摘要
由于限制性内切酶在重组表达时具有切割宿主DNA的细胞毒性,生产难度大,其供应一直被国外企业垄断。实验室前期通过广谱甲基化保护,初步实现了部分限制性内切酶的国产化,但工艺仍停留在低密度摇瓶培养水平,产量无法满足市场需求。研究以限制性内切酶Pst Ⅰ为对象,通过单因素试验筛选了诱导温度、诱导时间、诱导剂浓度、补充碳源、pH值与诱导时机等多个发酵条件。结果表明,在37℃下,开始发酵后6 h,添加终浓度1.4 mmol/L IPTG进行诱导发酵8 h,以甘油为补充碳源,控制发酵体系pH为8.0时Pst Ⅰ的表达量最高,Pst Ⅰ蛋白产量为0.0129 g/L,为未优化的2.15倍,同时酶产率为1.926×10^(6)U/L,为未优化的3.74倍。
Restriction endonuclease is one of the most important enzymes in modern biomedicine.Due to the cytotoxicity of restriction endonucleases in digesting host DNA during recombinant expression, the production of restriction endonucleases was difficult, and foreign companies had monopolized their supply.Previously, our laboratory had produced several restriction endonucleases successfully through broad methylation protection.However, the production process relied on low-density shake flask culture, and the yield was unable to meet the market demand.In this study, we optimized the fermentation conditions of recombinant restriction endonuclease Pst Ⅰ.Several fermentation conditions such as induction temperature, induction time, induction agent concentration, supplementary carbon source, pH, and induction timing were screened by single-factor test.The results showed that the highest expression of Pst Ⅰ was achieved at the following conditions: fermentation temperature at 37 ℃,6 h after the start of fermentation with the addition of a final concentration of 1.4 mmol/L IPTG for 8 h, pH of the fermentation system at 8.0,and using glycerol as a supplemental carbon source.The yield of Pst Ⅰ protein was 0.0129 g/L,2.15 times as much as before optimization, while active enzyme yield was 1.926×10^(6)U/L,3.74 times as much as before optimization.
作者
张建
张新亚
李婷婷
罗志丹
许恒皓
卢辰
ZHANG Jian;ZHANG Xinya;LI Tingting;LUO Zhidan;XU Henghao;LU Chen(Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening,Jiangsu Ocean University,Lianyungang 222005,China;Co-Innovation Center of Jiangsu Marine Bio-industry Technology,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Institute of Marine Resources Development,Lianyungang 222005,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2022年第5期100-105,共6页
Food and Fermentation Industries
基金
国家自然科学基金项目(31501763)
连云港市高新区科技项目(ZD201919)。
关键词
限制性内切酶
PstⅠ
重组表达
restriction endonuclease
PstⅠ
recombinant expression
