过氧化物酶A4-Prx在毕赤酵母中的优化表达

Fermentation Optimization of a Novel Recombinant Peroxiredoxin in Pichia pastoris for Zearalenone Detoxification

摘要:

以来源于不动杆菌Acinetobacter sp. SM04的过氧化物酶A4-Prx的毕赤酵母工程表达菌株GS115/pPIC9K-A4-Prx为研究对象，优化其表达培养条件以提高该菌株对于目的蛋白A4-Prx的表达量。本论文首先研究了培养基成分与诱导条件对表达量的影响，结果表明培养基的pH、甘氨酸浓度和诱导温度对外源蛋白的产量均有显著影响。采用Box-Behnken设计，利用Design Expert软件进行二次回归分析得到了目的蛋白的最优表达条件为：诱导培养基pH 7.0、甘氨酸浓度为0.11%及诱导温度30 ℃。在此优化条件下重组蛋白的理论表达量达129.87 mg/L，约为未优化下的2倍，实验验证实际表达量达128.94 mg/L，且重组表达的过氧化物酶A4-Prx对酒糟蛋白饲料（DDGS）和食品中的玉米赤霉烯酮毒素（Zearalenone，ZEA）具有高效降解能力。本研究为过氧化物酶的工业化高密度发酵奠定了基础，推动生物降解ZEA研究的进展。

Abstract:

In order to maximize the expression of a novel peroxiredoxin purified from Acinetobacter sp. SM04 in recombinant Pichia pastoris GS115/pPIC9K-A4-Prx, a single-factor test coupled with response surface methodology (RSM) was used to adjust the induction condition. The results showed that pH value, temperature and glycine concentration were significant influencing factors for A4-Prx expression, and the optimized induction conditions were as follows: pH 7, 0.11% glycine at 30 ℃, and the theoretic yield of A4-Prx could reach to 129.873 mg/L. Then further experiments under the optimized induction conditions illustrated that the concentration of the novel peroxiredoxin synthesized by Pichia pastoris GS115/pPIC9K-A4-Prx was 128.941 mg/L and had a significantly ZEA-degrading effect on DDGS and some food materials. This study laid the foundation for the high-density industrial fermentation of peroxiredoxin as well as the development of ZEA degradation.