雅致放射毛霉AS3.2778酸性蛋白酶的纯化及性质研究

Purification and Properties of Acidic Proteases from Actinomucor elegans AS3.2778

毛霉蛋白酶对大豆蛋白有较高的水解效率以及对蛋白水解物具有良好的脱苦效果,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,采用硫酸铵盐析、羧甲基琼脂糖凝胶阳离子交换层析及苯基琼脂糖凝胶疏水层析对其进行分离纯化,从雅致放射毛霉AS3.2778的发酵麸曲中纯化得到两个酸性蛋白酶组分Pa1和Pa2。电泳分析并结合凝胶色谱确定纯化的两个组分均是单体酶,其分子质量分别在35ku和33ku。对其性质研究发现:Pa1在pH5.5、50℃有最大催化活性,Pa2在pH4.5、50℃有最大催化活性;两者在温度低于40℃,酸性的环境(pH4.0～7.0)中有很好的稳定性;浓度10μmol/L的Pepstatin A可以完全抑制这两种蛋白酶的活性,表明Pa1和Pa2属于天冬氨酸蛋白酶家族;金属离子对两者的活性影响不显著,其中仅Cu2+、Zn2+对Pa1有弱的抑制作用,而Mn2+、Co2+、Cu2+对Pa2有弱的抑制作用;Pa1和Pa2具有相同的肽键选择性,并且与雅致放射毛霉碱性蛋白酶Pb1存在一定的肽键选择互补性;Pa2的N端氨基酸序列为:GTGTVPVTDY,这与来源于根霉属微生物的酸性蛋白酶有很高的同源性。

Proteases from Mucor have a good market prospect in the production of soybean polypeptides for their high effectiveness in hydrolyzing soy protein and debittering hydrolyzed soy protein. In this work, we isolated and purified two acid proteases, Pal and Pa2 fromActinomucor elegans AS3.2778-fermented wheat bran by ammonium sulfate fractional precipitation, CM-Sephadex cation-exchange chromatography and phenyl-Sephadex hydrophobic chromatography. Pal and Pa2 were identi- fied by SDS-PAGE analysis as monomeric enzymes with respective molecular weight of 35 ku and 33 ku. Pal had the highest activity at pH 5.5 and 50 ℃, and Pa2 at pH 4.5 and 50 ℃. Both enzymes showed good stability at temperatures below 40 ℃ and in acidic environment （pH 4.0 -- 7.0） and were entirely inhibited by 10 μ mol/L Pepstatin A, indicating that they belong to the aspartic protease family. Pal and Pa2 were weakly inhibited by Cu2＋ and Zn2＋ and Mn2＋, Co2＋ and Cu2＋, respectively. The two enzymes had the same peptide bond selectivity complementary to that of alkaline protease Pbl from Actinomucor elegans AS3.2778. The N-terminal amino acid sequence of Pa2 was GTGTVPVTDY, which was highly homologous to that of acid proteases from Rhizopus published in the NCBI database.