豆血红蛋白在毕赤酵母中的表达条件优化

Optimization of leghemoglobin expression conditions in Pichia pastoris

摘要:

【背景】豆血红蛋白可赋予素肉制品类似牛肉的红褐色质地，已被美国食品药物监督管理局批准作为人造素肉的着色剂，近年来受到广泛关注。【目的】优化毕赤酵母产豆血红蛋白的培养条件，提高毕赤酵母产豆血红蛋白的产量。【方法】首先通过单因素试验研究蛋白胨种类、大豆蛋白胨浓度、铁盐种类及血红素浓度在诱导阶段对毕赤酵母产豆血红蛋白的影响；然后通过Plackett-Burman试验设计筛选出对豆血红蛋白产量影响最大的3个因素，再通过最陡爬坡法确定3个因素的变化范围，对3个因素进行响应面分析；最后根据响应面结果进行摇瓶发酵和发酵罐高密度发酵。【结果】单因素试验发现：用4%大豆蛋白胨作为主要氮源、甲醇诱导浓度为1.5%、血红素浓度为5 μmol/L时发酵效果较好，经过响应面优化后得到蛋白胨浓度为51.48 g/L、pH 5.66、培养基装液量35.84 mL/250 mL是最优发酵条件。在此优化条件下，LegH摇瓶发酵产量为0.191 mg/mL，与预测值(0.183 mg/mL)比较接近。采用5 L发酵罐进行高密度发酵，LegH产量最高达到0.384 mg/mL。【结论】优化了毕赤酵母表达豆血红蛋白的发酵条件，获得了较高产量，通过罐上发酵的进一步优化，具有一定的工业化前景。

关键词:豆血红蛋白;毕赤酵母;响应面法;培养基优化;高密度发酵

Abstract:

[Background] Leghemoglobin (LegH) can endow vegetarian meat products with beef-like reddish-brown color and meat texture, which has been approved by Food and Drug Administration as a colorant for vegetarian meat and has attracted extensive attention in recent years.[Objective] To optimize the expression conditions and increase the yield of LegH in P.pastoris. [Methods] Firstly, single-factor experiments were conducted to study the effects of peptones, peptone concentration, iron salts and hematin concentration on LegH production during the induction stage. Secondly, the three factors that have the greatest impact on LegH production were screened out through the Plackett-Burman design. Thirdly, the variation ranges and the optimum levels of the three factors were determined via the steepest ascent design and the response surface methodology (RSM), respectively. Finally, according to the RSM results, shake flask fermentation and high-density fermentation in a 5-L tank were carried out. [Results] The single-factor experiments demonstrated that with 4% soybean peptone as the main nitrogen source, the methanol concentration of 1.5%, and the hematin concentration of 5 μmol/L, the yield of LegH was higher. The RSM-optimized fermentation conditions were peptone concentration of 51.48 g/L, pH 5.66, and medium volume of 35.84 mL/250 mL. Under the optimized conditions, the yield of LegH in the shake flask was 0.191 mg/mL, close to the predicted value (0.183 mg/mL), and that in the 5-L tank reached 0.384 mg/mL. [Conclusion] Through optimization of the fermentation conditions for P.pastoris, higher yields of LegH were obtained,which had certain prospects for industrialization after further improvement in the tank fermentation.

Key words:leghemoglobin;Pichia pastoris;response surface methodology;medium optimization;high-density fermentation