重组植酸酶appA-2QN在毕赤酵母中的高效表达  

High Level Expression of a Recombinant Phytase app A-2QN in Pichia pastoris

作　　者：宋玛丽[1] 王茜[1] 杜军[1] 赵峰梅[2] 梁爱华[1] 

机构地区：[1]山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,山西太原030006 [2]山西省生物研究所,山西太原030006

出　　处：《华北农学报》2015年第2期55-58,共4页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31372199;31272100);山西省回国留学人员科研资助项目(2012-重点1)

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。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 concentra-tion of methanol during fermentation. The results showed that the OD600nm of the cell reached 313 after 147 h fermen-tation 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.

关 键 词：重组植酸酶 毕赤酵母 高密度发酵 高效表达 

分 类 号：Q786[生物学—分子生物学]