R 122L突变提高重组人阴离子型胰蛋白酶的稳定性的研究  

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] 

机构地区：[1]生物反应器工程国家重点实验室,华东理工大学,上海200237

出　　处：《中国生化药物杂志》2014年第2期60-64,67,共5页Chinese Journal of Biochemical Pharmaceutics

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.

关 键 词：重组人阴离子型胰蛋白酶 点突变 R122L 稳定性 

分 类 号：R394.3[医药卫生—医学遗传学]