双水相系统纯化重组苯丙氨酸脱氢酶的条件研究  

Recovery and Purification of Recombinant Phenylalanine Dehydrogenase from Interface in Aqueous Two-phase Systems

作　　者：李进军[1] 赵晨[1] 王辂[1] 李端华[1] Li Jin-jun;Zhao Chen;Wang Lu;Li Duan-hua(Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province,Department of Pharmacy,Chengdu University,Chengdu 610052)

机构地区：[1]成都大学药学院,抗生素研究与再评价四川省重点实验室,成都610052

出　　处：《国外医药（抗生素分册）》2022年第6期421-424,I0003,共5页World Notes on Antibiotics

基　　金：四川省科技厅国际合作项目“针对新型污染物-耐药基因的酶法清除技术研究”(2020YFH0013);四川省科技厅国际合作项目“基于噬菌体技术的皮炎湿疹新治疗方案研究”(2021YFH0016);成都市科技局国际合作项目“酶法清除耐药基因的关键技术研究”(2020-GH02-00031-HZ)。

摘　　要：目的 研究重组大肠埃希菌表达的苯丙氨酸脱氢酶在双水相系统中的分配,并对其影响因素进行单因素考察,优化出最佳的双水相系统。方法 采用高效液相色谱法检测,依据纯度和收率来优化纯化条件。研究了聚乙二醇(PEG)分子量,聚乙二醇(PEG)浓度,硫酸铵浓度,氯化钠浓度,pH对苯丙氨酸脱氢酶在双水相中分配的影响。结果 优化的双水相系统为:PEG4000 10%,硫酸铵14%,氯化钠10%且pH 6.5,此系统下的酶活收率、纯化倍数及酶比活分别为162%、578及9280 U/mg。结论 在双水相系统的中间层提取苯丙氨酸脱氢酶是一种新的纯化方式,此方法简单易行,收率较高,纯度好且处理量大,仅通过一步实验就达到初级纯化和浓缩的目的,是一种经济有效的纯化方法,为下步工业化生产奠定基础。Objective To investigate the partition of recombinant E.coli phenylalanine dehydrogenase(PheDH)in aqueous two-phase systems(ATPS), the factors which influence the partition in the system were also examined with One-Factor-at-a-Time method, then the optimal ATPS system was obtained. Method Combined with HPLC, all the samples in the process were analyzed to optimize the purification conditions. Results The best optimal ATP system was 10%(w/w) PEG-6000, 14%(w/w)(NH4)2SO4 and 10%(w/w) NaCl at pH 6.5. The activity recovery, purification factor and specific activity values were of 162%、578、9280 U/mg, respectively. Conclusion Data showed that to extract recombinant dehydrogenase(PheDH) from the interface of the ATPS is an effective way, and also is a new method, which has several advantages such as simplicity of operation, high purification, high handling capacity, and the primary purification and concentration is carried out in a single-step operation, which make it an economical and attractive technology for industrial production of recombinant dehydrogenase(PheDH).

关 键 词：苯丙氨酸脱氢酶(PheDH) 双水相系统(ATPS) 中间层 硫酸铵 聚乙二醇(PEG) 纯化 

分 类 号：R917[医药卫生—药物分析学]