黑曲霉菊糖酶的纯化及性质

黑曲霉(Aspergillus niger)319发酵液经硫酸铵分级沉淀、DEAE-纤维素52离子交换层析和Sephadex G-100分子筛层析,得到了电泳纯的菊糖酶组分。提纯倍数为67,收率为25.5％。菊糖酶的最适pH为5.0,最适温度为60℃。此酶为单亚基蛋白,凝胶过滤法测得分子量为28000,含糖13.9％,用等电聚焦法测得等电点为5.4,该酶对温度有较高的稳定性,对pH的稳定范围较窄。Hg²⁺、Pb²⁺和Cu²⁺对该酶有强烈的抑制作用。此酶对菊糖有较强的底物专一性,产物为果糖,但它也可作用于蔗糖,I/S值为0.348。当以菊糖为底物时,K_m为6.25mmol/L,V_m为67.11 μmol·mg~(-1)·min~(-1)。

PURIFICATION AND PROPERTIES OF INULINASE FROM ASPERGILLUS NIGER

The main component of inulinase was purified from fermentation broth of Aspergillus niger 319 to homogeneity by using ammonium sulfate fraction, ion-exchange chromatography on DEAE-cellulose column and Sephadex G-100 gel filtration. The specific activity was as 67 folds at the fermentation broth, and the yield was 25.5%. The inulinase, containing 13.92% of carbohydrate, was a monomer protein with a molecular weight of 28,000 Dalton; and its isoelectric point was pH 5.4. The optimal pH and temperature of the inulinase was pH 5.0 and 60 degrees C, respectively. The enzyme was strongly inhibited by heavy metal ions of Hg2+, Pb2+ and Cu2+. The optimal substrate for the enzyme was inulin and the product was only fructose, but it also had invertase activity with the I/S of 0.348. The Km and Vm of the inulinase was 6.25 mmol/L and 67.11 mumol.mg-1.min-1, respectively.