Picrophilus Torridus嗜热酯酶的克隆表达及性质研究
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摘要
目的目的以Picrophilus torridus DSM9790基因组为模板,克隆嗜热酯酶EstPt 1的基因,实现其在大肠杆菌BL21(DE3)中的表达,并对重组EstPt 1进行酶学性质表征。方法利用PCR扩增,获得EstPt 1基因,连接到pET-28a(+)质粒,将重组质粒转化入大肠杆菌BL21(DE3)中,构建重组工程菌株。通过聚丙烯酰胺凝胶电泳(SDS-PAGE),对硝基苯酚法(p NPB),薄层色谱(TLC)和高效液相色谱法(HPLC)对EstPt 1的性质进行研究。结果 EstPt 1成功表达,分子量为35 kD;最适反应温度为85℃,最适pH 9.0,有良好的热稳定性和酶活,能在24 h内完全降解5 mmol/L邻苯二甲酸二丁酯(DBP)。结论重组EstPt 1有很强的高温耐受性和稳定性,以及良好的DBP降解活性。
Objective To clone and express the gene of thermophilic esterase EstPt 1 in E. coli BL21(DE3), and to characterize the enzymatic property of recombinant EstPt 1, using Picrophilus torridus DSM9790 genome as template. Methods The gene encoding EstPt 1 was obtained by PCR amplification using genomic DNA of Picrophilus torridusDSM9790 as template; then it was connected to plasmid pET-28 a(+) to construct recombinant engineering strain. The properties of EstPt 1 were characterized by SDS-PAGE, p NPB method, TCL and HPLC. Results EstPt 1 was successfully expressed and its molecular weight was determined to be 35 kD. It showed good thermostability and enzymatic activity, with optimum reaction temperature of 85 ℃ and optimum pH of 9.0. It could completely degrade 5 mmol/L dibutyl phthalate(DBP) in 24 h. Conclusion Recombinant EstPt-1 has high temperature tolerance, stability and good DBP degradation activity.
Objective To clone and express the gene of thermophilic esterase EstPt 1 in E. coli BL21(DE3), and to characterize the enzymatic property of recombinant EstPt 1, using Picrophilus torridus DSM9790 genome as template. Methods The gene encoding EstPt 1 was obtained by PCR amplification using genomic DNA of Picrophilus torridusDSM9790 as template; then it was connected to plasmid pET-28 a(+) to construct recombinant engineering strain. The properties of EstPt 1 were characterized by SDS-PAGE, p NPB method, TCL and HPLC. Results EstPt 1 was successfully expressed and its molecular weight was determined to be 35 kD. It showed good thermostability and enzymatic activity, with optimum reaction temperature of 85 ℃ and optimum pH of 9.0. It could completely degrade 5 mmol/L dibutyl phthalate(DBP) in 24 h. Conclusion Recombinant EstPt-1 has high temperature tolerance, stability and good DBP degradation activity.
引文
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[2]王嘉翼,樊双虎,任超,等.一株黄色杆菌的分离鉴定及对邻苯二甲酸酯的降解研究[J].生物技术通报,2018,34(10):32-40.
[3]Wang Z,Deng D Y,Yang L L.Degradation of dimethyl phthalate in solutions and soil slurries by persulfate at ambient temperature[J].J Hazard Mater,2014,271:202-209.
[4]Wu J,Liao X,Yu F,et al.Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp.strain M673 and functional analysis of its expression product in Escherichia coli[J].Appl Microbiol Biotechnol,2013,97(6):2483-2491.
[5]Ahmadi E,Yousefzadeh S,Ansari M,et al.Performance,kinetic,and biodegradation pathway evaluation of anaerobic fixed film fixed bed reactor in removing phthalic acid esters from wastewater[J].Sci Rep,2017,7:41020.
[6]Chang B V,Lu Y S,Yuan S Y,et al.Biodegradation of phthalate esters in compost-amended soil[J].Chemosphere,2009,74(6):873-877.
[7]吴学玲,代沁芸,梁任星,等.利用高效降解菌株强化修复土壤中DBP及其细菌群落动态解析[J].中南大学学报,2010,42(5):1188-1194.
[8]Zhang X Y,Fan X,Qiu Y J,et al.Newly identified thermostable esterase from Sulfobacillus acidophilus:properties and performance in phthalate ester degradation[J].Appl Environ Microbiol,2014,80(22):6870-6878.
[9]Kruger N J.The Bradford method for protein quantitation[J].Methods Mol Biol,1994,32:9-15.