来自基因工程菌E.coli BL21/pET-DsbA-MalQ的麦芽糖转糖基酶的分离纯化及酶学性质研究

Purification and Characterization of Amylomaltase from Genetic Engineer Strain E.coli BL21/pET-DsbA-MalQ

通过6-His的分离标签,联合采取硫酸铵沉淀,生物半透膜透析和亲和层析等方法,纯化了目标酶。通过使用凝血酶切割DsbA-MalQ融合蛋白,证明酶切后酶蛋白仍具有麦芽糖转糖基酶活性。该酶的最适温度为37℃,最适pH值为6.5。当处理温度小于40℃时,酶的活力基本保持在最高活性的80%以上,相对稳定;在酶的pH耐受方面,在pH5.5～8.0范围内较稳定。金属离子和EDTA对酶活性的影响实验表明,EDTA对酶活的影响不大,但Zn2+、Cu2+、Hg2+、Ag+对酶蛋白有较强的抑制作用,而Ca2+、Mg2+、Mn2+等对表达的酶有一定的激活作用。在37℃,pH6.5时以麦芽三糖为底物测得酶促反应的Km值为0.378mmol/L,Vmax值为1.979mmol/(L·min)。

The fusion protein was purified by Ni-NTA His-Bind affinity chromatography and cut with thrombin. The purified protein had 4- α -glucanotmnsferase activities. Its optimum pH and optimum temperature were 37 ℃ and pH 6,5, respectively. When the enzyme was treated at different temperature or pH, the enzyme activity would show different. When the treated temperature was over 40 ℃ or the treated pH below 4.5 or over 9.0, the enzyme activity would decrease quickly. Influences of ions and EDTA on enzyme activity were studied. The results showed that ions Zn^2＋, Cu^2＋, Hg^2＋, Ag~ inhibited enzyme activity, ion Ca^2＋, Mg^2＋, Mn^2＋ increased the enzyme activity, while EDTA showed no such effects on the enzymes. The Km and Vmax were 0.378 mmol/L and 1.979 mmol/（L · min）, respectively.