一种新颖的毕赤酵母酸性蛋白酶基因的克隆和异源表达以及酶学性质研究  

Cloning and heterologous expression of a novel Pichia pastoris acid protease gene and study on its enzymatic properties

作　　者：马纳纳 徐冬冬[1] 黄伟谦[1] 游子娟 罗晓春[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《广东农业科学》2015年第12期141-146,共6页Guangdong Agricultural Sciences

基　　金：广东省科技计划项目(2012B020311003;2013B010404003)

摘　　要：采用基因克隆方法获得酸性蛋白酶基因,在毕赤酵母KM71表达系统中进行表达,并将重组酵母酸性蛋白酶r Pr A经过浓缩后研究其酶学性质,初步探究了r Pr A对大豆蛋白和酪蛋白的水解情况。结果表明:经过异源表达后的重组毕赤酵母酸性蛋白酶分子量约为44 ku,在p H3.0的发酵条件下酶活最高,可达46.92U/m L。该酸性蛋白酶的最适p H值为3.0,最适温度为35℃。Mn2+对该酶活性有激活作用,多种金属离子对该酶有抑制作用;0.1 mmol/L的Pepstain A即可完全抑制酶活,说明此重组酶的活性中心有天冬氨酸残基。对大豆蛋白和酪蛋白的初步水解试验可知,当E/S为4 000 U/g时,此重组酶对大豆分离蛋白和酪蛋白的水解度分别为9.0%和8.34%。优化发酵条件以及与多种蛋白酶进行复配,该重组蛋白酶将在蛋白水解中有很好的应用前景,有望应用到畜禽饲料加工行业。This study obtained a novel acidi protease gene by cloning, and it was expressed in Pichia pastoris KM71, then the enzymatic properties of this recombinant yeast acid protease rPrA were studied after being concentrated. Besides, its degree of hydrolysis to soy protein isolate （SPI） and casein was initially researched. The results showed that the molecular weight of heterologous expression of recombinant P. pastoris acid protease was about 44 ku,and it could get the highest enzyme yield of 46.92 U/mL under the fermentation condition of pH 3.0. The optimum pH for the acidi protease was 3.0, the optimum temperature for rPrA was 35℃. Although Mn2＋ had an active rote in its activity, a variety of metal ions could inhibit its activity. 0.1 mmol/L pepstain A could completely inhibit its activity, proving that rPrA had aspartic acid residues in its active site. The hydrolysis to SPI and casein experiment showed that, when E/S was 4 0130 U/g, its hydrolysis degrees to SPI and casein were 9.0% and 8.34%, respectively. The results indicated that by optimizing fermentation conditions and functioning with other proteases, rPrA would have a great utility potential in protein hydrolysis, and it was expected in apply to the feed processing industry.

关 键 词：毕赤酵母 重组酵母酸性蛋白酶 酶学性质 蛋白水解 

分 类 号：S188[农业科学—农业基础科学]