| Amylase-2 was purified by two methods, and the characteristics of Amylase-2 were analyzed. The Starch-PAGE method for detecting amylase activity was optimized. Amylasecomponents showed transparent bands clearly when substrate concentration in resolving gel was 3.0% and renatured time was 3h. The amylase concentration influenced amylase components zone. There were four amylase components in mouldstarter and mature koji by Starch-PAGE. They were named Amylase-1、Amylase-2、Amylase-3 and Amylase-4 respectively, and Amylase-2 possessed the highest activity among them.Amylase-2 was purified to electrophoretic homogeneity by preparative electrophoresis ,it had a molecular weight of 52.0kD estimated by SDS-PAGE.Amylase-2 was also purified to homogeneity by sequential ammonium sulfate precipitation, DEAE-Cellulose-52 Fast Flow chromatography and Sephadex-G-100 gel filtration. The apparent molecular weight was 52.0kD.The specific activity was increased with the purification fold of 11. 35 and yield of 7.76%.Enzymatic properties showed that the enzyme exhibited the maximum activity at 50℃and pH6.2.Furthermore,the enzyme performed stable at 40℃and the pH range of acid-stable.The enzyme activity was activated by K+,Ca2+,Mg2+,Zn2+ ,and Mn2+ activated the enzyme markedly.Ba2+,Pb+,Cu2+ inhibited enzyme activity,and Hg2+ inhibited strongly, however, Na+,Fe2+ had no effect.The Km of Amylase-2 was 0.149mg/mL and Vmax was 6.14mg/min when enzyme activity was determined with soluble starch as substrate at 40℃. The substrate specificity results showed that the optimal substrate of the enzyme was wheat or sweet potato starch. Amylase-2 was considered asα- Amylase according to the results of enzyme hydrolysates by TLC . |
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