重组大肠杆菌高效分泌表达β-木糖苷酶发酵条件的优化

Optimization of fermentation conditions of recombinant β-xylosidase extracellularly expressed in E.coli

【目的】嗜热拟青霉β-木糖苷酶基因在大肠杆菌中高效分泌表达重组β-木糖苷酶摇瓶发酵条件优化,及5 L发酵罐放大培养。【方法】通过单因素试验对诱导剂种类及其添加量、诱导起始OD600、培养温度、培养时间进行优化研究。【结果】摇瓶优化结果表明:2%乳糖为诱导剂、培养温度为33°C、OD600控制在0.8?0.9时诱导为最佳产酶条件,在此条件下培养48 h后胞外酶活达到103.9 U/mL,胞外分泌的比例高达99%以上。进行5 L发酵罐放大培养,发酵48 h胞外酶活达到最高值392.5 U/mL,蛋白含量为10.1 g/L。【结论】该重组大肠杆菌高效分泌β-木糖苷酶,具有较好的工业化生产前景。

[Objective] The fermentation conditions for production of recombinant β-xylosidase from Paecilomyces thermophila expressed in E. coli were optimized and produced in a 5 L fermentor. [Methods] Single factor experiments were used to investigate the effect of different inducers and their concentrations, initial cell density, cultivation temperature and time on the expression of recombinant β-xylosidase. [Results] The results showed that the β-xylosidase could be induced by IPTG and lactose, lactose was better than that of IPTG. Under the optimal conditions by adding 2% lactose at a initiated 006oo of 0.8-0.9 and incubation for 48 h at 33 ℃ the ratio of extracellular enzyme reached over 99% and enzyme activity was 103.9 U/mL. Further optimization of the culture conditions were performed in 5 L fermentor, the highest β-xylosidase activity of 392.5 U/mL and extracellular protein concentration of 10.1 g/L were obtained after 48 h. [Conclusion] The recombinant β-xylosidase with high level production will be useful in various industrial applications.