来源于海洋细菌Altererythrobacter luteolus SW109T的新型酯酶E29的克隆表达及其酶学性质

Cloning, expression and characterization of a novel esterase (E29) from a marine bacterium Altererythrobacter luteolus SW109T

摘要:

【目的】克隆表达一个来源于海洋细菌的酯酶E29,并研究其酶学性质。【方法】从细菌Altererythrobacter luteolus SW109^T 中筛选并扩增出一个酯酶基因,将其克隆至pSMT3 载体上,并将重组质粒转化至大肠杆菌BL21(DE3)中进行异源表达,分析表达产物的酶学性质。【结果】氨基酸序列分析结果表明,酯酶E29 属于脂类水解酶第二家族(Family II)。酶学性质分析结果显示,酯酶E29 的最适反应底物为对硝基苯酚丁酸酯,最适反应温度为45 ℃,最适反应pH 为8.5;10 mmol/L 的Co²⁺和Mn²⁺及15%的异丙醇和乙腈能强烈抑制酯酶E29 的活性,1%的SDS 能使酶失活,甘油的存在能促进酶活性。【结论】酯酶E29 是一个海洋来源的新型酯酶,其具有较高的酶活力值、较宽的底物谱以及对部分有机溶剂和金属离子较好的耐受性,在工业方面具有潜在的应用价值。

Abstract:

[Objective] We cloned, expressed and characterized a novel esterase E29 from a marine bacterium. [Methods] An assumed esterase gene, which was predicted via the genome of Altererythrobacter luteolus SW109^T and amplified by PCR, was cloned into expression vector pSMT3. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). Subsequently, E29 was obtained via heterogenetic expression and characterized. [Results] The amino acids sequence analysis revealed E29 represents a new member of Family II of lipolytic enzyme. According to biochemical characterization, the maximum hydrolysis activity was obtained using p-nitrophenyl butyrate as substrate, at 45 ℃ and pH 8.5. The activity of E29 decreased in solution containing 10 mmol/L Co²⁺ or Mn²⁺, or 15% isopropanol or acetonitrile. The esterase was inactivated in 1% SDS solution. The addition of glycerol can promote the activity of E29 strikingly. [Conclusion] E29 is a novel marine esterase. Due to its high enzyme activity, wide substrates selectivity as well as tolerance of some organic solvents and metal irons, E29 has potential application in industry.