文献类型：期刊文献

中文题名：R 122L突变提高重组人阴离子型胰蛋白酶的稳定性的研究

英文题名：The research of site R 122L mutate improve the stability of recombinant human anionic trypsin

作者：马强[1];吴倩[1];李素霞[1]

机构：[1]生物反应器工程国家重点实验室,华东理工大学,上海200237

年份：2014

卷号：34

期号：2

起止页码：60

中文期刊名：中国生化药物杂志

外文期刊名：Chinese Journal of Biochemical Pharmaceutics

收录：北大核心:【北大核心2011】；

语种：中文

中文关键词：重组人阴离子型胰蛋白酶;点突变;R122L;稳定性

外文关键词：recombinant human anionic trypsin;site-directed mutagenesis;R 122 L;stability

摘要：目的研究自切位点突变R122L对重组人阴离子型胰蛋白酶稳定性的影响。方法在大肠杆菌中表达重组人阴离子型胰蛋白酶和R122L突变体,并进行纯化,对纯化后酶及其突变体的稳定性进行对比研究。结果mhT2(R122L)发挥催化活力的最适pH为7~11;低温及酸性环境中稳定性较好,其活力在pH 7.6条件下同样能被典型的金属离子螯合剂(如EDTA)、还原剂(如β-ME)、变性剂、Fe3+及丝氨酸蛋白酶抑制剂所抑制,以N-苯甲酰-L-精氨酸(n-benzoyl-l-arginine ethyl ester,BAEE)作为底物时的米氏常数为0.010mmol/L。结论相比于hT2野生型,R122L突变体蛋白最适pH范围增大,耐热性能也有所提高,与底物BAEE的亲和力增强,同时对于Fe3+、金属离子螯合剂、还原剂以及变性剂等的耐受力也有增强,稳定性大大提高。
Objective The stability and other characteristics of the active recombinant human anionic trypsin(hT 2)with site-mutation R 122 L(mhT 2)were investigated.Methods An active human anionic trypsin and its R 122 L mutate were produced with E.coli BL 21(DE 3)and purified with ion-exchange chromatography.The properties of mutant were studied and compared with the wild type.Results The optimal pH for mhT 2 was 7~11.mhT 2 was active over a broad temperature range(4℃~80℃)and owned a little better thermal stability than the wild type.The inhibition of typical metal chelating agent(EDTA),Fe 3+,denaturant,reducer(β-ME)on activity of mhT 2 was the same as the wild type.Michaelis constant Km of mhT 2 was 0.010 mmol/L with BAEE as a substrate,a little lower than wild type.Conclusion Compared with the wild type,the R 122 L site mutate significantly enhanced tolerance to acidic pH、denaturants、reductions and autolysis.