产胸苷磷酸化酶菌株初筛方法的建立及其应用

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OD_290nm远大于胸苷OD_290nm,从而导致发酵液OD_290nm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。

Establishing Pre-screening Method to Select Thymidine Phosphorylase Strains and Its Application

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 25mmol/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 OD_290nm of 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 20s, after stopping 5min irradiate 10s, 15s or 20s 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.