小麦内质网氧化还原酶在毕赤酵母中的表达

Expression of Wheat Endoplasmic Reticulum Oxidoreductin 1 in Pichia pastoris

为开发健康天然的新型酶制剂型面粉改良剂,利用毕赤酵母重组表达了小麦内质网氧化还原酶1(wEro1);将wero1基因亚克隆至毕赤酵母表达载体pPICZαA,实现了wEro1在毕赤酵母(Pichia pastoris)GS115中的分泌表达,优化诱导表达条件后wEro1的产量可达(53.24±2.11)U/mL;应用硫酸铵沉淀和阴离子交换层析纯化了wEro1,对电泳纯wEro1进行酶学性质研究.结果表明:wEro1具有催化二硫键异构酶(PDI)的巯基氧化活性,且最适pH为7.5,最适温度为30℃;在低温和中性pH条件下,该酶具有较好的稳定性.将毕赤酵母重组wEro1添加到面粉中,考察其对面粉加工品质的影响,发现毕赤酵母重组wEro1改善面粉粉质特性的能力比大肠杆菌重组wEro1更强.

Wheat endoplasmic reticulum oxidoreductin 1(wEro1)was expressed by Pichia pastoris recombinant to develop a healthy and natural novel biological flour improver.The wero1 gene was subcloned into the Pichia pastoris expression vector pPICZαA,and the secretory expression of wEro1 was realized in Pichia pastoris GS115.The yield of wEro1 reached(53.24±2.11)U/mL after optimizing the induced expression conditions.The expressed products were purified by ammonium sulfate precipitation and anion exchange chromatography,and the enzymatic properties of wEro1 were investigated.The reasults show that wEro1 possesses thiol oxidizing activity catalyzing disulfide isomerase(PDI)with an optimum pH of 7.5 and an optimum temperature of 30℃;the enzyme has good stability under low temperature and neutral pH conditions.Pichia pastoris recombinant wEro1 was added to flour to investigate its effect on flour processing quality.It was found that the ability of Pichia pastoris recombinant wEro1 to improve flour silty properties was stronger than that of E.coli recombinant wEro1.