摘要： 对来源于大肠杆菌的植酸酶基因 appA 进行突变获得植酸酶基因突变体 appA -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/ appA -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OD_600nm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2.03×10⁵ U/mL。结果表明,通过高密度发酵提高了产植酸酶appA-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。

关键词: 重组植酸酶, 毕赤酵母, 高密度发酵, 高效表达

Abstract: The gene appA 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 appA -2QN produced in shaking-flask 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/ appA -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 OD_600nm of 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/mL.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.

Key words: Recombinant phytase, Pichia pastoris, High cell-density fermentation, High level expression

中图分类号: 

• Q786