Kim Sae-Jin, Park Hye Won, Shin Chang-Hun, Kim Chan-Wha
School of Life Sciences and Biotechnology, Korea University, Seoul.
Biosci Biotechnol Biochem. 2009 Feb;73(2):299-303. doi: 10.1271/bbb.80507. Epub 2009 Feb 7.
A cryopreservation condition for D-amino acid oxidase (DAAO)-overexpressing Escherichia coli (E. coli BL21(DE3)/pET-DAAO) was established. Ten percent was the optimum concentration of glycerol as a cryoprotectant, and its diffusion into stationary phase cells was superior to that into log cells. The results also showed that rather than fast cooling, a slow cooling method was appropriate to our recombinant E. coli. In addition, 15 min was the best equilibration period, at which higher than 90% of recovery rates were maintained at all test points. Most importantly, the relative recovery rates, product yield, and fermentation pattern of the cell banks (CBs) constructed according to our cryopreservation method did not change over 12 months, confirming that our method not only permits exceptional cryopreservation, but offers prolonged productivity. Taken together, our results demonstrating a cryopreservation method for E. coli BL21(DE3)/pET-DAAO provide insight into an improvement in the industrial production of DAAO.
建立了过表达D-氨基酸氧化酶（DAAO）的大肠杆菌（E. coli BL21(DE3)/pET-DAAO）的冷冻保存条件。作为冷冻保护剂，甘油的最佳浓度为10%，其扩散到稳定期细胞中的效果优于对数期细胞。结果还表明，对于我们的重组大肠杆菌，缓慢冷却方法比快速冷却更合适。此外，15分钟是最佳平衡期，在此期间所有测试点的回收率均保持在90%以上。最重要的是，根据我们的冷冻保存方法构建的细胞库（CBs）的相对回收率、产物产量和发酵模式在12个月内没有变化，这证实了我们的方法不仅能实现优异的冷冻保存，还能延长生产效率。综上所述，我们关于大肠杆菌BL21(DE3)/pET-DAAO冷冻保存方法的研究结果为DAAO的工业化生产改进提供了思路。
