacc是多铜多酚氧化还原酶漆酶家族（pfam02578 Cu-oxidase_4）成员，具有新特性。对Anox_Lacc与该家族六个同源物的多序列分析揭示了四个保守的铜配体和几个完全保守的新残基。Anox_Lacc富含亮氨酸、谷氨酰胺和赖氨酸，且丙氨酸、精氨酸、甘氨酸和丝氨酸残基较少。Anox_Lacc的氨基酸组成偏向可能导致了UARK-01漆酶活性具有特殊的热化学性质。木质素利用和超嗜热碱性漆酶的产生都是嗜热栖热放线菌属中的新发现。
