米曲霉酸性蛋白酶基因在毕赤酵母中的异源表达及酶学性质

Heterologous expression and characterization of Aspergillus oryzae acidic protease in Pichia pastoris

摘要:

酸性蛋白酶作为一类重要的天冬氨酸蛋白酶，被广泛应用于食品、医药和皮革等领域。为推动酸性蛋白酶的研究及应用，通过对发酵豆制品样品进行宏基因组测序，从中获得米曲霉酸性蛋白酶基因pepA，在毕赤酵母GS115中进行异源表达，并对重组酶PepA进行酶学性质分析。结果显示毕赤酵母发酵上清液中酸性蛋白酶的活性为50.62 U/mL。SDS-PAGE验证PepA的分子量约为50 kDa，且发酵上清液几乎无杂蛋白。PepA的最适pH值为4.5，最适温度为50 ℃，Mn2+和Cu2+对其具有激活作用，而Fe3+、Fe2+与Ca2+则具有抑制作用。上述研究结果可为米曲霉酸性蛋白酶的异源表达及其相关工业应用提供指导。

Abstract:

Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn2+ and Cu2+ enhanced the activity of PepA, whereas Fe3+, Fe2+ and Ca2 had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.