Abstract:

Engineering strain was acquired by transforming directly evolved plasmid from the incubated conservation bacterium E. coli BL21（DE3）pLysS/PET-15b-dhaT’-24 to the host cell E. coli BL21（DE3）pLysS. The lactose induced engineering strain was fermented to acquire 1,3-propanediol oxidoreductase（PDOR）with 182 U/mL activity. The optimal reaction pH was 10 and the pH stabile range was 7.0—9.0. The optimal reaction temperature was 55 ℃ and stabile temperature range was 30—45 ℃. 3-Hydroxypropionaldehyde（3-HPA）was catalysed by the PDOR to produce 1,3-propanediol（1,3-PD）. The reaction was coupled with another reaction of glycerol dehydrogenase（GDH，acquired from another engineering strain）to realize NADH regeneration. Thus，1,3-PD coupling enzymatic catalysis was constructed. Due to the two enzymes from engineering strains showed suitable characteristics，the reaction was continued for 34 hours and 63.4% translation rate of 3-HPA，64.6% 1,3-PD production rate were acquired.

摘要：

培养定向进化后的质粒保藏菌E. coli BL21（DE3）pLysS /PET-15b-dhaT’-24 并进行质粒抽提，将抽提的质粒转化入感受态宿主细胞E. coli BL21（DE3）pLysS中得产1,3-丙二醇氧化还原酶的工程菌。工程菌经乳糖诱导后进行发酵培养获得酶活为182 U/mL的1,3-丙二醇氧化还原酶，最适反应pH值为10，pH值稳定范围为7.0～9.0，最适反应温度为55 ℃，温度稳定范围为30～45 ℃。利用工程菌产的1,3-丙二醇氧化还原酶进行转化3-羟基丙醛为1,3-丙二醇的反应，同时偶联甘油脱氢酶（由另一工程菌制备）转化甘油的反应进行辅酶NADH的再生，实现了1,3-丙二醇的双酶耦合的连续反应。由于来源于工程菌的双酶酶学性质相适应，反应连续进行34 h后，底物3-羟基丙醛的转化率达63.4%，产物1,3-丙二醇的产率达64.6%。