一株碱性淀粉酶产生菌Bacillus flexus XJU-3的分离鉴定及酶学特性分析(英文)

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

【目的】从新疆石河子市一处碱性工业污水（pH11．0）分离、鉴定高产碱性淀粉酶菌株，并对其所产酶酶学特性进行研究。【方法】在碱性淀粉酶分离培养基上对所分离菌株进行筛选，分离到一株高产碱性淀粉酶菌株，并将其编号XJU-3。应用生理生化试验，脂肪酸含量，16SrDNA序列以及（G＋C）mol％含量等方法对菌株进行鉴定，同时对XJU-3所产碱性淀粉酶的生物学特性进行研究。【结果】XJU-3可在pH4．0～12．5的LB培养基上生长，最适生长温度37℃。16SrDNA序列构建的系统进化树表明XJU-3与Bacillus flexus类聚在一起，且序列同源性为99％。该菌产生的淀粉酶最适pi110．0，最适温度40℃，且在pH9．0～13．0内有较高活性和稳定性。Co^2＋和Mg^2＋能明显提高酶的活性。【结论】XJU-3被鉴定为Bacillus flexus，由于XJU-3与B．flexus DSM 1320^γ在尿素水解和优势脂肪酸含量上有差异，且具有宽范围pH耐受性，因此XJU-3被认为是B．flexus的一个新菌株。XJU-3所产的碱性淀粉酶酶学特性良好，具有极大的工业应用潜力。

[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 homology, 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℃. 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 Co^2＋ and Mg^2＋. [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 species B.flexus. The enzymatic properties of XJU-3 alkaline amylase indicated its potential in industrial applications.