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(pNPB),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 torridus DSM9790 as template;then it was connected to plasmid pET-28a(+)to construct recombinant engineering strain.The properties of EstPt 1 were characterized by SDS-PAGE,pNPB 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.
作者
杜晓韵
张兴群
张晓彦
DU Xiao-yun;ZHANG Xing-qun;ZHANG Xiao-yan(College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China;State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 201620,China)
出处
《食品与药品》
CAS
2019年第3期169-173,共5页
Food and Drug
基金
国家自然科学基金(21506055)
国家自然科学基金(51863020)