工程菌发酵产酶及其在1,3-丙二醇耦合酶催化制备中的应用

Enzyme from engineering strain and application in 1,3-propanediol coupling-enzymatic catalysis

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%。

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.