Abstract: Alkaline protease is a widely used protease preparation and plays a crucial role in many fields. Therefore, it is important to discover high-quality alkaline protease genes and overexpression successfully for the needs of industrial applications. In this study, the alkaline protease genes, pa1 and pa2 from Cordyceps fumosorosea and Beauveria bassiana, respectively, were ligated to the expression vector pPIC9, and successfully expressed in Pichia pastoris GS115 with high yield. The purified recombinant alkaline protease PA1 and PA2 both showed optimum activities at pH 8.5 and 60 ℃, which distinguished from other enzyme counterparts. The two enzymes showed good thermostability at 50 ℃, and both retained about 70% of activity after incubating for 10 min at 60 ℃. PA1 and PA2 had wide pH ranges and retained more than 80% of activity after incubation for 1 h from pH 4.0 to 11.0. In addition, PA1 and PA2 also exhibited some resistance to surfactants and reducing agents. All these favorable enzymatic properties made PA1 and PA2 attractive for potential applications in industrial.

Key words: alkaline proteases, Pichia pastoris GS115, cloning and expression, characterization

摘要： 碱性蛋白酶作为广泛应用的蛋白酶之一，具有重要的工业应用价值。发掘优质的碱性蛋白酶基因，实现其高效表达，有助于更好的满足工业应用需求。分别将Cordyceps fumosorosea和Beauveria bassiana来源的碱性蛋白酶基因pa1及pa2与表达载体pPIC9连接，并在毕赤酵母GS115中实现高效表达。对于纯化后的重组蛋白PA1及PA2的酶学性质进行测定，二者最适pH均为8.5，最适温度均为60 ℃，与同类酶相比具有一定的优势；PA1及PA2的温度稳定性较好，50 ℃下酶活保持稳定，60 ℃下处理10 min，二者均保持70%左右的酶活；PA1及PA2的pH耐受范围宽泛，在pH 4.0~11.0的环境中处理1 h，均能保持80%以上的活性。此外，PA1及PA2对于表面活性剂和还原剂也表现出一定的耐受性。这些性质都表明PA1及PA2是具有工业应用潜力的优质碱性蛋白酶。

关键词: 碱性蛋白酶, 毕赤酵母, 克隆表达, 酶学性质