一种快速筛选重组耐高温β-葡聚糖酶大肠杆菌的方法

A Rapid Screening Method for Isolating High Temperature Resistant β-glucanase Producing Strain of Recombinant Escherichia coli

目的:建立一种快速筛选重组耐高温葡聚糖酶大肠杆菌(Escherichia coli)菌株的方法,期望以此为高通量筛选模型,通过体外定向进化后得到在不改变耐高温性能的同时提高其纤维素酶解活性的突变体。方法:以刚果红染色法为基础,带有耐高温葡聚糖酶基因cel12B重组质粒的E.coli菌株依次经过通过菌落的原位尼龙膜转印、裂解、底物和酶的高温反应、显色、脱色一系列步骤,建立了完整的筛选流程,并考察了诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)用量和加入方式、转印前重组菌的培养时间、平板上的菌落密度、筛选平板中琼脂的用量、筛选平板的孵育温度与时间对筛选效果的影响。结果:当在诱导平板上预先涂上4μL5mg/mL诱导剂IPTG,于37℃放置2-3h后,再将适当菌液涂在平板上于同样温度诱导15h并控制每个平板上的菌落密度为50个/板,筛选平板中琼脂浓度为20g/L,筛选平板的孵育温度为70℃、时间为3h时产生的透明圈形状规则、大小合适、分布均匀,筛选效果最好。

Objective： The author has established a rapid screening method for isolating high temperature resistant β-glucanase producing strain of recombinant Escherichia coli. Using this high throughput screening model, those mutants which have higher activity without changing the high temperature resistance of recombinant enzyme will be expected to be obtained by directed evolution in vitro. Methods： Based on the method of Congo Red staining, the recombinant E. coli strains carrying cell2B gene encoding a thermostable β-glucanase were transferblotted to another plate in situ with nylon membrane and lysed sequentially, then the substrate CMC reacted with the released recombinant enzyme under high temperature. Finally, the resulting plate was stained by Congo Red and destained, thus, the whole screening procedure was established. In the process, the effect of the amount of IPTG in inductive plate, adding method of IPTG, culture time of recombinant before transblotting, colony density in plate, agar amount in screening plate, incubation temperature and incubation time in screening plate on transparent circles was studied. Results：The optimal conditions for obtaining regular suitable transparent circles in shape were as follows： the amount of inducer IPTG in inducer plate was 4 μL at a concentration of5 mg/mL, and the plate was incubated for 3 b at 37℃ , then the recombinant cells in plate where the colony density was 50 colonies/plate were cultured for 15 h at 37℃ , and the concentration of agar in plate was 20 g/L, incubation temperature and incubation time in screening plate was 70℃, 3 h respectively. Conclusion：The screening method for isolating thermostable β-glucanase Producing Strain of Recombinant Escherichia coli is fast, accurate, quite operational.