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首页> 外文期刊>Protein Expression and Purification >High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris
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High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris

机译：在Pichia Pastoris中重组曲霉的高水平表达，纯化和酶促表征重组曲霉碱性蛋白酶

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摘要

An alkaline protease (Ap) was cloned from Aspergillus sojae GIM3.33 via RT-PCR technique. A truncated Ap without the signal peptide was successfully expressed in the Pichia pastoris KM71 strain. The following describes the optimal process conditions for the recombinant engineering of a strain expressing a recombinant Ap (rAp) in a triangular flask: inoculum concentration OD600 value 20.0 in 40 mL working volume (in 500 mL flasks), methanol addition (1.0%; volume ratio), 0.02% biotin solution (60 mu L), and YNB primary concentration (13.0 g/L). Under these conditions, the protease activity of rAp in the fermentation broth reached 400.4 +/- 40.5 U/mL after induction for three days. The rAp was isolated and purified, and its enzymatic characteristics were tested. Its optimal pH was 10.0, and it remained stable in a pH range of 7.0-10.0. Its optimal temperature was 45 degrees C and it retained 50% activity at 40 degrees C for 60 min. The rAp activity was significantly inhibited by PMSF, Zn2+ and Fe2+ and the rAp had a broad substrate specificity for natural proteins and synthetic peptide substrates, and preferred substrates at PI position with large hydrophobic side-chain groups. Compared to Papain (8.7%) and Alcalase (12.2%), the degree of hydrolysis of rAp to soy protein isolate was 16.5%; therefore, rAp was a good candidate for the processing of food industry byproducts.
机译：通过RT-PCR技术从Aspergillus Sojae GIM3.33中克隆碱性蛋白酶（AP）。没有信号肽的截短的AP在Pichia Pastoris KM71菌株中成功地表达。以下描述了在三角形烧瓶中表达重组AP（RAP）的菌株的重组工程的最佳过程条件：Inoculum浓度OD600值20.0，在40ml工作体积（500ml烧瓶中），加入甲醇加入（1.0％;体积;体积比例），0.02％的生物素溶液（60μl）和YnB初级浓度（13.0g / L）。在这些条件下，在诱导后，发酵液中的RAP的蛋白酶活性达到400.4 +/- 40.5 U / mL三天。将RAP被隔离并纯化，并测试其酶促特性。其最佳pH值为10.0，其在7.0-10.0的pH范围内保持稳定。其最佳温度为45℃，保留为45℃。 40℃的50％活性60分钟。 PMSF，Zn2 +和Fe2 +显着抑制了RAP活性，并且RAP对天然蛋白质和合成肽基材具有宽的底物特异性，以及具有大型疏水侧链基的PI位置处的优选基板。与木瓜蛋白酶（8.7％）和alcalase（12.2％）相比，Rap对大豆蛋白分离物的水解程度为16.5％;因此，RAP是加工食品工业副产品的良好候选人。

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