【摘要】 对来源于大肠杆菌的植酸酶基因app A进行突变获得植酸酶基因突变体app A-2QN,利用酵母表达系统在摇瓶培养条件下表达后,该植酸酶突变体显示出良好的热稳定性。为提高该植酸酶的表达量,降低植酸酶的生产成本,对表达该酶的重组酵母菌GS115/app A-2QN进行了高密度发酵研究,通过控制发酵过程中碳源(甘油)的添加使得菌体生长达到一定密度,从而实现高效表达。在诱导蛋白质表达阶段,发酵液中甲醇的含量影响植酸酶的表达量,利用变色酸分光光度法对甲醇含量进行了检测分析。结果表明,在5 L发酵罐中进行高密度发酵147 h后,菌体浓度OD600nm达到313,通过将甲醇浓度控制在2.5%左右,经过诱导102 h后,蛋白达到了较高的表达量,为7.06 g/L,酶活性(发酵效价)为2.03×105U/m L。结果表明,通过高密度发酵提高了产植酸酶app A-2QN的酵母工程菌的细胞生长密度和蛋白质表达量,揭示该重组酵母菌株具有良好的表达稳定性。更多还原

【Abstract】 The gene app A for phytase from Escherchia coli was modified by means of site directed mutagenesis to improve its thermal stability. Using yeast expression system the mutant phytase app A-2QN produced in shakingflask culture showed an increased thermal stability. To increase its expression level and lower production costs,an approach of the high cell-density fermentation of recombinant yeast GS115 / app A-2QN was carried out in this study.During fermentation the concentration of glycerin was controlled to promote the cell growth and achieve the high cell-density fermentation. The method of chromotropic acid spectrophotography was used to determine the concentration of methanol during fermentation. The results showed that the OD600 nmof the cell reached 313 after 147 h fermentation in a 5 L fermentor. After inducing of 102 h with methanol at a concentration of 2. 5%,the expression level of phytase reached 7. 06 g / L. The phytase activity was 2. 03 × 105 U / m L. These results indicated that the cell density and the expression level of target protein were significantly improved by high density fermentation. The recombinant yeast strain has favorable expression stability.更多还原