Isolation and identification of an alkaliphilic Bacillus flexus XJU-3 and analysis of its alkaline amylase

Abstract:

[objective] To isolate and identify a new Bacillus strain capable of growing under highly alkaline conditions as alkaline amylase producers and to characterize its enzymatic properties. [Methods] The isolates were sampled from alkaline sewage in Shihezi city, Xinjiang and screened by plating them on the amylase agar medium depending on the halo zone diameter. The alkaline amylase producer with best enzymatic activity was designated as XJU-3. XJU-3 was identified by its physiological and biochemical characteristics, 16S rDNA sequence homol-ogy, and the content of its major cellular fatty acids. [Results] XJU-3 was a Gram-positive, spore-forming, aerobic, motile rod alkaliphilic bacterium. It can grow at a broad range of pH (4.0-12.5) in Luria broth medium and its optimum growth was at pH 10 and 37°C. Its NaCl tolerance was up to 15%. Its major cellular fatty acids were anteiso-C15:0 and iso-C15: 0. Comparative 16S rRNA gene sequence analyses showed that XJU-3 was most closely related to Bacillus flexus, with 99% similarity. The genomic DNA (G+C) content of our isolate was 39.13 mol %. XJU-3 produced extracellular alkaline amylase, and its maximal enzyme activity was observed at 40℃ and pH 10.0. More than 70% of the enzymatic activity was remained at pH 13.0. The enzyme activity was strongly enhanced with the presence of Co2+ and Mg2+. [Conclusion] The strain XJU-3 was confirmed as B. flexus. Owing to its excellent pH tolerance, the kinds of major cellular fatty acids, and several phenotypic characteristics that were different from the description of the reference strain, the strain was further classified as a new variant of the spe-cies B. flexus. The enzymatic properties of XJU-3 alkaline amylase indicated its potential in industrial applications.