重组大肠杆菌耐热α-淀粉酶的分离纯化及其酶学性质研究  

Purification and Enzymological Characterization of Recombinant E.coli Thermostable Alpha-Amylase

作　　者：张洪斌[1] 张强[1] 胡雪芹[1] 

机构地区：[1]合肥工业大学化学工程学院,安徽合肥230009

出　　处：《食品科学》2009年第21期217-220,共4页Food Science

基　　金：安徽教育厅重点项目(KJ2008A067);合肥工业大学博士基金项目(GDBJ2008-021)

摘　　要：对重组大肠杆菌耐热α-淀粉酶分离纯化及其酶学性质进行研究,结果表明:该酶分子量约为90kD,最适温度为60～70℃,最适pH值为6.6,酶学动力学常数Km值为143.52mmol/L;酶活力在pH5.4～7.8较为稳定;4℃保存2周酶活力仅下降一半,35℃保温3d有50%以上的酶活力,70℃以上酶失活很快;Mn2+对酶催化作用有较大的促进,K+、Ca2+有微弱的促进作用,Mg2+对催化反应无影响,Cu2+的抑制作用最强,其他金属离子Co2+、Zn2+、Fe2+对酶催化作用有不同程度的抑制作用;有机离子对酶催化作用均是抑制作用,其中抑制作用最强的是SDS。After accomplishing fermentation, recombinant E.coli cells were disrupted via ultrasonic treatment for the release of thermostable α-amylase from them. Subsequently, the enzyme was purified by GST affinity chromatography and subjected to enzymological characterization. This enzyme exhibited a molecular weight of 170 kD, an optimum temperature ranging 60- 70 ℃, an optimum pH value of 6.6 and a Km value of 0.14352 mol/L. Stable activity was observed at pH 5.4-7.8. In addition, only 50% activity was lost after 2 weeks of storage at 4 ℃, 35 ℃ storage for 3 d led to an activity loss of more than 50%,whereas this enzyme was inactivated rapidly when the storage temperature was more than 70 ℃. Mn^2＋ revealed a strong promotion effect on the enzyme activity, K^＋ and Ca^2＋ only a weak promotion effect, Mg^2＋ no effect, Cu^2＋ the strongest inhibition effect, and other metal ions, including Co^2＋, Zn^2＋, Fe^2＋ various inhibition effects. Organic ions such as EDTA, SDS, urea and Tris were all inhibitors of this enzyme, among which SDS was the strongest one.

关 键 词：重组大肠杆菌 Α-淀粉酶 纯化 性质 

分 类 号：Q939.97[生物学—微生物学]