背景:生物催化生产L-膦丝菌素(L-PPT)是目前最有前途的方法。在这项工作中,我们使用共表达D-氨基酸氧化酶和过氧化氢酶的大肠杆菌菌株(E.大肠杆菌DAAO-CAT)将生物催化D-PPT氧化为PPO,然后使用共表达谷氨酸脱氢酶和甲酸脱氢酶的第二个大肠杆菌菌株(E.大肠杆菌GluDH-FDH)将生物催化的PPO还原为L-PPT。
结果:我们比较了不同浓度的IPTG或乳糖对5L发酵罐中蛋白质表达和酶活性的影响。大肠杆菌DAAO-CAT的最佳诱导条件为0.05mMIPTG,28℃诱导18小时DAAO和CAT的比酶活性分别为153.20Ug-1和896.23Ug-1,分别。大肠杆菌GluDH-FDH的最佳诱导条件为0.2mMIPTG,在28℃诱导19小时GluDH和FDH的比酶活性分别为41.72Ug-1和109.70Ug-1,分别。大肠杆菌DAAO-CAT生物催化200mMD-PPT4h,时空产率为9.0g·L-1·h-1,转化率超过99.0%。然后用大肠杆菌GluDH-FDH将220mMPPO转化为L-PPT3h,时空产率为14.5g·L-1·h-1,转化率超过99.0%。据我们所知,这是生产L-PPT最有效的生物催化反应。
结论:我们发现与乳糖相比,IPTG在大肠杆菌DAAO-CAT和大肠杆菌GluDH-FDH的酶活性和生物量方面具有优势,IPTG更环保。我们的数据暗示,IPTG在经济可行性和扩大工业发酵的有效性方面可以替代乳糖。
BACKGROUND: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT.
RESULTS: We compared the effects of different concentrations of
IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM
IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g-1 and 896.23 U g-1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM
IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g-1 and 109.70 U g-1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production.
CONCLUSIONS: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and
IPTG is more environmentally friendly. Our data implicated that
IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations.