雅致放射毛霉AS3.2778碱性蛋白酶的制备及应用

毛酶蛋白酶对大豆蛋白具有较高的水解效率以及对蛋白水解物良好的脱苦效果，因此在大豆多肽的制备方面显示出很好的应用前景。为了开发这一蛋白酶系，采用离子交换，疏水层析及凝胶层析对其进行了分离纯化，从雅致放射毛霉As3.2778的发酵麸曲中分离纯化出一种蛋白酶，其纯度提高了22.7倍，酶活回收16.1%，最终比酶活可达到6094u/mg。电泳分析发现，该蛋白酶在还原及非还原条件下均显示出单一条带，表明该酶已达到电泳纯，并且是一单体蛋白，其分子量大约在32KDa。酶谱分析表明，纯化的蛋白酶仅为原发酵麸曲中三种蛋白酶组分中的一种。以大豆蛋白为底物，探讨了纯化后的蛋白酶对大豆蛋白的水解效果。结果显示，该蛋白酶在55℃，pH8.0～10.0的条件下对大豆蛋白显示出较强的水解能力。对比实验发现，纯化的毛酶蛋白酶对大豆蛋白的水解效果优于Papain、Alcalase及Protamex，表明该蛋白酶具有更为广泛的肽键选择性，对大豆蛋白亲和力更强，在大豆蛋白肽链上存在相对较多的酶切位点。

Preparation and Application of one Alkaline Protease from Actinomucor elegans AS3.2778

Proteases from mucor had shown a good market prospect in the production of soy-polypeptides for their high hydrolysis efficiency to soy protein and debittering effect to hydrolysate. To explore these proteases, using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography, one protease was purified from the fermented wheat bran by Actinomucor elegans AS3.2778. After these procedures, the protease was purified 22.7 folds with a total yield of 16.1% and the final specific activity was 6094u/mg. SDS- polyacrylamide gel electrophoresis shown that the enzyme was homogeneous after purification and it was a single chain polypeptide with a molecular weight of 32kDa. Using SPI as substrate, the purified protease had relative high activity at 55℃ and pH8.0～10.0. Hydrolysis experiments shown this protease had a relative high hydrolysis efficiency to SPI than papain,alcalase and protamex，and indicated the purified protease had more extensive peptide bonds selectivity, a relative high affinity to SPI and more cleavage points on SPI than papain, alcalase and protamex.