Abstract:

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 ℃. Mn2+ revealed a strong promotion effect on the enzyme activity, K+ and Ca2+ only a weak promotion effect, Mg2+ no effect, Cu2+ the strongest inhibition effect, and other metal ions, including Co2+, Zn2+, Fe2+ 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.

Key words: recombinant E.coli, α-amylase, purification, characterization