来源于海洋细菌Altererythrobacter epoxidivorans CGMCC1.7731T的新型碱性酯酶E22的酶学性质

摘要

[目的]克隆表达海洋细菌Altererythrobacter epoxidivorans CGMCC 1.7731T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pET-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(FamilyⅡ).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55C,并在60C孵育2h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Ba2+或Ca2+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 using p-nitrophenyl butyrate as substrate.It was an alkaline esterase that had highest activity at pH 10.5.The optimal reaction temperature was 55 C.After incubated at 60 C 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.