In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L₉(3³) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5 L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71 μg/ml in the shake-flask, and 613.47 μg/ml in the 5 L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280 rpm for 4 days at 26 °C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.

为了获得用于生产重组灵芝免疫调节蛋白（rFIP-glu）的更好的发酵参数，研究了工程化的毕赤酵母GS115的发酵时间，温度，甲醇浓度和培养基的初始pH，同时对培养基的免疫调节活性进行了研究。确认了rFIP-glu。L ₉（3 ³）首先采用正交实验在摇瓶水平上优化各种发酵参数。随后在5 L发酵罐中验证了优化的发酵参数。在鼠巨噬细胞RAW264.7细胞上评估了rFIP-glu的生物活性，包括细胞活力和肿瘤坏死因子-α（TNF-α）mRNA。结果表明，当将毕赤酵母在甲醇浓度为1.0％和初始pH值为6.5，并在26°C下以280 rpm持续摇动4天。体外生物活性测定表明，rFIP-glu对RAW264.7细胞具有明显的毒性，并具有诱导巨噬细胞RAW264.7细胞中TNF-αmRNA表达的能力。总之，在最佳发酵参数下，工程化毕赤酵母显示出良好的发酵特性。它可能是进一步研究的候选工业菌株。