来源于海洋细菌Altererythrobacter epoxidivorans CGMCC 1.7731~T的新型碱性酯酶E22的酶学性质  

Characterization of a novel alkaline esterase(E22) from a marine bacterium Altererythrobacter epoxidivorans CGMCC 1.7731~T

作　　者：戎振 霍颖异[1] 徐曹狄 简书令 王春生[1] 许学伟[1] 

机构地区：[1]国家海洋局第二海洋研究所国家海洋局海洋生态系统与生物地球化学重点实验室,浙江杭州310012

出　　处：《微生物学通报》2017年第7期1622-1630,共9页Microbiology China

基　　金：国家自然科学基金项目(No.41506183)~~

摘　　要：【目的】克隆表达海洋细菌Altererythrobacter epoxidivorans CGMCC 1.7731~T中的酯酶基因e22,并研究其酶学性质。【方法】分析菌株的全基因组序列,筛选获得一个酯酶基因e22,将其克隆至p ET-28a载体上,并转化至大肠杆菌BL21(DE3)细胞中表达,研究纯化后表达产物的酶学性质。【结果】通过氨基酸序列分析,确定酯酶E22属于脂类水解酶第二家族(Family Ⅱ)。酶学性质研究结果表明,该酶最适反应底物为对硝基苯酚丁酸酯(C4);最适反应pH 10.5,为碱性酯酶;最适反应温度为55°C,并在60°C孵育2 h后仍保留超过50%的活性,显示了良好的热稳定性;1%甲醇、1%Triton X-100或0.1%SDS对酯酶E22的活性无显著影响,而10 mmol/L的Ba^(2+)或Ca^(2+)则对其活性有抑制作用。【结论】E22是一个新型海洋来源酯酶,具有耐碱性、热稳定性、有机溶剂和去垢剂耐受性等优良特性,在工业生产中具有较好的应用潜力。[Objective] To clone and express an esterase gene e22 from marine bacterium Altererythrobacter epoxidivorans CGMCC 1.7731T and characterize the enzyme. [Methods] Screening of the genome of strain CGMCC 1.7731T obtained an esterase-encoding gene, e22. The gene was amplified by PCR and cloned into the expression vector pET-28a. Then, the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） for heterogenetic expression. After purified by affinity chromatography, the enzyme was characterized. [Results] The amino acids sequence analysis indicated E22 belongs to Family II of lipolytic enzyme. The enzymatic property assay revealed that E22 had maximum activity usingp-nitrophenyl butyrate as substrate. It was an alkaline esterase that had highest activity at pH 10.5. The optimal reaction temperature was 55 ℃. After incubated at 60 ℃ for 2 h, E22 still remained over 50% activity, which showed its moderate thermal stability. The activity of E22 was not significantly affected by the presence of 1% methanol, 1% Triton X-100 or 0.1% SDS. On the contrary, the addition of 10 mmol/L Ba2＋ and Ca2＋ would apparently inhibit the catalytic capacity of E22. [Conclusion] E22 is a novel marine esterase with excellent properties such as thermostability, and alkaline, organic solvent and detergent tolerance. These characteristics suggest its application prospects in the industrial production.

关 键 词：海洋细菌 碱性酯酶 酶学性质 克隆表达 

分 类 号：Q936[生物学—微生物学]