脂肪酶在进化过程中具有菌属特征,新嗜热脂肪酶的开发是脂肪酶研究和应用的重要方向。从嗜热微生物发酵产物或利用宏基因组方法分离发现新嗜热脂肪酶的操作过程较为繁琐。该文基于生物信息学方法和异源表达技术,对海洋热泉来源的Caldisericum exile基因组数据进行筛选和进化分析,发现BAL81435.1(GenBank ID)可能编码新的嗜热脂肪酶。将该序列克隆到pET28(a)载体上,诱导表达后对该酶进行分离纯化和酶学性质表征。试验证实BAL81435.1编码新的嗜热脂肪酶,其底物为长碳链(C12以上)的对硝基苯磷酸酯,最适底物为C16的长碳链,最适pH值为8.8,最适温度为60 ℃。该嗜热脂肪酶在4~60 ℃具有较好的温度耐受性,对大部分有机溶剂、金属离子、变性剂及高浓度的NaCl有较好的耐受性,但对表面活性剂敏感。该研究表明BAL81435.1编码新的嗜热脂肪酶,为嗜热脂肪酶的进一步开发奠定基础。
Lipases possess unique genus features during bacterial evolution. Discovery of new thermophilic lipases represents an important field for lipase research and application. The current methods of isolation and identification of new thermophilic lipases from thermophilic microbial fermentation products and through metagenomic methods are laborious. Based on bioinformatics methods and heterologous expression techniques, the Caldisericum exile genomic data from marine hot springs were screened and evolutionarily analyzed, and it was found that BAL81435.1 (GenBank ID) may encode new thermophilic lipase. The sequence was cloned into pET28(a) vector, and the properties of recombinant lipase were characterized after purification. BAL81435.1 encodes a new thermophilic lipase whose substrates are long-chain (>C12) para-nitrophenyl phosphate (p-NPP), with maximum specificity detected at chain length of C16. Its optimal pH was 8.8 and optimal temperature was 60 ℃. This lipase is thermally-stable between 4 ℃ and 60 ℃ and tolerant to most organic solvents, metal ions, denaturing agents and high salinity, but surfactant-sensitive. This study discovers a novel thermophilic lipase encoded by BAL81435.1, which lays foundation for prospective lipase study and exploitation.
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