| 马纳纳，徐冬冬，黄伟谦，等.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[J].广东农业科学,2015,42(12):141-146 |
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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 |
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| DOI： |
| 中文关键词: 毕赤酵母 重组酵母酸性蛋白酶 酶学性质 蛋白水解 |
| 英文关键词: Pichia pastoris recombinant acid protease enzymatic properties proteolysis |
| 基金项目: |
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| 摘要点击次数: 1293 |
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| 中文摘要: |
| 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rPrA 经过浓缩后研究其酶学性质，初步探究了rPrA 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pH3.0 的发酵条件下酶活最高，可达46.92
U/mL。该酸性蛋白酶的最适pH 值为3.0，最适温度为35益。Mn2+对该酶活性有激活作用，多种金属离子对该酶
有抑制作用曰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。 |
| 英文摘要: |
| 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 kuand 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
role 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 4 000 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. |
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