泡盛曲霉产菊粉酶的分离纯化和酶学性质

Isolation,Purification and Characterization of Inulinase from Awamori

利用(NH4)2SO4分级沉降、透析袋、DEAE-纤维素离子交换层析、Sephadex凝胶层析和SDS-PAGE电泳分析对泡盛曲霉(Aspergillus awamori)分泌的菊粉酶粗酶液进行分离纯化,探究了菊粉酶的酶学性质,得到菊粉酶Ⅰ、菊粉酶Ⅱ、菊粉酶Ⅲ。菊粉酶的Ⅰ纯化倍数为10.81倍,酶比活力为261.13U/mg;菊粉酶Ⅱ的纯化倍数为14.19倍,酶比活力为400.72U/mg;菊粉酶Ⅲ的纯化倍数为13.92倍,酶比活力为392.99U/mg,表明泡盛曲霉产胞外菊粉酶具有三种同工酶成分。纯化的菊粉酶经SDS-PAGE检测,显示为单一条带。经测定该酶主要表现外切菊粉酶活力,最适温度为60℃,在20—60℃范围内具有良好的温度耐受性,最适p H为5.0,p H5.0—7.0之间具有较稳定的酸碱耐受性。以菊粉为底物,测得动力学常数Km=12.80mmol/m L、Vmax=31.87mg/(m L/min)。

Inulinase crude enzyme solution from awamori was seperated and purified by Ammonium sulfate sedimentation, dialysis bags, DEAE - cellulose ion exchange chromatography, sephadex and SDS - PAGE and the charaterization of inulinase was explored. Inulinase Ⅰ, Ⅱ ,Ⅲ were pu- tiffed. Inulinase Ⅰ was purified 10.81 folds with specific activity of 261.13U/rag, while Inulinase Ⅱ was purified 14.19 folds with specific activity of 400.72U/mg and lnulinase Ⅲ was purified 13.92 folds with the specific activity of 392.99U/mg. The purified enzyme was identified with a molec- ular mass by SOS- PAGE. The enzyme was determined mainly other tangential Inulinase vitality, the optimum temperature was 60℃ ,and it had a good temperature resistance within the range of 20 - 60 ℃. The optimum pH was 5.0, and it maintained more stable pH tolerance between pH 5.0 - 7.0. It was measured with Km = 12.80mmol/ml, Vmax = 31.87mg/（mL/min）.