摘要
建立了一种快速简便地筛选高产胸苷磷酸化酶菌株的初筛方法及其在紫外诱变育种中的应用。结果显示,在液体培养基中,添加25mmol/L胸苷不会影响菌体生长且菌体中的胸苷磷酸化酶能催化胸苷生成胸腺嘧啶,在同等浓度下产物胸腺嘧啶OD290nm远大于胸苷OD290nm,从而导致发酵液OD290nm显著升高,据此建立产胸苷磷酸化酶短乳杆菌的初筛方法。在此基础上结合紫外诱变条件的优化,确定初次紫外照射时间为20 s,停止5 min后再照射10 s、15 s、20 s,控制紫外致死率在80%左右,以此条件构建突变文库。通过初筛和复筛确定突变株EB27、EA42酶活分别达到1.025 U/mg湿菌体和0.916 U/mg湿菌体,较初始菌株提高了50%和35%,且具有良好的遗传稳定性。
A suitable screening method to select thymidine phosphorylase high-product strains was quickly established,which was applied to the UV mutagenesis breeding. The results showed that adding 25 mmol / L or less thymidine in the liquid medium did not affect the cell growth. The thymidine can be converted into thymine by the catalysis of thymidine phosphorylase in the bacterial and the OD290 nmof product thymine is much higher than thymidine under the same concentration which can lead to significant increase of the fermentation broth.Based on the above principles,effective pre-screening method was established. Furtherly,the UV mutagenesis time was optimized as follows： initial mutagenesis time was 20 s,after stopping 5min irradiate 10 s,15s or 20 s to create mutant library by controlling the mortality rate about 80%. Through pre-screening and re-screening,the thymidine phosphorylase activity of EB27,EA42 could reach 1. 025 U / mg wet cells and 0. 916 U / mg wet cells,which was increased by 50% and 35% than that of the original strain. The high-product strains remained stable after transferred for 5 times.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2014年第12期78-83,共6页
China Biotechnology
关键词
短乳杆菌
初筛方法
紫外诱变
胸苷磷酸化酶
Lactobacillus brevis Pre-screening method UV mutagenesis Thymidine phosphorylase
