青霉菌B01 产菊粉酶特性的研究及菊粉酶系分析

研究了pH 值、温度、底物浓度、金属离子对青霉菌(Penicillium sp.)B01 所产胞外菊粉酶活性的影响以及酶活随反应时间的变化情况。结果表明，pH4.6 时菊粉酶活力最大，最适反应温度是55℃。在pH3.6～5.4 的范围内，60℃以下酶活较稳定。Ca2+ 对菊粉酶有激活作用，而Cu2+ 对其活性强烈抑制。采用活性聚丙烯酰胺凝胶电泳法(PAGE)对该酶粗液进行蛋白质分析，获得较清晰的6 条蛋白谱带；2,3,5- 氯化三苯基四氮唑(TTC)活性染色结果显示，只有3 条谱带表现为具有菊粉酶活性。对酶解产物用薄层层析(TLC)和高效液相色谱(HPLC)法的分析结果，进一步证实这3 条谱带具有菊粉酶活性，并确定其主要为外切型菊粉酶，产物主要为果糖。

Characteristics and Enzyme System of Inulinase from Penicillium sp.B01

With inluin as the reaction substrate, the effects of pH value, temperature, substrate concentration, metal ion and reaction time on the activity of inulinase from Penicillium sp.B01 were investigated. The results showed that the maximal inulinase activity may be observed at 55 ℃ and pH 4.6; under the conditions of pH 4.6 to 5.4 and below 60 ℃, the inulinase activity is stable; Ca2+ promotes the inulinase activity, but Cu2+ significantly inhibits it. Moreover, the crude inulinase of Penicillium sp.B01 was separated by native-polyacrylamide gel electrophoresis (native-PAGE), and six distinct protein bands were obtained. Then the seperated inulinase was tested with inulin and triphenyl tetrazolium chloride (TTC) was used for activity staining. But only three bands were found to have inulinase activity. The analysis of reaction product by TLC and HPLC further proved that these three bands have the exoinulinase activity, and confirmed that the main composition of reaction product is fructose.